The detection, characterization, and monitoring of SARS-CoV-2 recombinant variants is a challenge to public health authorities worldwide. Recombinant variants, composed of two or more SARS-CoV-2 lineages, often have unknown impacts on transmission, immune escape, and virulence in the early stages of emergence. We examined 4,213 SARS-CoV-2 recombinant SARS-CoV-2 genomes collected between 2020 and 2022 from California to describe regional and statewide trends in prevalence. Many of these recombinant genomes, such as those belonging to the XZ lineage, or novel recombinant lineages, likely originated within the state of California. We discuss the challenges and limitations surrounding Pango lineage assignments, the use of publicly available sequence data, and adequate sample sizes for epidemiologic analyses. Although these challenges will continue as SARS-CoV-2 sequencing volumes decrease globally, this study enhances our understanding of SARS-CoV-2 recombinant genomes to date while providing a foundation for future insights into emerging recombinant lineages.

L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compare activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimised and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the ‘in-gel’ staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria.

Background: Despite a range of available treatments, there is a remaining unmet need for patients with severe post-partum hemorrhage (sPPH). Objective: This manuscript evaluated efficacy and safety of recombinant activated factor VIIa (rFVIIa) in sPPH management. Methods: An open-label, multi-center, randomized controlled trial (RCT; NCT00370877) and four observational studies (OS; OS-1 [NCT04723979], OS-2, OS-3 and OS-4) were analyzed regarding efficacy (need for subsequent invasive procedures, including uterine compression sutures, uterine or iliac artery ligation, arterial embolization or hysterectomy) and safety (incidence of thromboembolic events [TE] and maternal mortality) of rFVIIa for sPPH. The RCT, and OS-1 and OS-2, included a control group of women who did not receive rFVIIa (with propensity score-matching used in OS-1 and OS-2), whereas OS-3 and OS-4 provided descriptive data for rFVIIa exposed women only. Results: A total of 446 women exposed to rFVIIa and 1,717 non-exposed controls were included. In the RCT, fewer rFVIIa exposed women (50% [21/42]) had an invasive procedure versus non-exposed women (91% [38/42]; odds ratio 0.11, 95% confidence interval 0.03–0.35). In OS-1, more rFVIIa exposed women (58% [22/38]) had an invasive procedure versus non-exposed women (35% [13.3/38]; odds ratio 2.46, 95% confidence interval 1.06–5.99). In OS-2, 17% (3/18) of rFVIIa exposed women and 32% (5.6/17.8) of non-exposed women had an invasive procedure (odds ratio 0.33, 95% confidence interval 0.03–1.75). Across all included women, TEs occurred in 1.5% (0.2% arterial and 1.2% venous) of rFVIIa exposed women and 1.6% (0.2% arterial and 1.4% venous) of non-exposed women with available data. Conclusions: The positive treatment effect of rFVIIa in the RCT was not confirmed in the OS. However, the safety analysis did not show any increased incidence of TEs with rFVIIa treatment.

Background: Approximately 10-20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated if immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations can be overcome by immunization with VVX001 (i.e., Alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by ELISA and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by CFSE staining and subsequent FACS analysis. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG1 subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the NTCP attachment site. IgG reactivity to sub-viral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A-H. A pronounced reactivity of CD3+CD4+ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusion: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.

Abstract: Plasma hemopexin (HPX) is the key antioxidant protein of the endogenous clearance pathway that limits the deleterious effects of heme released from hemoglobin and myoglobin. During intra-vascular hemolysis, heme partitioning to protein and lipid increases as the plasma concentration of HPX declines. Therefore, the development of HPX as a replacement therapy during high heme stress could be a relevant intervention for hemolytic disorders. A logical approach to enhance HPX yield involves recombinant production strategies from human cell lines. The present study focuses on a biophysical assessment of heme binding to recombinant human HPX (rhHPX) produced in the Expi293FTM (HEK293) cell system. In this report, we examine rhHPX in comparison with plasma HPX using a systematic analysis of protein structural and functional characteristics related to heme binding. Analysis of rhHPX by UV/Vis absorption spectroscopy, circular dichroism (CD), SEC-HPLC and catalase-like activity demonstrated a similarity to HPX fractionated from plasma. In particular, the titration of HPX apo-protein(s) with heme was performed for the first time using a wide range of heme concentrations to model HPX-heme interactions to approximate physiological conditions (from extremely low to more than 2-fold heme excess). The CD titration data showed an induced bisignate CD Soret band pattern typical for plasma and rhHPX versions at low heme-to-protein molar ratios and demonstrated that further titration is dependent on the amount of protein-bound heme to the extent that the arising opposite CD couplet results in a complete inversion of the observed CD pattern. The data generated in this study suggests more than one binding site in both plasma and rhHPX. Further, our study provides a useful analytical platform for detailed characterization of HPX-heme interactions and potentially, novel HPX fusion constructs.

Fowlpox virus (FPV) has been used as a vector for many years. To date, surprisingly, very little data exist on the persistence of fowlpox vaccines in chicken tissues, or what kind of immune cells respond to the vaccination at the site of inoculation. Although both humoral and cellular mediated immunity (CMI) play a part in overall immunity against FPV, little is known regarding the cell-mediated immune responses to FPV infection. The main aim of this paper was to measure persistence of the recombinant fowlpox vaccine in the skin tissues following vaccination. The results showed that the recombinant FPVs do not persist for long, and are cleared by 6 days post-vaccination (dpv). The response of immune cells (macrophages, B cells, CD4+ and CD8+ T cells) that infiltrating the site of vaccination was estimated.

Recombinant laboratory plasmids (RLPs) are common in biological research and freely shared among academic research laboratories (ARLs), a practice required by many research funding agencies. However, the generation of accurate, reproducible results in experiments utilizing RLPs can be hampered by a lack of accompanying sequence information and metadata. This culture of RLP sharing without knowledge of sequence or etiology is accepted by publishers, not regulated by governments, and outside the realm of bio-industry. In addition, no centralized infrastructure currently exists to collate such data, which at the moment is fragmented across companies, non-profits, and governments and thus is not easily accessed or enacted toward threat assessment. The ubiquity, free exchange, and dual-use risk of RLPs exemplifies a biosecurity threat and elevates the need to characterize their composition to facilitate improved biorisk management by the academic community. A number of common sense solutions are available to create a culture that addresses the biosecurity gap posed by RLP sharing. Culture shift in RLP management will require new norms, effective data management for collation of RLP sequences and metadata, and an incentive structure that encourages sequencing by stakeholders. The next generation of researchers must initiate and champion this shift with support from funding agencies and endorsement from governments and international organizations. Coordination of efforts and stakeholders will require international public/private collaboration, a structure that will be critical to ensure widespread utility as well as the ability of lower-resourced partners to participate, contribute, and benefit.

Industrial effluents contain contaminants that pose threats to the environment and human health. Bioremediation is an environmentally friendly, sustainable, and cost-effective approach to degrade or transform pollutants using microbial enzymes. Laccase is a versatile enzyme that oxidizes a wide range of substrates, such as dyes, phenolic compounds, pesticides, and polycyclic aromatic hydrocarbons (PAHs), with the consumption of molecular oxygen. Laccase can also remove heavy metals by forming complexes with organic ligands or reducing them to fewer toxic forms. However, the application of laccase in bioremediation is limited by its low production, stability, and specificity. Recombinant technology has been employed to enhance the expression, activity, and stability of laccase in various bacterial hosts. Immobilization techniques have been developed to improve the reusability and stability of laccase in different environmental conditions. This review summarizes the recent developments in the production optimization of recombinant laccase enzymes and their role in the bioremediation of industrial effluents. It also discusses the challenges of laccase-based biocatalytic systems for environmental cleanup.

With the COVID-19 pandemic now ongoing for close to a year, people all over the world are still waiting for a vaccine to become available. The initial focus of accelerated global research and development efforts to bring a vaccine to market as soon as possible was on novel platform technologies that promised speed but had limited history in the clinic. In contrast, recombinant protein vaccines, with numerous examples in the clinic for many years, missed out on the early wave of investments from government and industry. Emerging data are now surfacing suggesting that recombinant protein vaccines indeed might offer an advantage or complement to the nucleic acid or viral vector vaccines that will likely reach the clinic faster. Here, we summarize the current public information on the nature and on the development status of recombinant subunit antigens and adjuvants targeting SARS-CoV-2 infections.

Recombinant α1-microglobulin (A1M) is proposed as protector during 177Lu-octreotate treatment of neuroendocrine tumors, which is currently limited by bone marrow and renal toxicity. Co-administration of 177Lu-octreotate and A1M could result in a more effective treatment by protecting healthy tissue, but, the radioprotecting action of A1M is not fully understood. The aim of this study was to examine the proteomic response of kidneys and bone marrow early after 177Lu-octreotate and/or A1M administration. Mice were injected with 177Lu-octreotate and/or A1M, while control mice received saline or A1M vehicle solution. Bone marrow, kidney medulla, and kidney cortex were sampled after 24 hours or 7 days. Differential protein expression was analyzed with tandem mass spectrometry. Dosimetric estimation was based on 177Lu activity in kidney. PHLDA3 was the most prominent radiation responsive protein in kidney tissue. In general, no statistically significant difference in expression of radiation-related proteins was observed between the irradiated groups. Most canonical pathways were identified in bone marrow from the 177Lu-octreotate+A1M group. Altogether, tissue-dependent proteomic response followed exposure to 177Lu-octreotate alone or together with A1M. Combining 177Lu-octreotate with A1M did not inhibit the radiation induced protein expression early after exposure, and late effects should be further studied.

A large variety of fermentation products are used in food and feed production, but also in other industries, and many of these products are produced with genetically modified microorganisms (GMMs). In food and feed production, prominent examples are amino acids, vitamins, food and feed enzymes, colorants, non-caloric sweeteners and human milk oligosaccharides. From a regulatory perspective, fermentation products are typically produced under containment. This means that premises, equipment and work processes need to be designed to prevent or at least minimize release of GMMs into the environment. The fermentation products themselves should not contain any live cells of the GMM. Over the past years, there have been concerning developments, particularly in the European Union, stipulating that also absence of recombinant DNA might be interpreted as a regulatory requirement for fermentation products produced with GMMs. In this paper, we (i) attempt to place these developments into the historical context, (ii) sketch the potential negative repercussions for the food and feed industries, (iii) elaborate on the safety of recombinant DNA, and (iv) postulate that recombinant DNA should remain an integral part of the safety assessment of fermentation products but should not be misconstrued as a criterion for regulatory classification of products of biotechnology.

This study aimed to produce single-chain recombinant eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Chinese hamster ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit plays contributes on high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M) in which a linker including the eCG β-subunit CTP region (amino acids 115 to149) was inserted between the β-subunit and α-subunit of wild-type single chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells. Single cells expressing each protein were isolated from 10 and 7 clones, respectively. Secretion increased gradually during the cultivation period and peaked at 4,000–5,000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34–40 kDa, while that of eel FSH-M increased substantially, with two bands at 39–46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker showed potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well-known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum. Cytoplasm obtained from different E. coli strains was examined as an example of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

Bovine herpesvirus (BoHV)-5 is a worldwide distributed pathogen usually associated with a lethal neurological disease (meningoencephalitis) in dairy and beef cattle resulting in important economic losses due to the cattle industry. Using recombinant glycoprotein D of BoHV-5 (rgD5), we evaluated the long-duration humoral immunity of the recombinant vaccines in a cattle model. Here we report that two doses of intramuscular immunization, particularly with the rgD5ISA vaccine, are superior to iBoHV-5ISA immunization in the induction of long-lasting antibody responses. Recombinant gD5 antigen elicited tightly mRNA transcription of the Bcl6 and the chemokine receptor CXCR5 which mediate memory B cells and long-lived plasma cells in germinal centers (GCs). In addition, using an in-house Enzyme-Linked Immunosorbent Assay (ELISA) we observed higher and earlier responses of rgD5-specific IgG antibody and the upregulation of mRNA transcription of IL2, IL4, IL10, IL15 and IFN-γ cytokines in rgD5 vaccinated cattle, indicating a mixed immune response. We further show that rgD5 immunization provides protection against both BoHV -1 and -5. Our findings indicate that the rgD5-based vaccine represents an effective vaccine strategy to induce an efficient control of alpha-herpesviruses.

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eCG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the eel LH-wt and eel LH-M plasmids were isolated from five and nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5,000–7,500 ng/mL on day 9. The molecular weight of eel LH-wt was 32–36 kDa while that of eel LH-M increased to approximately 38–44 kDa, indicating the detection of two bands. Treatment with peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC50 value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new rec-eel LH analog which contains the eCG β-subunit CTP linker comprising O-linked glycosylation sites, had more potent activity than the wild-type in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

The pandemic SARS-CoV-2 continues to evolve to give rise to variants of concern that can escape vaccine-induced immunity. As such, more effective vaccines are urgently needed. In this study, we evaluated virus-like particle (VLP) as a vaccine platform for SAS-CoV-2. The spike, envelope, and membrane proteins of the SARS-CoV-2 Wuhan strain were expressed by a single recombinant baculovirus BacMam and assembled into VLPs in cell culture. The morphology and size of the SARS-CoV-2 VLP as shown by transmission electron microscopy was found to be similar to the authentic SARS-CoV-2 virus particle. In a mouse trial, two intramuscular immunizations of the VLP BacMam with no adjuvant elicited spike-specific binding antibodies in both sera and bronchoalveolar lavage fluids. Importantly, BacMam VLP -vaccinated mouse sera showed neutralization activity against SARS-CoV-2 spike pseudotyped lentivirus. Our results indicated that the SARS-CoV-2 VLP BacMam stimulated spike-specific immune responses with neutralization activity.

In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. Soon, a recombinant virus (MYXV-Tol or ha-MYXV) was identified, harboring a 2.8 kb insertion containing evolved duplicates of M060L, M061L, M064L, and M065L from MYXV. Since 2017, 1616 rabbits and 82 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ ha-MYXV strains. A subset (20%) of the positive samples was tested for the insert with MYXV being detected in rabbits and recombinant MYXV in hares. Recently, two wild rabbits found dead in South Portugal, showing skin oedema and pulmonary lesions tested positive for the 2.8 Kb insert. Sequencing showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs of both rabbits by electron microscopy, and isolation in RK13 cells showed virus infectivity. Despite the analysis of recombinant MYXV genomes may predict its ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, which raises serious concerns for the future of the Iberian wild leporids and emphasizes the need to continue monitoring MYXV and recombinant MYXV in both species.

Growth factors, chemokines and cytokines guide tissue regeneration after injuries. However, their applications as recombinant proteins are almost non-existent due to the difficulty of maintaining their bioactivity in the protease-rich milieu of injured tissues in humans. Safety concerns have ruled out their systemic administration. The vascular system provides a natural platform for circumvent the limitations of the local delivery of protein-based therapeutics. Tissue selectivity in drug accumulation can be obtained as organ-specific molecular signatures exist in the blood vessels in each tissue, essentially forming a postal code system (“vascular zip codes”) within the vasculature. These target-specific “vascular zip codes” can be exploited in regenerative medicine as the angiogenic vasculature forming in the regenerating tissues has a unique molecular signature. The identification of vascular homing peptides capable of finding these unique “vascular zip codes” after their systemic administration provides an opportunity for the target-specific delivery of therapeutics to tissue injuries. Therapeutic proteins can be “packaged” together with homing peptides by expressing them as multi-functional recombinant proteins. These multi-functional recombinant proteins provide an example how molecular engineering gives a compound an ability to home to regenerating tissue and enhance its therapeutic potential. Regenerative medicine has been dominated by the locally applied therapeutic approaches despite these therapies are not moving to clinical medicine with success. There might be a time to change the paradigm towards systemically administered, target organ-specific therapeutic molecules in future drug discovery and development for regenerative medicine

Poliovirus 2A protease (PV2Apro) plays a vital role in viral replication and down-regulation of host cell protein synthesis. In order to understand more concerning PV2Apro, the protein was over-expressed in bacteria following amplification using sense and antisense primers and cloning in pET15b. Several expression hosts were tested and BL21 (DE3) pLysS cells gave the best expression of PV2Apro with minimal unwanted protein expression following IPTG induction. The 2Apro protein was purified to homogeneity using column chromatography, its solubility determined and its molecular weight and composition determined by MALDI-TOF mass spectrometry. The protease was found in the insoluble fraction and the purified protein had a slightly lower molecular weight than predicted. Moreover, three dimensional structure was modelled using template 1z8r with 58% identity and validated using ramachandran plot. Results revealed that most of the residues lie in favoured and allowed regions. These findings could help in a better understanding of PV2Apro structure and inhibition thus, highlighting potential targets for antiviral drug development.

Verticillium wilt (VW) is an important and widespread disease of cotton and once established is long lived and difficult to manage. In Australia, the non-defoliating pathotype of Verticillium dahliae is the most common, and extremely virulent. Breeding cotton varieties with increased VW resistance is the most economical and effective method of controlling this disease and is greatly aided by understanding the genetics of resistance. In this study, VW resistance was investigated in 240 F7 recombinant inbred lines (RIL) derived from a cross between MCU-5 that has good resistance and Siokra 1-4 which is susceptible. Using a controlled environment bioassay, we found that resistance based on plant survival or shoot biomass was complex but with major contributions from chromosomes D03 and D09, with genomic prediction analysis estimating a prediction accuracy of 0.73 based on survival scores compared to 0.36 for shoot biomass. Transcriptome analysis of MCU-5 and Siokra 1-4 roots uninfected or infected with V. dahliae revealed that the two cultivars displayed very different root transcriptomes and responded differently to V. dahliae infection. Ninety-nine differentially expressed genes were located in the two mapped resistance regions and so are potential candidates for further identifying the genes responsible for VW resistance.

Objective To observe the effect of roxadustat on blood pressure and micro-inflammatory response in patients with hemodialysis anemia, and to provide a viable technique for the treatment of hemodialysis anemia patients. Methods A total of 100 hemodialysis anemia patients admitted to the Nephrology Department of our hospital from July 2020 to July 2021 were enrolled and randomly divided into an observation group (roxadustat) and a control group (human recombinant erythropoietin (rhEPO). The clinical efficacy, blood lipid metabolism, inflammatory factor levels, iron metabolism-related indicators, blood biochemical indicators, anemia indicators, adverse reactions and blood pressure were recorded and compared. Results There was no significant difference in baseline data and serum indicators (P>0.05). The study group demonstrated superior performance to the control group in terms of clinical efficacy, blood lipid metabolism, inflammatory factor levels, iron metabolism-related indicators, blood biochemical indicators, anemia indicators, adverse reactions and blood pressure (all P<0.05). Conclusion Compared with rhEPO treatment, roxadustat exhibits a promising outcome in treating anemia and has slight impact on blood pressure of patients.

99mTc is a well-known radionuclide widely used and readily available for medical imaging. Despite this, the 99mTc isotope is rarely utilized for tumour imaging due to the lack of agents that can selectively deliver it to tumours. However, the use of immunologically compatible recombinant proteins, which combine metal-binding sites and tumour-specific ligands, shows promise for utilizing 99mTc in the early detection of tumours. To illustrate this, a protein called E1b, containing a metal binding center with ELP repeats, tumour-specific ligands recognizing integrin αvβ3 and nucleolin, as well as a short Cys-rich sequence, was artificially designed. This protein was produced in E. coli and purified to homogeneity using metal-chelate chromatography. In experiments, C57Bl/6 and Balb/C mice with subcutaneously grafted lung carcinoma (LLC) or mammary gland adenocarcinoma (Ca755, EMT6, or 4T1) were used to test 99mTc-E1b selectivity and the kinetics of isotope retention. The mice were intravenously injected with approximately 80 MBq of 99mTc in complex with E1b, which had been freshly purified using gel-chromatography. The biodistribution of the labeled protein was then studied using the SPECT/CT imaging method within 24 h. The ratio of radionuclide distribution in the tumour versus the adjacent normal tissue (T/N) steadily increased over the 24-h period, reaching 15.7±4.2 for EMT6, 16.5±3.8 for Ca755, 6.7±4.2 for LLC, and 7.5±3.1 for 4T1.

The use of recombinant bovine somatotropin (rbST) leads to an increase, in variable amounts, of milk production in buffalo, but there is a lack of information on the influence of rbST on their metabolism. This study looked at the effects of a single 500 mg dose of rbST on lipid profile, liver and kidney function and physical, chemical and cellular constitution of milk in 14 buffalo over 14 days, from the 100th day of lactation, and compared them with 14 animals in a control group. From the 1st day after rbST, there was a rise in beta hydroxybutyrate (β-HBO), possibly due to the higher dry matter intake or by biotransformation of NEFA into β-HBO. Treatment did not influence blood glucose, non-esterified fatty acids (NEFA), triglycerides, cholesterol, total protein, albumin, AST, GGT, bilirubin, urea or creatinine. In 71.3% of the buffalo there was a gradual increase in milk production with maximal response in the first week that was then followed by a gradual decrease, whilst in 21.4%, the increase in production occurred between 7 and 10 days, and only 7.1% of animals did not respond. On the 3rd, 5th, 7th and 10th days after treatment, an increase was found in milk production between the two groups equal to 1.04, 1.52, 1.42 and 1.06 liters, respectively. In relative terms this means an increase in milk production, respectively, of 15.1%; 21.0%; 19.8 % and 15.1 %. The constitution of the milk showed no difference in the amounts of fat, lactose, total solids or somatic cell count; however, on the 3rd day after rbST, there was a decrease in protein; notably, from the 5th day, protein values show no statistical difference. It can be concluded that the application of rbST in buffalo from the 100th day of lactation is metabolically safe, since the treatment neither caused imbalances in fat metabolism nor overloaded the liver or renal function, and the changes in milk composition were transient and limited to a decrease in milk protein.

Background: Visceral leishmaniasis (VL) is s critical public health problem in over ninety countries. The control measures adopted in Brazil have been insufficient when it comes to preventing the spread of this neglected disease. In this context, a precise diagnosis of VL in dogs and humans could help to reduce the number of cases of this disease. Distinct studies for the diagnosis of VL have used single recombinant proteins in serological assays; however, results have been variable, mainly in the sensitivity of the antigens. The development of multiepitope-based proteins could be relevant in solving such a problem. Methods: A chimeric protein (rMELEISH) was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat shock proteins HSP70 and HSP 83.1, and tested in ELISA for the detection of L. infantum infection in humans and dogs. Results: rMELEISH was able to discriminate between VL cases and cross-reactive diseases and healthy samples, with sensitivity and specificity values of 100% as compared to the use of a soluble Leishmania antigenic extract (SLA). Conclusions: Preliminary data suggest that the rMELEISH protein presents a potential to be tested in future studies against a larger serological panel for the diagnosis of canine and human VL.

As recommended in the ICH Guidelines (S5-R2 and S6-R1), and based on bioethical concerns, we chose bovine embryos (BE) to check the in vitro embryo development considering the use of different amounts of rHGAL-1 as supplementations of in vitro embryo culture (IVP) mediums. Based on procedures for commercial BE in vitro production, using oocytes aspirated from slaughterhouse ovaries, the rHGAL-1 supplementation performed in two experiments (#01 on the oocyte maturation - IVM medium supplemented and experiment #2 on culture step IVC, supplemented SOF medium). There were IVP commercial procedures done, with 3 IVP batches per experiment and distributed the oocytes in four groups of treatment (one control group and three different dosages of rHGAL-1 to supplement both IVM and SOF mediums, using (2, 20 and 40µg.mL-1 respectively). A total of 962 (experiment 1) and 1,213 (experiment 2) oocytes were aspirated and submitted to IVP procedure. There was no damage to in vitro bovine embryos growth, considering cleavage percentage (%CLE), blastocysts development on day 7 (BlD7, BxD7, BhD7), or hatching blastocysts maturation on day 8 (BhD8%), regardless of rHGAL-1 supplementation. The immunohistochemistry assay with D8 embryos cultivated with rHGAL-1 supplementation on the culture medium (SOF medium) could demonstrate the presence of exogenous GAL-1, distributed in mass cell and trophoblastic cells, and the profile observed is dependent of exogenous supplementation and it was more evident in hatched embryos. The findings reassure the use of a reasonable amount of rHGAL-1 for in vitro embryonic development and make using rHGAL-1 in assisted reproduction in humans more reliable and safer.

One of the most interesting groups of fatty acid derivates is the group of conjugated fatty acids, from which the most researched are conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), which are associated with countless health benefits. Sex pheromone mixture of some insect species, including tobacco horn-worm (Manduca sexta), are typical for the production of uncommon C16 long conjugated fatty acids with two and three conjugated double bonds, as opposed to C18 long CLA and CLNA. In this study, M. sexta desaturases MsexD2 and MsexD3 were expressed in multiple strains of Y. lipolytica with different genotypes. Experiments with supplementation of fatty acid methyl esters into the medium resulted in production of novel fatty acids. Using GCxGC-MS 20 new fatty acids with two or three double bonds were identified. Fatty acids with conjugated or isolated double bonds or combination of both were produced in trace amounts. Results of this study prove that Y. lipolytica is capable of synthesizing C16 conjugated fatty acids. Further genetic optimization of the Y. lipolytica genome and optimization of the fermentation process could lead to increased production of novel fatty acid derivatives with biotechnologically interesting properties.

Avian infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis, which causes considerable economic losses to the poultry industry worldwide. It is imperative to develop safe and efficient candidate vaccines to control IBV infection. In the current study, recombinant baculoviruses co-expressing S1 and N proteins, mono-expressing S1 or N proteins alone of IBV were constructed and prepared into subunit vaccines rHBM-S1-N, rHBM-S1 and rHBM-N. The levels of immune protection of these subunit vaccines were evaluated by inoculating specific pathogen-free (SPF) chickens at 14 days of age, boosting with the same dose 14 days later, and following challenge with a virulent GX-YL5 strain of IBV 14 days post-booster (dpb). The commercial vaccine strain H120 was used as a control. The IBV-specific antibody levels as well as the percentages of CD4+ and CD8+ T lymphocytes were detected within 28 days post-vaccination (dpv). The morbidity, mortality, and re-isolation of virus from the tracheas and kidneys of challenged birds were evaluated at 5 days post-challenge (dpc). The results showed that the IBV-specific antibody levels and the percentages of CD4+ and CD8+ T lymphocyte in rHBM-S1-N group were higher than those of rHBM-S1 and rHBM-N groups, especially the cellular immunity response. At 5 dpc, the mortality, morbidity and virus re-isolation rate of rHBM-S1-N were slightly higher than those of H120 group, but were lower than those of rHBM-S1 group and rHBM-N group. The present study demonstrated that the protection of recombinant baculovirus co-expressing S1 and N proteins was better than that of recombinant baculoviruses mono-expressing S1 or N protein alone. Thus, the recombinant baculovirus co-expressing S1 and N proteins could serve as a potential IBV vaccine and this demonstrates that the bivalent subunit vaccine including the S1 and N proteins might be a strategy for the development of an IBV subunit vaccine.

Lectins, characterized by their carbohydrate-binding ability, have an extensive practical application. However, their industrial use is limited by low yields, and few active recombinant lectins have been reported. In this study, the algal lectin BPL-3 (Bryopsis plumosa lectin 3) was successfully produced using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong hemagglutination activity using human erythrocytes, similar to that of native BPL3. rD2BPL3 had a similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N-acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 had binding activity to the beta conformation. The process developed in this study was suitable for the quality-controlled production of high amounts of soluble recombinant lectins.

(1) The study using the quails of the Manchurian golden breed was aimed at the efficacy of the food supplement based on micro-incapsulated phytase of Obessumbacterium proteus in the recombinant yeast producer Yarrowia lipolytica at a dose of 500 FYT/kg of the feed. (2) The effect of the supplement in the poultry diets on the productivity of young and adult quails was assayed; (3) It was found that the keeping the quails on the diets using the microencapsulated phytase O. proteus provided the highest live weight, the highest intensity of egg production, and the slaughter yield of animals, the lowest feed conversion compared to those in control groups and the groups getting a commercial phytase supplement based on Aspergillus ficuum. Taken together the data obtained in our experiments indicate a high degree of assimilation of phytate-containing products by the quails when using the encapsulated phytase compared to both the control and commercial preparation of phytase of the PhyA class. It can be concluded that it is a highly expedient and promising supplement based on the microencapsulated phytase of class D for feeding, which shows the best kinetic features compared to the phytases of PhyA and PhyC classes when working in the chyme of the poultry.

D-Glucaric acid is an important bio-based building block of polymers and is a high value-added chemical that can be used in a variety of applications. In the present study, the Udh target genes from Pseudomonas putida and Pseudomonas syringae were used together to construct the expression vector pETDuet-2×Udh. The transformants of BL21(DE3) with vector pETDuet-2×Udh was applied to produce glucaric acid from glucuronic acid. After optimization of induction conditions, the highest Udh expression were achieved when 0.4 mmol L-1 isopropyl-β-d–thiogalactoside (IPTG) was added to cell cultures at an OD600 value of 0.6 followed by culture at 26℃ for 6 hours. The production of glucaric acid substantially reached 5.24 ± 0.015 g L-1 in fed-batch cultures in a 30 L tank. In the present study, a new system for glucaric acid production was established, which was more economic and friendly to environment.

Background: This study investigated the biological effects on circulating monocytes after challenge with SARS-CoV-2 recombinant spike protein. Methods: Whole blood collected form seven ostensibly healthy healthcare workers was incubated for 15 min with 2 and 20 ng/mL recombinant spike protein of Ancestral, Alpha, Delta and Omicron variants. Samples were analyzed with Sysmex XN and DI-60 analyzers. Results: Cellular complexity (i.e., presence of granules, vacuoles and other cytoplasmic inclusions) increased in all samples challenged with the recombinant spike protein of Ancestral, Alpha and Delta variants, but not in those containing Omicron. The cellular content of nucleic acids was constantly decreased in most samples, achieving statistical significance in those containing 20 ng/mL of Alpha and Delta recombinant spike proteins. The heterogeneity of monocyte volumes significantly increased in all samples, achieving statistical significance in those containing 20 ng/mL of recombinant spike protein of Ancestral, Alpha and Delta variants. The monocyte morphological abnormalities after spike protein challenge included dysmorphia, granulation, intense vacuolization, platelet phagocytosis, development of aberrant nuclei and cytoplasmic extrusions. Conclusions: The SARS-CoV-2 spike protein triggers important monocyte morphological abnormalities, more evident in cells challenged with spike protein of the more clinically severe Alpha and Delta variants.

An HPLC method for indicating stability was developed, created, and confirmed for the quantification measurement of recombinant human erythropoietin in large quantities as well as dosage forms. An isocratic separation was accomplished using a Thermo Biobasic C18 (250 mm 4.6 mm i.d., 300Ao, 5 m) column with a flow rate of 1.0 mL min-1 as well as a UV detector to monitor the eluate at 278 nm. Acetonitrile, 0.05 mM potassium dihydrogen phosphate (80:20 v/v), and orthophosphoric acid adjusted to 4.0 made up the mobile phase. The drug was exposed to oxidative stress, hydrolysis, and photodegradation to simulate stress conditionsRecombinant Human Erythropoietin, the parent compound, was eluted at roughly 6.675 minutes, as well as all byproducts have been entirely disconnected inside an overall analytical run time of about 15 minutes. To identify quantification limits of 0.05 and 0.2 g mL-1, respectively, the method was linear over a concentration range of 1-6 g mL-1 (r = 0.9989). Recombinant Human Erythropoietin can be measured accurately, selectively, sensitively, and precisely using this method both in dosage form and in bulk. Stress-induced degradation products did not interact with the identification of Recombinant Human Erythropoietin, indicating that the assay is stable.

Staphylococcus aureus mastitis remains a major challenge for dairy farming. Here, 24 mice were immunized and divided into four groups: G1: control; G2: Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) DNA vaccine; G3: F0F1 ATP synthase subunit α (SAS), succinyl-diaminopimelate (SDD), and cysteinyl-tRNA synthetase (CTS) recombinant proteins; and G4: SAS+SDD+CTS plus GM-CSF DNA vaccine. The lymphocyte subpopulations and the intracellular interleukin-17A (IL-17A) and interferon-γ production in the draining lymph node cells were immunophenotyped by flow cytometry. The immunophenotyping and lymphocyte proliferation was determined in spleen cells cultured with and without S. aureus stimulus. Immunization with S. aureus recombinant proteins generated memory cells in draining lymph nodes. Immunization with the three recombinant proteins plus GM-CSF DNA led to an increase in the percentage of IL-17A+ cells among overall CD44+ (memory), T CD4+, CD4+ T CD44+ CD27-, γδ TCR, γδ TCR+ CD44+ CD27+ and TCRVγ4+ cells. Vaccination with S. aureus recombinant proteins associated with GM-CSF DNA vaccine downregulates TH2 immunity. Immunization with the three recombinant proteins plus the GM-CSF DNA led to a proliferation of overall memory T, CD4+ and CD4+ TEM cells upon S. aureus stimulus. This approach fostered type 3 immunity, suggesting the development of a protective immune response against S. aureus.

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS-CoV, SARS-CoV-2 does not bind human DPP4.

Antibody-mediated targeting is an efficient strategy to enhance the specificity and selectivity of polymer nanomedicines towards the target site, typically a tumor. However, direct covalent coupling of an antibody with a polymer usually results in a partial damage of the antibody binding site accompanied with a compromised biological activity. Here, an original solution based on well-defined non-covalent interactions between tris-nitrilotriacetic acid (trisNTA) and hexahistidine (His-tag) groups, purposefully introduced to the structure of each macromolecule, is described. Specifically, trisNTA groups were attached along the chains of a hydrophilic statistical copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA), and at the end or along the chains of thermo-responsive di-block copolymers based on N-isopropylmethacrylamide (NIPMAM) and HPMA; His-tag was incorporated to the structure of a recombinant single chain fragment of an anti-GD2 monoclonal antibody (scFv-GD2). Static and dynamic light scattering analyses confirmed that mixing of polymer with scFv-GD2 led to the formation of polymer/scFv-GD2 complexes; those prepared from thermo-responsive polymers formed stable micelles at 37 °C. Flow cytometry and fluorescence microscopy clearly demonstrated antigen-specific binding of the prepared complexes to GD2 positive murine T-cell lymphoma cells EL-4 and human neuroblastoma cells UKF-NB3, while no interaction with GD2 negative murine fibroblast cells NIH-3T3 was observed. These non-covalent polymer protein complexes represent a new generation of highly specific actively targeted polymer therapeutics or diagnostics.

Introduction: Adverse events related to vaccines are reactions that can lead to serious complications, in the present that we live at the publication of this article with the need to generate high rates of vaccination have been presented in the case of vaccine agents against COVID-19 adverse events, with thrombotic events being the most important. Case Presentation: This is a 37-year-old female patient who presents 11 days after ChAdOx1-S vaccination against COVID-19 presents with intense headache associated with symptoms of intracranial hypertension, she was initially taken to simple cranial tomography showing presence of cerebral edema and indirect signs of cerebral venous thrombosis, it is proposed to carry out cerebral angiography which showed the presence of thrombosis of both transverse venous sinuses, no hypercoagulability states were found in the laboratories, the immunological tests carried out for antiphospholipid syndrome were negative, the thrombotic event was associated with the ChAdOx1-S vaccine, later the patient was taken to thrombectomy by Neuroradiology, achieving complete channeling of both transverse venous sinuses. Conclusions: Adverse events associated with vaccination have been described with multiple vaccine agents, this component against COVID-19 is also an event to consider, knowing the pathophysiology of these events is essential to be able to reduce these risks and select the least risky agent. Despite this risk, vaccination remains the only cost-effective measure to reduce the morbidity and mortality associated with COVID-19.

Recombinant human collagen peptide (RCP) is a recombinantly created xeno-free biomaterial enriched in RGD (arginine-glycine-aspartic acid) sequences, with good processability that is being investigated for regenerative medicine applications. Recently, the biocompatibility and osteogenic ability of β-TCP/RCP (RCP granules combined with β-tricalcium phosphate (TCP) submicron particles) were demonstrated. In the present study, β-TCP/RCP was implanted into experimental periodontal tissue defects (three-walled bone defect) created in beagle dogs to investigate tissue responses and subsequent regenerative effects. Micro computed tomography image analysis at 8 weeks postoperatively showed that the amount of new bone after β-TCP/RCP graft was significantly greater (2.2 fold, P&lt;0.05) than that of the control (no graft) group. Histological findings showed that the transplanted β-TCP/RCP induced active bone-like tissue formation including TRAP-positive and OCN-positive cells as well as bioabsorbability. Ankylosis did not occur, and periostin-positive periodontal ligament-like tissue formation was observed. Histological measurements revealed that β-TCP/RCP implantation formed 1.7-fold more bone-like tissue and 2.1-fold more periodontal ligament-like tissue than the control, and significantly suppressed gingival recession and epithelial downgrowth (P&lt;0.05). These results suggest that β-TCP/RCP is effective as a periodontal tissue regenerative material.

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and performed further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen is secreted normally from the patient’s hepatocytes but had accelerated degradation by plasmin in the circulation.

Breast cancer (BC) is among the leading causes of mortality from cancer in women. Many of the available anticancer drugs have various side effects. Therefore, researchers are seeking novel anticancer agents particularly from natural compounds and in this regard, snake venom is still one of the main sources of drug discovery. Previous studies showed potential anticancer effects of Cytotoxin II (CTII) from Naja naja oxiana against the different types of cancers. In this study, a pET-SUMO-CTII vector was transformed into SHuffle® T7 Express, an Escherichia coli strain, for recombinant protein expression (rCTII) and the cytotoxic effects of this protein was assessed in MCF-7 cells. The flow cytometry assay was applied to measure the apoptosis and cell cycle. Also, mRNA levels of the Bax, Bcl2, P53, caspase-3, caspase-8, caspase-9, caspase-10, matrix metalloproteinases (MMP)-3, and MMP-9 were analyzed by quantitative real-time PCR to determine the underlying cellular pathways affected by rCTII. The results of this study showed that treatment with 4 µg mL-1 of rCTII enhanced apoptosis through the intrinsic and extrinsic pathways. Also, the increase of the cells' proportion in the sub-G1 phase as well as a reduction in S phase was observed. In addition, the expression of MMP-3 and MMP-9 was decreased in the treated group in comparison to the control group that may contribute to the reduced migratory ability of tumor cells. These experimental results indicate that rCTII has anti-proliferative potential, and so this protein could be a potential drug for BC therapy in combination with other drugs.

One of the main objectives of developing new anti-cancer vaccine strategies is to effectively induce CD8+ T cell-mediated anti-tumor immunity. Live recombinant vectors, notably Listeria mono-cytogenes, have been shown to elicit a robust in vivo CD8+ T cell response in preclinical settings. Significantly, it has been demonstrated that Listeria induces inflammatory/immunogenic cell death mechanisms such as pyroptosis and necroptosis in immune cells that favorably control immunological responses. Therefore, we postulated that the host's response to Listeria-based vec-tors and the subsequent induction of CD8+ T cell-mediated immunity would be compromised by the lack of regulatory or effector molecules involved in pyroptosis or necroptosis. To test our hy-pothesis, we used recombinant L. monocytogenes carrying the ovalbumin gene (LM.OVA) to vac-cinate wild-type (WT), caspase-1/11-/-, gsdmd-/-, ripk3-/-, and mlkl-/- C57Bl/6 mice. We performed an in vivo cytotoxicity assay to assess the efficacy of OVA-specific CD8+ T lymphocytes in eliminat-ing target cells in wild-type and genetically deficient backgrounds. Furthermore, we evaluated the specific anti-tumor immune response in mice inoculated with B16F0 and B16F0.OVA mela-noma cell lines. Our findings demonstrated that while caspase-1/11 and GSDMD deficiencies in-terfere with the rapid control LM.OVA infection, of the KO seems to contribute to the early acti-vation of OVA-specific CTL responses. In contrast, individual deficiency of each one of these pro-teins negatively impact the generation of long-lasting effector CD8+ T cells.

Rabbit hemorrhagic disease virus disease 2 (RHDV2) emerged in the United States in 2018 and has spread in both domestic and wild rabbits nationwide. The virus has a high mortality rate and can spread rapidly between infected individuals. Vaccination against RHDV2 provides the best protection against disease and should be considered by all rabbit owners. Here, we investigate the duration of immunity provided by vaccination with the Medgene Platform conditionally licensed commercial vaccine 6 months following the initial series. Rabbits received either the vaccination or a placebo and were challenged with RHDV2 6 months later. All vaccinated rabbits survived challenge whereas 18/19 non-vaccinated controls succumbed to infection within 10 or fewer days post-challenge. These results demonstrate lasting immunity following vaccination with the Medgene RHDV2 vaccine.

Soy leghemoglobin (LegH) has been gaining interest over the last years as an efficient flavor and aroma compound in plant-based meat substitutes. Hence, in the following article we demonstrate the methods for LegH production using a recombinant Komagataella phaffii strain. Multiple fed-batch fermentation with an alternative to BSM medium, where glucose was used as the main carbon source, were implemented and the growth kinetics, e.g., maximal specific biomass growth - 0.239 g·g−1·h−1, biomass yield from substrate - 0.298 g·g−1 and maximal specific substrate consumption rate - 0.81 g·g−1·h−1, were identified. Leghemoglobin production resulted in a yield of 0.513 mg·gDCW 1, while the highest biomass density achieved in the study was 121.80 gDCW·L 1. The applied medium showed potential for additional optimization studies, as in contrast to BSM, made it possible to separate pH control from nitrogen supply, does not affect medium turbidity measurements and does not induce metabolite synthesis during yeast biomass growth.

Background: Chagas disease is still a neglected disease considered a public health problem. Early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome the problems currently faced. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. Methods: The rTC was constructed based on amino acid sequences from different combinations of Trypanossoma cruzi antigens in the same polypeptide and tested in ELISA for detecting different Chagas disease. Results: rTC1 was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, and healthy samples, with 96.6% sensitivity and 97.96% specificity, respectively. Conclusion: These data suggest a preliminary study that rTC1 has the po-tential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.

Gelsolin is an actin-binding protein that is competitively bound by lysophosphatidic acid (LPA), a possible biomarker for the early detection of ovarian cancer. Our group has previously shown the usage of gelsolin 1-3 (a fragment of gelsolin composed of the first three of its six subdomains) along with actin to detect the presence of lysophosphatidic acid. This histidine-tagged gelsolin 1-3 fragment is synthesized in our lab using bl21 Rosetta cells but required further characterization. This study aims to provide sufficient characterization of gelsolin 1-3 synthesized in our lab via both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry using a Fourier-transform ion cyclotron resonance mass spectrometer (FTICR-MS). Both techniques show the presence of protein at the expected mass of approximately 41 kDa. Additionally, the dissociation of a smaller fragment of the complex when introduced into the gas phase which was equivalent to the approximate mass of one subdomain of gelsolin 1-3 further supports this conclusion.

A recent study revealed a persistent presence of the PP-Spike recombinant protein in 50% of individuals who received ModRNA injections, contrasting with unvaccinated controls, whether SARSCoV-2 infected or not, up to 187 days post-injection. The implications of these findings are discussed in the context of PP-Spike recombinant protein-induced hyperimmune/inflammatory reactions and thrombotic vasculitis as mechanisms to elucidate the serious side effects, long-term organ complications, and excess deaths observed after administering Covid ModRNA products. These results underscore the urgent need for a comprehensive reassessment of the risk-benefit profile of ModRNA injections, including an immediate halt in their use for any person, and a renewed emphasis on understanding and addressing their long-term effects on human health

In the face of climate change, bringing more useful alleles and genes from wild relatives of wheat is crucial to develop climate-resilience varieties. We used two populations of backcrossed recombinant inbred lines (BIL1 and BIL2), developed using two intra-specifically diverse Aegilops tauschii accessions from lineage 1 and lineage 2, respectively, and backcrossing to common wheat cultivar ‘Norin 61’. The study aimed to identify quantitative trait loci (QTL) associated with heat stress (HS) tolerance. The two BILs were evaluated under heat stress environments in Sudan for phenology, plant height (PH), grain yield (GY), biomass (BIO), harvest index (HI), and thousand kernel weight (TKW). Grain yield was significantly correlated with BIO and TKW under HS; therefore, stress tolerance index (STI) was calculated for these traits as well as for GY. A total of 16 heat tolerant lines were identified based on GY and STI-GY. The QTL analysis performed using inclusive composite interval mapping identified a total of 40 QTLs in BIL1 and 153 QTLs in BIL2 across all environments. We detected 39 QTLs associated with GY-STI, BIO-STI, and TKW-STI in both populations (14 in BIL1 and 25 in BIL2). The QTL associated with STI were detected on chromosomes 1A, 3A, 5A, 2B, 4B, and all the D-subgenomes. We found that QTL detected only under HS for GY on chromosome 5A, TKW on 3B and 5B, PH on 3B and 4B, and grain filling duration on 2B. The higher number of QTLs identified in BIL2 for heat stress tolerance suggests the importance of assessing the effects of intraspecific variation of Ae. tauschii in wheat breeding as it could modulate the heat stress responses/adaptation. Our study provides useful genetic resources for uncovering heat-tolerant QTLs for wheat improvement for heat stress environments.

A B S T R A C T Genital herpes disease is caused mainly by herpes simplex virus type 2 (HSV-2), which is sexually transmitted with a high prevalence in both developed and developing countries. No vaccine is currently available against genital herpes. In this study, we introduced and expressed two genes encoding HSV-2 glycoprotein D (gD) and VP16 protein and a GFP marker gene (control) in tobacco seedlings. Positive plant infection was assessed through characteristic tobacco mosaic virus disease symptoms on leaves and by monitoring the fluorescence emitted by the expressed GFP protein. Expression of the HSV-gD2 and VP16 antigens was verified by RT-PCR, ELISA and Western blot. As a proof of concept, the immunogenicity and the protection ability of the plant produced gD antigen was tested in a mouse model of genital herpes and compared to gD antigen produced in a mammalian expression system. This showed that the plant-gD preparation, when used in combination with a CpG oligodeoxynucleotide as adjuvant, was highly immunogenic and capable of inducing complete immunity to lethal vaginal HSV-2 challenge in mice. Thus, the data presented here may have implications for the development of a production system for highly immunogenic plant-based HSV-2 vaccine antigens.

Polyethylene glycol (PEG) is an inert, water soluble polymer, used for decades in pharmaceuticals. Although PEG is considered safe, concerns persist about the potential adverse effects of long-term exposure to PEG-containing therapies, specifically in children, following the introduction of PEGylated recombinant factor products used for the treatment of hemophilia. Given the absence of long-term surveillance data, and to evaluate the potential risk, we estimated PEG exposure in the pediatric population receiving US Food and Drug Administration-approved parenteral therapies with pediatric indications. We used a range of pediatric weights and doses based on prescribing information (PI) or treatment guidelines. PIs and reporting websites were searched for information about adverse events (AEs). For a child weighing 50 kg on the highest prophylactic dose of a FVIII product, the range of total PEG exposure was 40–21,840 mg/year; for FIX products, the range was 13–1342 mg/year; and for other products, the range was 383–26,743 mg/year, primarily as a derivative excipient. No AE patterns attributable to PEG were found for any of these products, including potential renal, neurological, or hepatic AEs. Our analyses suggest the pediatric population has had substantial exposure to PEG for several decades, with no evidence of adverse consequences.

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare glycoprotein-mediated entry efficiencies of: VSV G, SARS-CoV-2 S, EBOV GP, LASV GP, and CHIKV E we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase-PEST (NLucP) reporter gene, which we used in combination with the live-cell substrate Endurazine™ to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

Background: COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants are dominating. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. Objectives: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. Methods: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro by protein chemical methods, mass-spectrometry, circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study specific antibody and cytokine responses, safety and Omicron neutralization. Results: Defined and pure immunogens could be produced in large amounts as secreted and folded proteins in mammalian cells. Antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, RBD-specific IgG levels induced by the different vaccines were comparable but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were 7- and 2-fold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively and 6-fold higher than those of the bivalent vaccine. Conclusion: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

In mammals, the role of interleukin-18 (IL-18) in the immune response is to drive inflammatory and, normally therefore, anti-viral responses. IL-18 also shows promise as a vaccine adjuvant in mammals. Chicken IL-18 (chIL-18) has been cloned. The aim of this study was to investigate the potential of chIL-18 to act as a vaccine adjuvant in the context of a live recombinant Fowlpox virus vaccine (fpIBD1) against Infectious bursal disease virus (IBDV). fpIBD1 protects against mortality, but not against damage to the bursa of Fabricius caused by IBDV infection. The Fowlpox virus genome itself contains several candidate immunomodulatory genes, including potential IL-18 binding proteins (IL-18bp). We have knocked out (Δ) the potential IL-18bp genes in fpIBD1 and inserted (::) the cDNA encoding chIL-18 into fpIBD1 in the non-essential ORF030, generating five new viral constructs –fpIBD1::chIL-18, fpIBD1ΔORF073, fpIBD1ΔORF073::chIL-18, fpIBD1ΔORF214 and fpIBD1ΔORF214::chIL-18. The subsequent protection from challenge with virulent IBDV, as measured by viral load and bursal damage, given by these altered fpIBD1 strains, was compared to that given by the original fpIBD1. The results show that chIL-18 can act as a vaccine adjuvant, giving complete protection against challenge, with no detectable virus or damage in the bursa of Fabricius.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the Receptor-Binding Domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5 and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of high antibody cross-reactivity by ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants by sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

Nebulized thrombolysis offers locally targeted therapy with potentially lower bleeding risk than systemic administration for coronavirus disease 2019 (COVID-19) respiratory failure. In a proof-of-concept safety study, adult patients with COVID-19-induced respiratory failure and a <300mmHg PaO2/FiO2 (P/F) ratio, requiring invasive mechanical ventilation (IMV) or non-invasive respiratory support (NIRS) received nebulized rt-PA in two cohorts (C1 and C2), alongside standard of care during the first two UK COVID-19 waves. Matched historical controls (MHC; n=18) were used in C1. Safety co-primary endpoints were treatment-related bleeds and fibrinogen reduction to <1.0–1.5 g/L. A dose escalation strategy for improved efficacy with the least safety concerns was determined in C1 for use in C2; patients were stratified by ventilation type to receive 40–60 mg rt-PA per day for ≤14 days. Nine patients in C1 (IMV, 6/9; NIRS, 3/9) and 26 in C2 (IMV, 12/26; NIRS, 14/26) received nebulized rt-PA for a mean (SD) of 6.7 (4.6) and 9.1(4.6) days, respectively. Four bleeding events (one severe and three mild) in three patients were considered treatment-related. No significant fibrinogen reductions were reported. Greater improvement in mean P/F ratio from baseline to end of study was observed in C1 compared with MHC [C1; 154 to 299 vs MHC; 154 to 212). In C2, there was no difference in the baseline P/F ratio of NIRS and IMV patients. However, a larger improvement in P/F ratio was observed in NIRS patients [NIRS; 126 to 240 vs IMV; 120 to 188) and they required fewer treatment days (NIRS; 7.86 vs IMV; 10.5). Nebulized rt-PA appears to be well-tolerated, showing a trend of improved oxygenation and faster recovery in patients with acute COVID-19-induced respiratory failure requiring respiratory support; this effect was more pronounced in the NIRS group. Further investigation is required to study the potential of this novel treatment approach.

A SARS-CoV receptor-binding domain (RBD) recombinant protein was developed and manufactured under current good manufacturing practices in 2016. The protein known as RBD219-N1 when formulated on Alhydrogel®, induced high-level neutralizing antibodies and protective immunity with minimal immunopathology in mice after a homologous virus challenge with SARS-CoV (MA15 strain). In this report, we examined published evidence in support of whether the SARS-CoV RBD219-N1 could be repurposed as a heterologous vaccine against Coronavirus Infectious Disease (COVID)-19. Our findings include evidence that convalescent serum from SARS-CoV patients can neutralize SARS-CoV-2. Additionally, a review of published studies using monoclonal antibodies (mAbs) raised against SARS-CoV RBD and that neutralize the SARS-CoV virus in vitro, finds that some of these mAbs bind to the receptor-binding motif (RBM) within the RBD, while others bind to domains outside this region within RBD. This information is relevant and supports the possibility of developing a heterologous SARS-CoV RBD vaccine against COVID-19, especially due to the finding that the overall high amino acid similarity (82%) between SARS-CoV and SARS-CoV-2 spike and RBD domains is not reflected in RBM region (59%). However, the high similarity (94%) in the region outside of RBM offers the potential of conserved neutralizing epitopes between both viruses.

Bone morphogenetic protein (BMP) and platelet-derived growth factor (PDGF) are known to regulate and stimulate osteogenesis, both proteins playing an important role in bone homeostasis, constituting good candidates for osteoporosis´ treatment. Herein we aim to evaluate the effects of recombinant human BMP-7 (rhBMP7) and PDGF-BB (rhPDGF-BB) in oophorectomy-induced osteoporosis in rats. To this end, 40 Sprague-Dawley rats were submitted to oophorectomy surgery and treatments were started at the 100th day after the surgery, when all animals showed signs of osteoporosis. These peptide growth factors were applied intravenously once or twice a week and animals were followed for a total of five weeks. Two weeks after the end of the treatments, the animals were euthanized, the tissues were collected and Alkaline Phosphatase, X-ray, micro-CT and histology were assessed. The results indicate that the most promising treatments are: 20 µg/kg PDGF-BB + 30 µg/kg BMP-7 2x/week and 30 µg/kg BMP-7 2x/week, presenting an increase of 15% (p&lt;0.05) and 13% (p&lt;0.05) in bone volume fraction and 21% (p&lt;0.05) and 23% (p&lt;0.05) in trabecular number, respectively. In conclusion, rhPDGF-BB and rhBMP-7 are capable of inducing an increase in bone volume and density in this osteoporotic animal model, constituting interesting candidates for osteoporosis treatment.

To map Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV 2) spreading and evaluate immune responses variations against this virus, it was essential to locally set up efficient serological tests. The SARS-CoV2 immunogenic proteins were very expensive and not affordable mainly for Low and Middle Income Countries (LMICs). For this purpose, the commonly used antigen, Re-ceptor-Binding Domain (RBD) of Spike S1 protein (S1RBD), was produced using the the Baculovirus Expression Vector System (BEVS) . During the current study, the expression of S1RBD was monitored using western blot under different culture conditions. Different parameters were studied: Multiplicity Of Infection (MOI), cell density at infection and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3x 106 cells/mL and time post-infection (tpi or harvest time) of 72h and 72-96 h successively for expression in shake-flasks and 7L-bioreactor. A high production yield of S1RBD varying between 4mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in shake-flasks and in 7L-bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 patient sera evaluated by western blot. Thus, additional serological assays, such as ELISA, were developed using the purified S1RDB (published by other research groups).

Recently, we published that the monoclonal antibody (D12 mAb) recognizes gp63 of L. mexicana, it is responsible for COX activity. This D12 mAb exhibited cross-reactivity with Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba castellanii, and Naegleria fowleri. COX activity assays performed in these parasites suggested the potential presence of such enzymatic activity. In our investigation, we confirmed that wild-type recombinant gp63 exhibits COX-like activity, in contrast to a mutated recombinant gp63 variant. Consequently, our objective was to identify sequences orthologous to gp63 and subsequently analyze the binding of arachidonic acid (AA) to the putative active sites of these proteins. Given the absence of a crystallized structure for this protein in the Protein Data Bank (PDB), it was imperative to first obtain a three-dimensional structure by homology modeling, using leishmanolysin (PDB ID: LML1) as a template in the Swiss model. Database. The results obtained through molecular docking simulations revealed the primary interactions of AA close to the Zinc atom present in the catalytic site of gp63-like molecules of several parasites, predominantly mediated by hydrogen bonds with HIS264, HIS268 and HIS334. Furthermore, COX activity was evaluated in commensal species such as E. dispar and during the encystment process of E. invadens.

Porcine epidemic diarrhea virus (PEDV) has affected the pork industry worldwide and during outbreaks the mortality of piglets has reached 100%. Lipid nanocarriers are commonly used in the development of immunostimulatory particles due to their biocompatibility and slow-release delivery properties. In this study, we developed a lipid nanoparticle (LNP) complex based on glycyrrhizinic acid (GA) and tested its efficacy as an adjuvant in mice immunized with the recombinant N-terminal domain (NTD) of porcine epidemic diarrhea virus (PEDV) spike (S) protein (rNTD-S). The dispersion stability analysis (Z-potential–27.6 mV) confirmed the size and charge stability of the LNP-GA, demonstrating that the particles were homogeneously dispersed and strongly anionic, that favors nanoparticles binding with the rNTD-S protein, which showed a slightly positive charge (2.11 mV) by in silico analysis. TEM image of LNP-GA revealed nanostructures with a spherical-bilayer lipid vesicle (⁓100 nm). The immunogenicity of the LNP-GA-rNTD-S complex induced an efficient humoral response 14 days after the first immunization (P &lt; 0.05) as well as an influence on the cellular immune response by decreasing serum TNF-α and IL-1β concentrations, which was associated with an anti-inflammatory effect.

Research for novel vaccine adjuvants remains a critical need to improve the immune responses to a recombinant vaccine antigen. Emulsion adjuvants such as AS03 and MF59, particularly in the area of Influenza vaccines, have shown antigen dose sparing and allowed reduced immunizations. It was previously demonstrated that these emulsion adjuvants can be formulated using a simpler, low shear process of self-emulsification. The role of alpha tocopherol as an immunomodulator in emulsion adjuvants is evident from the success of AS03 in the on-going covid-19 pandemic. Although, it was a challenge to formulate alpha tocopherol with a low shear process to get closer to the AS03 composition, the self-emulsified version referred as self-emulsifying adjuvant systems (SE-AS) showed comparable immune responses to AS03 when co-administered with a Quadrivalent influenza virus (QIV) vaccine. In this paper, we first optimized the SE-AS with alpha tocopherol referred to as SE-AS44 to allow sterile filtration. We compared the in vitro cytokine profile with self-emulsifying adjuvant 160 (SEA160), a squalene-only self-emulsified adjuvant with composition similar to MF59. We compared SE-AS44 and SEA160 co-administered with a recombinant cytomegalovirus (CMV) pentamer antigen, which is a less immunogenic antigen, in vivo to compare the antibody and T-cell responses, in different adjuvanted groups, in C57BL/6 mice.

Achievement of solid fusion of the lumbosacral junction (L5-S1 level) is an important factor in adult spinal deformity (ASD) surgery. The purpose of this study is to explore the effectiveness and feasibility of injectable rhBMP-2 (a combination of Eschericia coli-derived rhBMP-2 and hydrogel type β-TCP carrier; NOVOSIS Inject) as a bone substitute for the fusion of lumbosacral junction. 20 patients (average age 69.1 years) diagnosed with ASD with sagittal imbalance who underwent surgical treatment including anterior lumbar interbody fusion (ALIF) in the L5-S1 level were evaluated. Injectable rhBMP-2 was applied in L5-S1 ALIF and followed-up for 1-year. Solid fusion rates and changes of clinical outcomes (Oswestry Disability Index [ODI], Visual Analog Scale [VAS] of back and leg) were measured and analyzed at 6 and 12 months after surgery. All postoperative adverse events were evaluated about the association with injectable rhBMP-2. Fusion rates were 68.4% and 100% at 6 and 12 months after surgery. Compared to baseline, ODI were improved to 45.8% and 63.7%, VAS(back) were improved to 69.2% and 72.8%, and VAS(leg) were improved to 49.2% and 64.8% at 6 and 12 months after surgery (p&lt;0.001, respectively). There were no adverse events associated with injectable rhBMP-2. Thus, injectable rhBMP-2 may be a suitable choice of a bone graft substitute when achieving solid interbody fusion in the lumbosacral junction.

Background: Early diagnosis of postoperative complications is an urgent task, allowing timely prescribing of appropriate therapy and reducing the cost of patient treatment. Prognostic models based on various data could help in the early diagnosis of postoperative complications after cardiac surgery. Objectives: The purpose of the study was to determine whether an integrated approach based on clinical data along with metabolites and biomarkers had greater predictive value than the models built on less data. Methods: The study included patients (n=62) admitted for planned cardiac surgery (coronary artery bypass grafting with cardiopulmonary bypass), who were retrospectively divided into two groups depending on the presence (n=26) or absence (n=36) of postoperative complications of all types. Clinical and laboratory data on the first day after surgery were analyzed. Additionally, patients' blood samples were collected before and on the first day after surgery to determine biomarkers (interleukin-6, procalcitonin, NT-proBNP, high-sensitivity troponin T) and metabolites (8 aromatic and 2 dicarboxylic acids were detected using gas chromatography-mass spectrometry). Results: Patients before surgery, compared with healthy donors, differed in the concentration of metabolites (p &lt; 0.05), and had concentrations of a number of biomarkers exceeding the corresponding reference values. Multivariate PLS-DA models, predicting the presence or absence of postoperative complications, were built using clinical data (ROC-AUC=0.71), concentrations of metabolites and biomarkers (ROC-AUC=0.60), and the entire data set (ROC-AUC=0.75). For comparison we built univariate models using the EuroScore2 (ROC-AUC=0.59) and SOFA (ROC-AUC=0.76) scales; concentrations of lactate (ROC-AUC=0.71), the dynamic changes of 4-hydroxyphenyllactic acid (ROC-AUC=0.69), and the sum of three sepsis-associated metabolites (ROC-AUC=0.70). The proposed complex model using entire data set was comparable to the SOFA scale, which confirms the expediency of searching for new predictive models based on a variety of factors.

Milk serves as a crucial source of natural bioactive compounds essential for human nutrition and health. The gastrointestinal digestion of milk proteins releases various biologically active peptides that positively impact human health and enhance physiological performance. With the growing production of cheese, Greek yogurt, and other dairy products, the volume of unutilized acid whey is significantly increasing. Acid whey, containing almost all whey proteins and approximately one-quarter of casein, presents a valuable raw material for generating peptides with potential health benefits. These peptides exhibit properties such as antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic, antihypertensive, antithrombotic, opioid, mineral-binding, and growth-stimulating activities, contributing to improved human immunity and the treatment of chronic diseases. Enzymatic hydrolysis of milk proteins has been developed as a method to produce these peptides, facilitating their use in food fortification. To incorporate whey into functional beverages, it undergoes fermentation by lactic acid bacteria with proteolytic activity. To specifically enrich these beverages with peptides exhibiting targeted bioactivities, it is essential to select lactic acid bacteria strains possessing the requisite protease profiles. Additionally, the sensory properties of the final product can be enhanced using starter cultures with diverse metabolic capabilities, even in the absence of added flavoring agents. Given the imperative to develop waste-free technologies and enhance the profitability of the dairy industry, there is a growing need to efficiently utilize and transform acid whey into a broad spectrum of valuable products and food additives. This approach is justified not only by the nutritional benefits of the bioactive components in whey but also by the commercial potential of safe and health-enhancing products.

The paper focuses on the application of a multilevel, integrative and territorial approach to the protection of cultural heritage through planning instruments in Serbia. It points out the problems of the sectoral approach and discusses international recommendations that can be integrated into the planning methodology. The case study is the area of Djerdap National Park, which has an already established international and national importance. The research suggests that the cultural heritage in the area, with the exception of the Roman limes, is still recognized at the level of individual localities. The study emphasizes the importance of aligning all levels of governance to safeguard the whole spatial entity rather than focusing solely on its particular areas. The interna-tional and national importance of the planning area contributes to the strengthening of awareness of heritage and its institutional protection. However, the vitality of the area, which is key to its sustainable development, comes from the local level. Planning documents contribute to the inte-gration of local values and local communities into development programs and projects.

In order to shorten the detection time and improve the average precision on embedded devices, A lightweight and high accuracy model is proposed for passion fruit in complex environments (backlight, occlusion, overlap, sunny, cloudy, rainy). Firstly, replacing the backbone network of YOLOv5 with a lightweight GhostNet model reduces the number of parameters and computation while improving detection speed. Secondly, a new feature branch is added to the GhostNet network, and the feature fusion layer in the neck network is reconstructed to effectively combine the lower-level and higher-level features, which not only improves the accuracy of the model but also maintains its lightweight. Finally, the knowledge distillation methods are used to transfer the knowledge from the more capable teacher model to the less capable student model, which significantly improving the detection accuracy. The improved model is denoted as G-YOLO-NK. The average accuracy of the G-YOLO-NK network is 96.00%, which is 1.00% higher than the original YOLOv5s model. Furthermore, the improved model size is 7.14MB, reduced to half of the original model, and the real-time detection frame rate is 11.25 FPS on the Jetson Nano. Compared to the state-of-the-art model, the proposed model outperforms them in terms of average precision and detection performance. The present work provides an effective model for real-time detection of passion fruits in complex orchard scenes, which can provide valuable technical support for the development of orchard picking robots and greatly improve the intelligence level of orchards.

The impact of interaction SS with MJO on rainfall along Java Island to East Nusa Tenggara has been studied using daily rainfall observation data from 2140 weather observation stations, which are also equipped with GSMaP, OLR, and ERA5 data. There were 61 SS events found, 17 during active MJO and 44 in inactive MJO. Rainfall increases more in the western study area when the active MJO and more in the eastern study area when the inactive MJO is due to SS. SS reduces rainfall in the Jakarta region, regardless of the activity or inactivity of the MJO. SS causes extreme rainfall to only occur in a small part of certain areas, so it tends to significantly reduce the possibility of extreme rainfall. In the southern part of the IMC, SS predominates over MJO in supporting increased water vapor transport. Large-scale synoptic circulations like the monsoon, SS, and MJO can interact with local land-sea wind circulation to produce spatiotemporal variability in rainfall on Java Island. Rainfall mostly increases in the afternoon and decreases in the morning when SS occurs, whether there is MJO or not. Convective instability analysis indicates that SS increases precipitation, most likely by lowering CIN and raising VIMFC.

The Simultaneous Localization And Mapping (SLAM) scanner is an easy and portable Light Detection And Ranging (LiDAR) data acquisition device. Its main output is a 3D point cloud covering the scanned scene. Regarding the importance of accuracy in the survey domain, this paper aims to assess the accuracy of two SLAM scanners: the NavVis VLX and the BLK2GO Scanner. This assessment is conducted for both outdoor and indoor environments. In this context, two types of reference data are used: the Total Station (TS) and the static scanner Z+F Imager 5016. To carry out the assessment, four comparisons are tested: cloud to cloud, cloud to mesh, mesh to mesh, and edge detection board assessment. However, the results of the assessments confirm that the accuracy of indoor SLAM scanner measurements (5 mm) is greater than that of outdoor ones (between 10 mm and 60 mm). Moreover, the comparison of cloud to cloud provides the best accuracy regarding direct accuracy measurement without manipulations. Finally, based on the high accuracy, scanning speed, flexibility, and the accuracy differences between tested cases, it is confirmed that SLAM scanners are effective tools for data acquisition.

In recent years, electroencephalogram (EEG) emotion recognition has emerged as a research hotspot in the field of artificial intelligence (AI). Despite advancements in EEG-based emotion recognition, room for enhancing performance persists due to EEG signal redundancy and limitations in feature extraction, leading to inefficient models and emotional information loss. To fully utilize EEG's emotional information and improve recognition accuracy while reducing computational costs, this paper proposes a Convolutional-Recurrent Hybrid Network with a dual-stream adaptive approach and an attention mechanism (CSA-SA-CRTNN). Firstly, the model utilizes a CSAM module to assign corresponding weights to EEG channels. Then, an adaptive dual-stream convolutional-recurrent network (SA-CRNN and MHSA-CRNN) is applied to extract local spatial-temporal features. After that, the extracted local features are concatenated and fed into a temporal convolutional network with a multi-head self-attention mechanism (MHSA-TCN) to capture global information. Finally, the extracted EEG information is used for emotion classification. We conducted binary and ternary classification experiments on the DEAP dataset, achieving 99.26% and 99.15% accuracy for arousal and valence in binary classification, and 97.69% and 98.05% in ternary classification, surpassing relevant algorithms. Additionally, the model's efficiency is significantly higher than other models, achieving better accuracy with lower resource consumption.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in the alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more response in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

Background: Educating college students about the importance of good sleep habits could lead to the adoption of healthier sleep routines, including sufficient sleep duration and sleep quality. The current pre-post study examines the impact of completing a sleep education program called “Sleep 101” on (1) sleep hygiene practices, (2) sleep quality, and (3) weekday and weekend sleep behaviors among college students. “Sleep 101” is an interactive online course developed to educate college students about the importance of sleep by addressing basic sleep physiology, the impact of sleep on mood, academic and physical performance, the interactions between sleep and various substances, and common sleep disorders. Methods: 338 college students were recruited from a midsized public university in the US and invited to participate in “Sleep 101”, of which 25 participants completed the baseline and post-intervention questionnaire. The Sleep Hygiene Index (SHI) was utilized to evaluate sleep hygiene practices, while standard questions were employed to assess weekday and weekend sleep behaviors and overall sleep quality. Results: Those who completed the study demonstrated a significant increase in weekday sleep duration and were less likely to engage in negative prior bedtime activities. In addition, 40% of those who completed baseline and post-intervention assessments noticed improvements in their sleep quality. Conclusion: These findings suggest that a brief online program is beneficial in improving sleep behaviors among college and could be a scalable approach to addressing current sleep issues and preventing future sleep problems in college students.

The profile of secondary metabolites occurring in apple cuticular layer is not only characteristic for particular apple cultivar, but also dynamically reflects various external factors in growing environment. In this study, the possibility to authenticate apple samples based on analysis of their cuticular layer extracts was investigated. Ultra-high-performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for obtaining metabolomic fingerprints. In total, 274 authentic apple samples, four cultivars harvested within the seasons 2020 to 2022 in the Czech Republic and Poland were analysed. The generated complex data processed by univariate and multivariate statistical methods enabled to build classification models for discrimination of apple cultivars as well as their geographical origin. The models showed very good performance in differentiation of Czech and Polish samples for three of four cultivars: ‘Gala’, ‘Golden Delicious’ and ‘Idared’, moreover, the validity of the models was verified over harvest seasons. The diagnostic markers, besides metabolites of triterpene biosynthetic pathway, mainly represented wax esters. ‘Jonagold’, which is known to be susceptible to mutations, was the only cultivar for which the unambiguous classification of geographical origin was not possible.

Superior fiber quality is one of the most important objectives in cotton breeding. To detect the genetic basis underlying fiber quality, an F2 population containing 413 plants was constructed by crossing Jifeng 914 and Jifeng 173, both of which have superior fiber quality, with Jifeng 173 being better. Five fiber quality traits were investigated in the F2, F2:3, F2:4, and F2:5 populations. Quantitative trait loci (QTL) mapping was conducted based on a high-density genetic map containing 11,488 single nucleotide polymorphisms (SNPs) and spanning 4,202.12 cM in length. Transgressive segregation patterns and complex correlations in the five tested traits were observed. A total of 108 QTLs were found, including 27 for fiber length (FL), 16 for fiber strength (FS), 24 for micronaire (MC), 17 for fiber uniformity (FU), and 24 for fiber elongation rate (FE). Chromosome A7 contained 12 QTL, ranking the first. No QTL was found on chromosome D1 and D11. Each QTL contributed 1.98%–21.45% to the phenotypic variation (PV), including 13 major effect QTLs that contributed more than 10% toward PV. Two QTLs could be repeatedly detected in three populations, including qFL-D3-2 in F2, F2:4, and F2:5 with 9.18%–21.45% of PV, qFS-A11-1 in F2:3, F2:4 and F2:5 with 6.05%–10.41% of PV. Another seven stable QTLs could be detected in two populations, including four major effect QTLs: qFL-A12-3, qFS-D10-2, qMC-D6-2, and qMC-D8-1. Fourteen QTL-overlapping regions were found, which might explain the complex correlations among the five phenotypic traits. Four regions on chromosome A11, D3, D6, and D10 covered by both stable and major effect QTLs are promising for further fine mapping. The genomic regions of the two QTLs detected in three populations and the four major effect QTLs contain 810 genes. Gene functional analysis revealed that the annotated genes are mainly involved in protein binding and metabolic pathways. Fifteen candidate genes in the qFL-D3-2 region are highly expressed in fiber or ovules during fiber initiation, elongation, secondary cell wall thickening, or maturation stages. qRT-PCR revealed that Ghir_D03G005440.1 and Ghir_D03G011310.1 may play a role in promoting fiber initiation, Ghir_D03G006470.1 may be beneficial for promoting fiber elongation. This study provides more information for revealing the molecular genetic basis underlying cotton fiber quality.

Currently there are fewer depth models applied to pepper picking detection, while the existing generalized neural networks have problems such as large model parameters, long training time, and low model accuracy.In order to solve the above problems, this paper proposes a Yolo-chili target detection algorithm for chili pepper detection. First, the classical target detection algorithm yolov5 is used as a benchmark model, and an adaptive spatial feature pyramid structure combining the attention mechanism and the idea of multi-scale prediction is introduced to improve the model's detection effect on occluded peppers and small target peppers. Secondly, a three-channel attention mechanism module is introduced to improve the algorithm's long-distance recognition ability and reduce the interference of redundant testers. Finally, the quantized pruning method is used to reduce the model parameters and realize the lightweight processing of the model. Applying the method to the homemade chili pepper dataset, the AP value of chili pepper reaches 93.11%; the accuracy rate is 93.51% and the recall rate is 92.55%.The experimental results show that yolo-chili is able to achieve accurate and real-time pepper detection under complex orchards.

The temporary immersion system bioreactor named GreenTray®[2], [3]presents advantages over the existing ones. Firstly, there is no need to use forceps to move the shoots or plantlets in or out of the recipient, neither the use of scalpel to divide them. Secondly, the basis of the shoot can remain in the GreenTray® and can sprout again in several cycles of growth. These two characteristics reduce the costs in the process of micropropagation. Besides, shoots or plantlets development is enhanced by the aeration of the recipient, thus enriching with CO2 the recipient atmosphere. The chapter describes and provides images and values of interest in (1) the micropropagation process of some commercial Prunus or Pyrus rootstocks; (2) the plantlet growth under autotrophic conditions; and (2) the inoculation of plants with pathogens that reproduce in vitro the symptoms observed in vivo, using the GreenTray® bioreactor.

Aiming at the problems of inaccurate segmentation of long object and information loss of small object in real-time semantic segmentation algorithm, this paper proposes a lightweight multi-branch real-time semantic segmentation network based on BiseNetV2. The new auxiliary branch makes full use of spatial details and context information to cover the long object in the field of view. Meanwhile, in order to ensure the inference speed of the model, the asymmetric convolution is used in each stage of the auxiliary branch to design a structure with low computational complexity. In the multi-branch fusion stage, the alignment and fusion module is designed to provide guidance information for deep and shallow feature mapping, so as to make up for the problem of feature misalignment in the fusion of information at different scales, and thus reduce the loss of small target information. In order to further improve the model’s awareness of key information, a global context module is designed to capture the most important features in the input data. The proposed network uses NVIDIA GeForce RTX 3080 Laptop GPU experiment, on the road street view data set Cityscapes and CamVid average occurring simultaneously ratio reached 77.1% and 78.4% respectively, with running speed of 127 frames/s respectively and 112 frames/s. The experimental results show that the proposed algorithm can achieve real-time segmentation and improve the accuracy significantly, showing good semantic segmentation performance

Excess nitrogen in agricultural drainage poses a serious threat to the water quality safety of the Yellow River basin. Utilizing aquatic plants to modify the rhizosphere microbial community structure and facilitate nitrogen transformation is a crucial strategy for mitigating regional water eutrophication. Our results revealed that the denitrification rate (DR), dissimilatory nitrate reduction to ammonium rate (DNRAR) and nitrogen fixation rate (NFR) of rhizosphere sediments exhibited a significant increase in artificially cultivated ditch. Compared with those in natural ditches, the richness of the bacterial community and the relative abundances of Bacteroidota, Verrucomicrobiota, Firmicutes, Anaeromyxobacter and Geobacter, which are involved mainly in the nitrogen cycle, in the rhizosphere sediments of artificially cultivated ditch were significantly greater due to improvements in environmental conditions. In artificially cultivated ditch, the dominant functional genes affecting DRNARs in the rhizosphere sediments of P. australis were nrfC and nrfA, while DRs were driven mainly by norB and napA, which were influenced by the nitrogen and carbon levels; and the dominant functional genes affecting NFRs in the rhizosphere sediments of T. orientalis were nifD, nifK and nifH. Our results provide a scientific basis for the use of aquatic plants for mitigating excess nitrogen levels in agricultural drainage.

Strawberry root rot caused by Neopestalotiopsis clavispora is one of the main diseases of strawberry and significantly impacts the yield and quality of strawberry fruit. There are now no effective control techniques available except for fungicide sprays, which may directly impact consumers. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional synthetic chemical fungicides. Trichoderma is widely used as a biological agent for controlling strawberry root rot. In order to provide resources for screening the highly effective biocontrol fungus for controlling strawberry root rot caused by Neopestalotiopsis clavispora, the biocontrol mechanism,the control effects of Trichoderma asperellum CMT10 against strawberry root rot and growth-promoting effects on strawberry seedlings were investigated by plate culture, microscopy observation and root drenching methods.The results showed that CMT10 had obvious competitive, antibiotic and hyperparasitism effects on Neopestalotiopsis clavispora CMGF3. The CMT10 could quickly occupy nutritional space, and the inhibition rates of CMT10 against CMGF3 in confrontation culture on 7 d was 65.49%. The inhibition rates of volatile metabolite and fermentation metabolite produced by CMT10 against CMGF3 were 79.67% and 69.84%, respectively. The mycelium of strain CMT10 can act as hyperparasites by making contacting, winding and penetrating hyphae of CMGF3. Pot experiment showed that the biocontrol efficacy of CMT10 on strawberry root rot caused by Neopestalotiopsis clavispora was 63.00%. CMT10 had obvious promotion effect on strawberry growth, the plant height, root length, total fresh weight, root fresh weight, stem fresh weight and root dry weight, with the promotion rates were 20.09%, 22.39%, 87.11%, 101.58%, 79.82% and 72.33%, respectively. Overall, this study showed the ability of Trichoderma asperellum CMT10 to control strawberry root rot and their potential to develop as novel biocontrol agents to replace chemical fungicides for eco-friendly, sustainable agriculture.

In this paper we study a class of impulsive delayed neural networks with parameter uncertainties and reaction-diffusion terms. Using the properties of M-matrices and some inequality techniques, some sufficient conditions are derived to guarantee the global exponential stability of the given system. The results obtained are presented by simple algebraic inequalities, which are certainly more concise than previous methods. Finally, the validity and usefulness of our results are illustrated by arithmetic examples.

Pyrops candelaria is one of the common pests of fruit trees, but the research on the pathogenic microorganisms it may carry is very limited. Therefore, it is essential to reveal the pathogenic microbes it carries and their potential hazards. This study found a new virus from the transcriptome of P. candelaria, which was first reported in P. candelaria and named as PyCaV (Pyrops candelaria associated virus). RACE and bioinformatics assay revealed that the full-length of PyCaV is 10855 bp with the polyA tail, containing a single open reading frame (ORF) encoding a polyprotein consisting of 3171 amino acid (aa). The virus has a typical Iflavirus structure, including two rhv domains, a RNA helicase domain (HEL), a 3C cysteine protease domain (Pro), and a RNA-dependent RNA polymerase domain (RdRp). Further phylogenetic analysis revealed that this virus belongs to family Iflaviridae and sequence alignements analysis suggedted PyCaV is new member in unassigned genus of family Iflaviridae. Further in-depth analysis of virus-infection-part showed that PyCaV is distributing in the whole longan lanternfly, including its head, chest, and abdomen, but more PyCaV was identified in the chest. The distribution of PyCaV in different parts of P. candelaria was further explored, which showed that more PyCaV were detected in its piercing-sucking mouthparts and chest viscera. Statistical analysis showed that the PyCaV infection was affected by time and locations.

Apis mellifera, especially weak ones, are highly vulnerable to Carpoglyphus lactis mites, which can rapidly infest and consume stored pollen, leading to weakened colonies and potentially colony collapse. This study aimed to ascertain and investigate the prevalence of this mite in honeybee colonies across 9 provinces in the Republic of Korea (ROK). A total of 615 honeybee colonies were collected, from 66 apiaries during the spring and 58 apiaries during the summer of 2023. A 1,242 bp segment of the the Cytochrome c oxidase subunit 1 (COI) gene was amplified using the polymerase chain reaction method. The detection levels of C. lactis in the honeybees were compared between winter and summer. Based on the COI sequence analysis, the nucleotide sequence similarity of C. lactis mites isolated in the ROK with those from China (NC048990.1) was found to be 99.5%, and with those from the United Kingdom (KY922482.1) was 99.3%. This study is the first report of C. lactis in Korean apiaries. The findings in this study demonstrate a significantly higher detection rate in winter, which is 4.1 times greater than that in summer (p &lt; 0.001). Furthermore, the results underscore the usefulness of molecular diagnostic techniques for detecting C. lactis mites.

With the burgeoning growth of online retail, the issue of counterfeit products has become a significant threat to both consumer trust and brand integrity, posing challenges to sustainable market practices. Online retail platforms are promoted to redesign their channel structures and enhance brand traceability to tackle counterfeits using blockchain technology. To investigate the interactions between blockchain technology and the channel structures of online retail platforms, we design a game theory model incorporating a manufacturer with(out) blockchain participation selling products via an online retail platform that operates an agency channel and/or a reselling channel. We show that the critical trade-off of blockchain participation comprises the brand assurance effect of the blockchain, the channel competition intensification effect, and the blockchain adoption cost. The platform is capable of strategically designing the channel structure to encourage (while discouraging) blockchain participation by the manufacturer. Our analysis highlights the complementarity between blockchain participation and the channel structure. Furthermore, the effect of the consumer’s belief in the reselling channel, the commission fee in the agency channel, and the substitutability between channels are investigated in depth. The research provides valuable insights for online retail platforms and policymakers seeking to foster a sustainable and trustworthy e-commerce ecosystem.

In this paper, we extend the KdVSKR equations in (1+1)-dimension and (2+1)-dimension to fractional KdVSKR equations with modified Riemann-Liouville derivative. The (2+1)-dimensional KdVSKR equation, which is a recent extension of (1+1)-dimensional KdVSKR equation, can model the resonances of solitons in shallow water. By means of the Hirota bilinear method, finite symmetry group method and consistent Riccati expansion method, many new interaction solutions have been derived. Soliton-cnoidal interaction solution of the (1+1)-dimensional fractional KdVSKR equation has been derived for the first time. For the (2+1)-dimensional frac-tional KdVSKR equation, two-wave interaction solutions and three-wave interaction solutions, including dark-soliton-sine interaction solution, bright-soliton-elliptic interaction solution, and lump-hyperbolic-sine interaction solution, have been derived. Impact of the fractional order on the shapes of these solutions has been illustrated by figures. Three-wave interaction solutions of fractional systems have not been reported in the existing references. The research idea in this paper can be applied to other fractional differential equations.

COX II containing a dual core CuA active site is one of the three core subunits of mitochondrial Cco, which plays a significant role in the physiological process. In this report, the full-length cDNA of COXⅡ gene was cloned from Sitophilus zeamais, which had an ORF of 684 bp encoding 227 amino acids residues. The predicted COXⅡ protein had a molecular mass of 26.2 kDa with pI value of 6.37, and multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXⅡ had high sequence identity 78.51% with the COXⅡ of other insect species, especially similarity to sitophilus oryzae. This gene was subcloned into the prokaryotic expression vector pET-32a, and induced by IPTG in E.coli Transetta (DE3) expression system. Finally the COXⅡ with 6-His tag was purified using affinity chromatography with Ni²⁺-NTA agarose. WB showed the recombinant COXⅡ was about 44 kD, and the concentration of fusion protein was 50μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXⅡ could catalyze the oxidation of substrate Cytc, and influenced by AITC. It was found that AITC could form a hydrophobic region with COXⅡ protein via molecular docking, besides, a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results will provide valuable information for elucidating the role of COXⅡ in Sitophilus zeamais responses to AITC, meanwhile, it will helpful to carry out a point mutation in AITC binding sites for the future research.

The properties of the annual, semiannual and triennial oscillations (AO, SAO and TO) in the middle atmosphere have been investigated using the TIMED/SABER temperature data. The Lomb-Scargle and wavelet spectra were used to determine the dominant oscillations in the background temperature field. The AO is prominent at the mid-latitudes. The AO amplitudes present an asymmetry between the two Hemispheres, being larger in the mesosphere than in the stratosphere. The SAO dominates the tropical regions, with three amplitude maxima at altitudes of 45, 75, and 85 km. The SAOs in the upper mesosphere (75 km) are out of phase with those in the mesopause (85 km) in the tropical regions, which can generate an enhancement of 11 K at each equinox, contributing to the lower mesospheric inversion layer. The TO is significant in the tropical region, with amplitude being maximum at 35, 45 and 85 km. Result shows that there may be potential interaction by the TO with SAO at 85km at the equator. The relation between ENSO and TO has also been discussed. The ENSO signal may modulate the amplitude of the TO, mainly in the lower stratosphere. The real origin of the TO may lie in the wave-mean-flow interaction.

As the world population grows, fulfilling the need for biomaterials in the healthcare system becomes increasingly important. Biomaterials, which include metals, polymers, ceramics, and composites, play an important role in the creation of medical devices that improve people's lives. The European Society for Biomaterials described biomaterials as non-biological materials used in medical devices to interact with biological systems. This definition has developed to encompass materials that treat, assess, or replace tissues or bodily functions. Biocompatibility, as defined by the FDA, is a key feature of biomaterials, as is the requirement to fulfil design requirements such as mechanical performance, geometry, and electrical control. Over the last decade, advances in regenerative medicine and tissue engineering have boosted biomaterials' usage in medical applications dramatically. Despite its promise, scientists, engineers, and physicians continue to face significant challenges due to the complexity of biomaterials. Biomaterials have various benefits, including biodegradability, non-toxicity, and biocompatibility, making them extremely useful for replacing, sustaining, or improving biological activities in damaged tissues. They are used in a wide range of applications, including tissue engineering implants, human tissue healing and regeneration, medication delivery systems, and biosensors. This study gives insights into the fundamental characteristics, case studies, and ethical issues associated with the usage and use of biomaterials, further examining their potential and significance in healthcare and medicine.

The paper describes the new method for image compression based on coordinated group signal transformation. The algorithm is a type of difference coding. Coordinated processing significantly simplifies the difference signal conversion scheme using a single group codec for all signals. The described method considers color channels as correlated signals of a multi-channel communication system. The specifics of the processed data required modification of the coordinated group processing algorithm. First, we changed how to calculate the difference signals to prevent data loss. Secondly, the group codec was supplemented with a neural network to improve the quality of reconstructed images. We considered the following types of neural networks: fully connected, recurrent, convolution, and convolution in the Fourier space. Based on the simulation results, it is best to use fully connected neural networks if the goal is to minimize processing delay time. These networks have a response time of 13 ms. On the other hand, if the priority is to improve quality in cases where delays are not critical, then convolution neural networks in the Fourier space should be used. These networks have a response time of 140 ms and are of significant interest.

The studied solar pot is a recent invention, which gained a utility model protection in 2021 from the Hungarian Intellectual Property Office under patent number 5489. The device is made for environmentally friendly cooking or heating (by utilizing solar energy) of foods and liquids. Its structure is similar to a double pipe heat exchanger, it has an outer mantle and an inner cooking tank. The goals of the paper are proposing a new physically-based mathematical model describing the solar pot and carrying out computer experiments with it, assembling an experimental system of the pot connected with a solar collector and performing measurements on it. Based on the results, the solar pot can successfully be used for cooking or sterilizing foods or liquids during the studied time period, in Hungary.

The research reported on this paper was aimed at improving the overall efficiency of a PDO certified artisanal cheese production process. Being a PDO certified foodstuff by the EU, it is considered to have properties and qualities determined by the geographical environment in which is made, with its production taking place in a specific and determined geographical location, in this case the Serra da Estrela region. In that sense, the authors conducted a mapping according to a systemic perspective of the processes involved in the context of manufacturing and distribution of certified Serra da Estrela cheese. Numerous methods were used throughout the process, such as a systemic design analysis, and techniques derived from ethnographic methods, which led to the collection of data in the field and consequently provided the immersion of the researcher in genuine work situations. Critical points were identified and emphasized in the systemic map with the purpose of encouraging initiatives to address and overcome the gaps and inefficiencies detected. The systemic design analysis triggered the development of design work. Observations following an ethnographic approach identified ergonomic risks in cheese making during the process of cutting excess chips, fostering the emergence of musculoskeletal disorders at the wrist. A tool that fits best to the task at hand was developed. A prototype of the new tool enabled collecting feedback from use in the work context, in order to inform product development. The domains of agricultural production and microbiology, considering the specific microorganisms developed trough the ripening process of the cheese, turned out to be aspects of high importance for the issue under focus, contributing to a broader understanding of the ripening process and its risks, simultaneously improving the efficiency and success of cheese production. If it were not for the systemic analysis, which served as a link between the boundaries of distinct domains such as the risk of microorganism contamination, ergonomics, energy efficiency, legislation and regulation policies, transportation challenges and economic viability, approached throughout the research. Simultaneously creating bridges between them, the various problems might not have been detected in the first place, as they are usually addressed in specialized disciplines, predetermined by the restrictions of each specific area of knowledge. As a consequence of the development of this research, which was based on an analysis that sought to establish possible connections between various disciplines and tried to constantly maintain a holistic perspective, making new connections and observing the issues from a new angle, apart from the already established methodologies. All this allowed to lay out the seeds for the development of a plan to tackle the critical points identified by the systemic analysis reported in this paper.

Abstract: Transcription factors are crucial pre-transcriptional regulatory mechanisms that can modulate the expression of downstream genes by binding to their promoter regions. DOF (DNA binding with One Finger) proteins are a unique class of transcription factors with extensive roles in plant growth and development. Our previous research indicated a decrease in iron content in chlorotic bamboo leaves. However, to our knowledge, genes related to iron metabolism pathways in bamboo species have not yet been studied. Therefore, in the current study, we identified iron metabolism-related (IMR) genes in bamboo and determined the transcription factors that significantly influence them. Among these, DOFs were found to have widespread effects and potentially significant impacts on their expression. We identified specific DOF members in Dendrocalamus latiflorus with binding abilities through homology with Arabidopsis DOF proteins, and established connections between some of these members and IMR genes using RNA-seq data. Additionally, molecular docking confirmed the binding interactions between these DlDOFs and the DOF binding sites in the promoter regions of IMR genes. The co-expression relationship between the two gene sets was further validated using q-PCR experiments. This study paves the way for research into iron metabolism pathways in bamboo and lays the foundation for understanding the role of DOF transcription factors in D. latiflorus.

This paper is focused on typical rural settlements in the Stara Planina Mountain area in Serbia. They are distinguished by their authenticity, identity and character, and they have still not been destroyed by inappropriate development as is the case in some other mountain areas in Serbia, where the effects of urbanization, globalization and homogenization are obvious. Consequently, there is a need to prevent these effects in mountain areas where there is a threat of disbalance between development and the inherited natural and cultural values. The paper starts from several important concepts that relate contemporary approaches to cultural heritage preservation, place morphology and planning future development. The concept definitions from recent documents from related fields are used. These concepts are then applied to the area of Stara Planina Mountain and its rural settlements. The necessity for an integrative approach to planning this area with significant natural and anthropogenic potential is pointed out, including its integration into international programs of sustainable development.

The wetlands in northeastern Bangladesh are home to some of the most prolific and valuable ecosystems on the planet, offering a wide range of benefits to both the local and wider communities. A limited number of research on this topic have been carried out in various wetlands in coastal hilsa, but none have been located in the northeastern region of Bangladesh. Thus, the goal of this study was to better understand the most commercially relevant species (anadromous hilsa) in northeastern Bangladesh as well as to assess the wetland ecosystem services more thoroughly with the contribution of their fish genetic resources. In addition to laboratory studies, fieldwork was done in the fishing settlements along the Surma River, Hakaluki Haor, and Tanguar Haor. The study classified 77 fish species into 21 subcategories of ecosystem services, of which 56% were not threatened, 17% were vulnerable, and 10% were highly endangered. The species Hilsa was found to be the most economically significant, accounting for 1% of the GDP overall. The study determined if the hilsa stock of the examined wetlands maintains a single or several subpopulations in comparison to other parts of the country when they migrate from saline water to northeastern Bangladesh (freshwater). But when compared to other groups, the FST score was between 0.000 to 0.017, indicating that the populations are identical and that there is genetic material sharing between them. Additionally, these statistics imply that the Sylhet haor and adjacent river systems contain a single stock of Hilsa fish. Nevertheless, a number of stresses and risks, including overexploitation, illicit and destructive fishing, siltation, and natural disasters like flash floods, have been linked to changes in the supply of hilsa and other ecosystem services. This study also emphasizes the necessity of policy attention and research to address the concerns.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC) which first emerged in 2012, persists and continues to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction of viral burdens by &gt;6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).

The continuous casting process of helical fiber reinforced metal matrix composites was proposed, and the experimental equipment was designed and manufactured. The relationship model be-tween the process parameters and the helical shape of the fibers in the metal matrix is analyzed by differential geometry. Helical carbon fiber reinforced lead matrix composites were prepared under different conditions. When the melting temperature and cooling intensity were increased, the helical radius of helical carbon fiber in lead matrix was increased. When the fiber rotation speed was increased, the helical radius was decreased, and the helical pitch was de-creased proportionally. The experimental results are consistent with the process model. Carbon fiber rein-forced aluminum composites were prepared. Scanning electron microscopy showed that the infiltration between the aluminum matrix and the carbon fiber was not complete. As the volume fraction of carbon fiber which was added in composites is too small, the mechanical properties of the composites have not been significantly improved.

This article presents an innovative approach to the design of a safe adaptive backstepping control system. Tailored specifically for underactuated marine robots, the system utilizes simple sensors for enhanced practicality and efficiency. Given their operation in diverse oceanic environments fraught with various sources of uncertainties, ensuring the system's safe and robust behavior holds paramount importance in the control literature. To address this concern, this paper introduces a control strategy designed to ensure robustness at both the kinematic and dynamic levels. By emphasizing the compensation for the system uncertainties, the design integrates a straightforward fuzzy system structure. To further ensure the system's safety, a funnel surface is defined, followed by the design of a suitable nonlinear sliding surface as a function of the funnel and tracking error. Specifically leveraging the Lyapunov function, the study formally establishes the Semi-globally Practically Finite-time Stability of the closed-loop system, validated through simulations conducted on underactuated marine robots.

Several types of guided bone regeneration materials are available with various properties and clinical indications. Among them, dentin-derived matrix from extracted teeth is an excellent alternative to traditional graft materials, such as allografts and xenografts. Different clinical preparation protocols exist: block form versus powder form, demineralized versus non-demineralized form. Out of these methods, demineralization protocols for dentin-derived matrix from extracted teeth have been noted for a wide range of successful clinical outcomes. The purpose of this animal study was to find out the most effective method of demineralizing dentin-derived matrix to result in the highest regenerative ability in the bone graft material in rabbit calvaria. The study conducted a comparative histomorphometric analysis of four groups in distinguished matrix conditions: a control group without graft placement, a non-demineralized dentin-derived matrix graft placement group, a 15-minute demineralized dentin-derived matrix graft placement group, and a 30-minute demineralized dentin-derived matrix graft placement group. Regenerative processes were evaluated three times at 2, 4, and 8 weeks post-graft placement. These groups of rabbits were evaluated randomly in a blinded setting to prevent bias. Among the four groups compared, the group with a 15-minute demineralized dentin-derived matrix graft showed the most efficient bone regeneration in the histomorphometric analysis. Based on this animal study, it was apparent that a 15-minute demineralization of dentin-derived matrix is the preferred preparation protocol to maximize the regenerative potential in the matrix.

Promoting green development has become an important way to optimize the ecological and economic structure and promote sustainable development. The Yangtze River Economic Belt is an important economic corridor in China, and it remains to be examined whether the implementation of environmental regulations has enhanced the green total factor productivity (GTFP) of cities. This paper calculates the green total factor productivity of 107 cities within the Yangtze River Economic Belt by using the super-efficient SBM model and the GML index with Chinese city panel data from 2007-2019 and measures the intensity of environmental regulations through textual analysis. The bidirectional fixed-effects model constructed in this paper suggests an inverted U-shaped relationship between environmental regulation and GTFP, which is confirmed by a series of robustness tests. Green technology innovation and advanced industrial structure play a mechanism role in this. In regions with lower levels of green technology and industrial structure, the inverted U-shaped relationship between environmental regulation and green total factor productivity is more significant. In addition, the phenomenon of GTFP rising and then falling because of environmental regulation is more obvious in cities in the middle and upper reaches of the Yangtze River and outside the urban agglomerations. Therefore, when formulating environmental regulation policies, it is necessary to balance the relationship between economic development and environmental protection and harmonize the environment and economic development of the entire Yangtze River Basin.

The main purpose of this study is the determination of the optimal data acquisition and processing parameters of electric and seismic tomographic techniques, for the investigation of complex geological environments. Two different study areas, in central-east Peloponnese and SW Attica were selected, where a detailed geological mapping and surface geophysical survey were carried out. The applied geophysical survey included the application of electrical resistivity tomography (ERT) and seismic refraction tomography (SRT). The geoelectrical measurements were acquired with different arrays and electrode configurations. Moreover, various types of seismic sources were used at seventeen shot locations along the seismic arrays. For the processing of geoelectrical data, clustered datasets were created increasing the depth of investigation and the discriminatory capability. The seismic data processing included: (a) creation of synthetic models and seismic records to determine the effectiveness and capabilities of the technique, (b) spectral analysis of the seismic records to determine the optimal seismic source type and (c) inversion of the field data to create representative subsurface velocity models. The results of the two techniques successfully delineated the complex subsurface structure that characterizes these two geological environments. The application of the ERT combined with the SRT, are the two dominant, high-resolution techniques for the elucidation of complex subsurface structures.

Cocoa (Theobroma Cacao, L.) represents an important market that gained relevance and became an esteemed commodity thanks to cocoa powder, chocolate, and other related products. This work has analyzed 59 cocoa powder samples from the European market. Three distinct subgroups were identified: organic or conventional, alkalized or not alkalized, and raw or roasted processing. The impact of the technological process was evaluated on the pH, color, and compositional traits, as content of biogenic amines and salsolinol. The phenolic fraction was also investigated trough both common and emerging methods. Results depict that the influence of the agronomical practices (organic/conventional) did not affect significatively (p&lt;0.05) the composition of cocoa powders; similarly, the roasting process was not discriminative for the compounds traced. On the other hand, the alkalinization process greatly impacted on color, and pH, no matter of cocoa provenience, obtention, or other processes, also resulting reducing the phenolic fraction of treated samples. Principal components analysis confirmed that the alkali process acts on pH, color, and phenolic composition, but not on other bioactives molecules (BAs and salsolinol). All samples resulted safe while alkalized powders have a great reduction in beneficial biocompounds. A novel strategy could be to emphasize non-alkalized powders in the label, to meet the demand for more beneficial products.

Oral squamous cell carcinoma (OSCC) is a highly prevalent and aggressive malignancy, with mortality rates that reach 60%, mainly due to an excessive diagnostic delay. MiRNAs, a class of crucial epigenetic gene-expression regulators, have emerged as potential diagnostic biomarkers, with >200 molecules reported to be dysregulated in OSCC. We had previously established an in silico methodology for the identification of the most disease-specific molecules by bridging genetics and epigenetics. Here, we identified the stage-specific miRNAs that govern the asymptomatic early stages of oral tumorigenesis by exploiting the reverse interplay between miRNA-levels and their target-genes’ expression. Incorporating gene expression data from our group’s experimental hamster model of sequential oral oncogenesis, we bioinformatically detected the miRNAs that simultaneously target/regulate >75% of the genes that are characteristically upregulated or downregulated in the consecutive stages of hyperplasia, dysplasia and early-invasion OSCC, while exhibiting the opposite expressional dysregulation in OSCC-derived tissue and/or saliva specimens. We found that all stages share the downregulation of miR-34a-5p, miR124-3p, and miR-125b-5p, while miR-1-3p is underexpressed in dysplasia and early-invasion. Malignant early-invasion stage is distinguished by the downregulation of miR-147a and the overexpression of miR-155-5p, miR-423-3p and miR-34a-5p. The identification of stage-specific miRNA molecules may facilitate their utilization as biomarkers for presymptomatic OSCC diagnosis.

Hydrogen sulfide is present in active or extinct volcanic areas (mofettas). The habitable premises in these areas are affected by the presence of hydrogen sulfide, which, even in low concentrations, gives off a bad to unbearable smell. If the living spaces considered are closed enclosures, then a system can be designed to reduce the concentration of hydrogen sulfide. This paper presents a membrane way to reduce the hydrogen sulfide concentration to acceptable limits using a cellulosic derivative-propylene hollow fiber based composite membrane module. The cellulosic derivatives considered were carboxymethyl–cellulose (NaCMC), cellulose acetate (CA), methyl 2–hydroxyethyl–cellulose (MHEC), and 2–hydroxyethyl–cellulose (HEC). In the permeation module, hydrogen sulfide is captured with a solution of cadmium that forms cadmium sulfide, usable as a luminescent substance. The composite membranes were characterized by SEM, EDAX, FTIR, FTIR 2D maps, thermal analysis (TG and DSC) and from the perspective of hydrogen sulfide air removal performance. To determine the process performances, the variables were: the nature of the cellulosic derivative–polypropylene hollow fiber composite membrane, the concentration of hydrogen sulfide in the polluted air, the flow rate of polluted air, and the pH of the cadmium nitrate solution. Pertraction efficiency was maximum for the sodium carboxymethyl–cellulose (NaCMC)-polypropylene hollow fiber membrane, a hydrogen sulfide concentration in the polluted air of 20 ppm, polluted air flow rate of 50 L/min, and a pH of 2 and 4.

The normal growth and development of skeletal muscle is essential for the health of the body. The regulation of skeletal muscle by intestinal microorganisms and their metabolites has been continuously revealed. Acetate is the predominant short-chain fatty acid synthesized by gut microbiota through the fermentation of dietary fiber, however, the underlying molecular mechanisms governing the interaction between acetate and skeletal muscle during rapid growth stage remain to be further elucidated; Here, specific pathogen-free (SPF) mice, germ-free (GF) mice, and germ-free mice supplemented with sodium acetate (GS) were used to evaluate the effects of acetate on skeletal muscle growth and development of young mice with gut microbiota deficiency; We found the concentration of serum acetate, body mass gain, succinate dehydrogenase activity and expression of myogenesis maker gene of skeletal muscle in the GS group was higher than GF group following sodium acetate supplement. Furthermore, the transcriptome analysis revealed that acetate activated biological processes regulating skeletal muscle growth and development in GF group that are inhibited due to gut microbiota deficiency. In vitro experiment, we proved that acetate up-regulated Gm16062 to promote skeletal muscle cell differentiation; Overall, our findings proved that acetate promote skeletal muscle growth and development in young mice via increase Gm16062 expression.