Paratuberculosis, or Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis affecting both domestic and wild ruminants. The agent was also found in wild mammals such as wild boar (Sus scrofa), however, the role of wild mammals in the epidemiology of MAP is unclear. During the research period, 941 free-ranging wild boar (S. scrofa) legally hunted in two locations in the Centre-eastern region of Portugal were examined. Ninety-seven wild boar exhibited one or more gross lesions and were tested for the presence of Mycobacterium avium subsp. paratuberculosis using acid-fast staining, mycobacterial culture, poly-merase chain reaction (PCR), and histopathological examination. Forty-five animals (46.4%, 95% CI: 36.5-56.3%) were identified as infected, as indicated by positive results in culture and/or PCR. The findings revealed that the most significant risk factor was being a juvenile compared to year-lings and adults (OR = 10.2, 95% CI: 2.2-48.0). Based on our results, 37.9% (n = 11) of the infected animals were considered suitable for human consumption. Our findings offer novel insights into mycobacterial infections in wild boar populations in Portugal and suggest that wild boar could be a source of human infection if zoonotic potential is considered.

Maximum likelihood estimation (MLE) is a popular method for parameter estimation in both applied probability and statistics but MLE cannot solve the problem of incomplete data or hidden data because it is impossible to maximize likelihood function from hidden data. Expectation maximum (EM) algorithm is a powerful mathematical tool for solving this problem if there is a relationship between hidden data and observed data. Such hinting relationship is specified by a mapping from hidden data to observed data or by a joint probability between hidden data and observed data. In other words, the relationship helps us know hidden data by surveying observed data. The essential ideology of EM is to maximize the expectation of likelihood function over observed data based on the hinting relationship instead of maximizing directly the likelihood function of hidden data. Pioneers in EM algorithm proved its convergence. As a result, EM algorithm produces parameter estimators as well as MLE does. This tutorial aims to provide explanations of EM algorithm in order to help researchers comprehend it. Moreover some improvements of EM algorithm are also proposed in the tutorial such as combination of EM and third-order convergence Newton-Raphson process, combination of EM and gradient descent method, and combination of EM and particle swarm optimization (PSO) algorithm.

Fungal infections caused by Candida spp. are responsible for high hospital morbidity and mortality rates. Therefore, there is a continuous search for new antifungal medicines, particularly ones with anti-Candida activity. This review analyzed articles published between 2002 and 2023 considering the anti-Candida activity of chemical compounds identified or isolated in plants of the Anacardiaceae family. In addition, the in-silico prediction of the isolated compounds described was performed. The analysis of 35 studies showed that extracts, essential oils, and compounds from the anti-Candida activity were frequently determined in vitro using the minimum inhibitory concentration (MIC). The most commonly tested species were Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, and C. guilliermondii, respectively. Essential oils were the most used form (37% of the studies). The isolated compounds with antifungal effects include cardanol, estragole, trans-anethole, β-caryophyllene, myrcene, catechin-3-o-rhamnoside, β-sitosterol-3-O-glucoside, 24Z-isomasticadienolic acid, oleanolic acid, and oleanolic aldehyde. The in-silico evaluation of those isolated compounds revealed the compounds' drug-likeness and possible antifungal activity. However, some of them showed high toxicity. In conclusion, compounds from the Anacardiaceae family show promise for developing new therapeutic antifungal drugs, especially in combination with conventional antifungals.

Conobea scoparioides (Plantaginaceae) is an herbaceous plant known as "pataqueira" that grows wild in seasonally wet areas of the Amazon region. It is used for aromatic baths and anti-protozoan remedies by the Brazilian Amazon native people. The main volatile compounds identified in the essential oil of "Pataqueira" were the phenolic monoterpenes thymol and thymol methyl ether and their precursors, the monoterpene hydrocarbons α-phellandrene and p-cymene. A hydrotalcite synthesized from blast-furnace slag exhibited a 3:2 (Mg/Al) molar ratio, and this layered double hydroxide (LDH) was evaluated as a catalyst in converting the main monoterpenes of the "Pataqueira" oil. This action significantly increased thymol content, from 41% to 95%, associated with the percentual reduction of other main components, such as thymol methyl ether, α-phellandrene, and p-cymene. The LDH reaction showed a strong tendency towards producing hydroxylated derivatives, and its behavior was similar to the hypothetical plant biosynthetic pathway, which leads to the production of the monoterpenes of "Pataqueira" oil. Thymol and its derivatives are potent antiseptics applied in pharmaceutical and hygienic products as antibacterial, antifungal, and antioxidant properties, among others. The present work reports a natural source with a high thymol content in aromatic plants from the Amazon, with evident economic value.

Fumaric acid is a chemical building block with many applications, namely in the polymer industry. The fermentative production of fumaric acid from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. The use of existing industrial side-streams as raw-material within biorefineries potentially enable production costs competitive against current chemical processes, while preventing the use of refined sugars competing with food and feed uses and avoiding purposely grow crops requiring large areas of arable land. However, most industrial side streams contain a diversity of molecules that will add complexity to the purification of fumaric acid from the fermentation broth. A process for the recovery and purification of fumaric acid from complex fermentation medium containing spent sulfite liquor (SSL) as carbon source was developed and is herein described. A simple two-stage precipitation procedure involving separation unit operations, pH and temperature manipulation and polishing through the removal of contaminants with activated carbon allowed the recovery of fumaric acid with 68.3% recovery yield with specifications meeting the requirements of the polymer industry. Further, process integration opportunities were implemented that allowed minimizing the generation of waste streams containing fumaric acid which enabled increasing the yield to 81.4% while keeping the product specifications.

Nowadays, animal breeders look for effective innovations to care animal health. Use of probiotics, postbiotics, parabiotics and the other beneficial natural substances are dominated. Mundticin-like substance EM 41/3 is thermo-stable bacteriocin (postbiotic) with a broad antimicrobial spectrum. In this study its effect in broiler rabbits was assessed. Rabbits represent food-derived animal and they are also suitable animal model. The parameters tested were analyzed by validated methods. Administration of MLS EM 41/3 produced by non-autochthonous strain Enterococcus mundtii EM 41/3 lead to significant increase of parameter immunity-phagocytic activity, while it did not influence microbiota composition. It also did not influence blood biochemistry and no oxidative stress was noted. Moreover, higher growth parameters were noted and also stimulated hydrolytic activity. Finally, no negative influence on rabbit meat quality was noted. Postbiotic MLS EM 41/3 administration in feeding strategy seems to be contributing fact for rabbit health status maintaining.

Mycobacterium avium ssp paratuberculosis (MAP) is the causative agent of Johne’s disease (JD) in animals and a suspected cause of Crohn’s disease (CD) in humans. Crohn’s Disease is a progressive, detrimental granulomatous enteritis of which common therapies are targeted to suppressing certain components of the immune system. The efficacy of these treatments can be variable and temporary with no clear process of selection for the individual patient. However, diagnosis of MAP has been elusive in patients with Crohn’s Disease. In animals it can be detected by several different techniques, including bacterial cell culture, polymerase chain reaction (PCR), and immunological targets. To move to an understanding of the possible pathogenicity of MAP and the sup-posed cause of CD in humans, it is necessary to demonstrate the presence of cell wall deficient mycobacterial (CWDM) forms of MAP in blood and tissue cultures from patients with CD. From this, more tailored and accurate therapies can be implemented and be developed to eradicate this infectious organism from humans. Current anti-mycobacterial antibiotics have limited efficacy as MAP is subtly different from tuberculosis. Newer methods, such as vaccination and gut microbiome remediation will be essential as part of combination therapies.

In this paper, we propose a methodology for analyzing the near-field coupling between two surface mount device (SMD) inductors using a 3-dimensional electromagnetic (3D-EM) model. To develop the 3D-EM model, we first extract the effective permeability of core magnetic material in the SMD using the loss tangent in the equivalent circuit model. Then the effective permeability is used in the magnetic material for the 3D-EM modeling of SMD inductor. The validity of the proposed 3D-EM model is confirmed by comparing the impedance and S-parameters obtained from both measured and EM-simulated values for the two near-field coupled SMDs. Finally, the near-field coupling effects between the two adjacent SMD inductors are visualized in terms of magnetic coupling path visualization (CPV) using the proposed 3D-EM model, which demonstrates its usefulness for near-field coupling analysis.

Kininogens are multidomain glycoproteins found in the blood of most vertebrates. They are important in the blood coagulation cascade pathways - in intrinsic pathway activation leading to thrombin generation partially independently from tissue factor dependent extrinsic pathway, connecting blood coagulation with the kallikrein-kinin system. Nothing is known about the shape on atomic level therefore the endeavor to obtain the good-quality spatial structure of kininogens is important for a better understanding of their role in disease and treatment. Application of cryo-EM is important for solving the spatial structure of kininogens, drawing new frontiers in understanding the function and opening new pathways for drug development.

1). BACKGROUND: Cell wall deficient mycobacteria (CWDM) have seldom been isolated in bacterial culture from human subjects. particularly in clinical disease, such as Crohn’s disease. Techniques have focused on the use of polymerase chain reaction (PCR) for the detection of CWDM in association with inflammatory bowel disease. (IBD). This paper is a report of the development of methods to demonstrate the presence of CWDM using the Ziehl-Neelsen (ZN) stain. This is in addition to the detection of the growth of CWDM in fluid media. 2). METHODS: The use of the ZN stain is suitable for the demonstration of CWDM in clinical samples and cultures, when the staining methods that were common prior to the 1970’s, are adopted. Specific culture media are developed and tested in parallel. Alterations in the concentration of basic fuchsin, and changes in the decolourising solutions are necessary. Decolourising solutions such as 20% concentrated sulphuric acid in water are preferable for detection of CWDM in cultures. 3). RESULTS: CWDM can be readily demonstrated using the simple methods outlined in this report. The ability to visualise the presence of CWDM opens the door to the development of specific fluid media for the isolation of CWDM in culture. 4). CONCLUSION: The ZN stain methods optimised for the demonstration of Mycobacterium tuberculosis complex (MTBC) are not suitable for research into infectious causes of inflammatory bowel disease (IBD), as well as for the detection of CWDM in latent tuberculosis. Revisiting the techniques for the detection of CWDM may also refocus attention on inflammatory diseases considered to be idiopathic, but having hallmarks of an infectious process.

Condition-specific competition, when environmental conditions alter the outcome of competition, can foster the persistence of resident species after the invasion of a competitively superior invader. We test whether condition-specific competition can facilitate the areawide persistence of the resident and principal West Nile virus vector mosquito Culex pipiens with the competitively superior invasive, Aedes albopictus, in water from different urban container habitats. (2) Methods: We tested the effects of manipulated numbers of A. albopictus on C. pipiens survival and development in water collected from common functional and discarded containers in Baltimore, Maryland, USA. The experiment was conducted with typical numbers of larvae found in field surveys of C. pipiens and A. albopictus and container water quality. (3) Results: We found increased densities of A. albopictus negatively affected the survivorship and development of C. pipiens in water from discarded containers but had little effect in water from functional containers. This finding was driven by water from trash cans, which allowed consistently higher C. pipiens survival and development and had greater mean ammonia and nitrate concentrations that can promote microbial food than other container types. (4) Conclusions: These results suggest that the contents of different urban containers alter the effects of invasive A. albopictus competition on resident C. pipiens, that trash cans, in particular, facilitate the persistence of C. pipiens, and that there could be implications for West Nile virus risk as a result.

Capsule endoscopy (CE) has been a widely used medical imaging tool for the diagnosis of gastrointestinal tract abnormalities like bleeding. But the CE captures a huge number of image frames that are time-consuming and tedious tasks for medical experts to diagnose manually. To address this issue, researchers focused on the computer-aided bleeding detection system to identify bleeding automatically in real-time. This paper presents a systematic review of the available state-of-the-art computer-aided bleeding detection algorithms for capsule endoscopy. The review was carried out by searching five different repositories: Scopus, PubMed, IEEE Xplore, ACM Digital Library, and ScienceDirect for all original publications on computer-aided bleeding detection published between 2001 and 2021. The PRISMA methodology was used to perform the review, and 112 full-texts of scientific papers were reviewed. The contributions of this paper are I) a taxonomy for computer-aided bleeding detection algorithms for capsule endoscopy is identified, II) the available state-of-the-art computer-aided bleeding detection algorithms including various color spaces (RGB, HSV, etc.), feature extraction techniques, and classifiers are discussed, and III) identify the most effective algorithm for practical use cases. Finally, the paper is concluded by providing future direction for computer-aided bleeding detection research.

Organic wastes and by-products from several activities, including food industries, farming, and animal husbandry, are a hygiene threat when aerobically decomposed. Therefore, their manage-ment is crucial for public health. In this direction, anaerobic digestion (AD) systems may be the solution by transforming waste into energy, which may decrease the environmental impact. However, their efficacy should be carefully examined. In this innovative study, we evaluated the physicochemical and microbial characteristics of liquid digestate (LD) retrieved from organic animal wastes in northern Greece using nanofiltration. Using treatment technologies including physical (solid-liquid separation, microfiltration and nanofiltration) and biological (anaerobic digestion), heavy metals and microbial (i.e., Salmonella spp., Escherichia coli, Enterococcus faecalis) concentrations reduced and nutrients were recovered. This work sets the basis for the efficient management of liquid digestate. Our method may enable the use of treated liquid digestate for unlimited irrigation water and other industrial applications of water. Apart from the sanitation process, the recovery of nutrients for soil fertilization seems to be a more sustainable way for fu-ture agricultural practices.

Soil moisture can be an important indicator of climate change in humid and semi-arid areas. This indicator can more efficiently propose different public policies related to climate change than just using precipitation and temperature data. Given the above, the objective of this study is to evaluate changes in soil moisture in the state of Pernambuco during the period 1961-2021, using the System of Hydrological Response Units for Pernambuco. In this study, two river basins in the state of Pernambuco that represent the different climatic conditions of the state will be chosen. The results show that in the coastal region, there is a tendency towards more saturated soils, and in the semi-arid region, there is a tendency towards drier soils. With these results, it is possible to conclude that public policy decisions for the economy, environment, and society must consider this vital water balance variable.

Dermal penetration is considered to mainly occur via passive diffusion. Therefore, a high concentration gradient of dissolved active ingredient between formulation and skin is needed to achieve an effective dermal penetration. The concentration gradient between formulation and skin can be changed by changing the amount of dissolved active ingredient in the formulation. In case of poorly soluble active ingredients this can be achieved by using nanosized drug material instead of larger sized material. The smaller size and the resulting larger surface increase the kinetic solubility and thus increase the concentration gradient of dissolved active ingredient between formulation and skin, which then results in an increased dermal penetration. In this study we demonstrated that the concentration gradient between formulation and skin can also be influenced by the number of particles being present in the formulation and by the dose (volume of formulation) applied to the skin. Particles of undissolved active ingredient are known to act as a reservoir by releasing dissolved molecules in the liquid phase of the formulation. It could now be demonstrated that the particles - once the liquid in which they are dispersed evaporates - can connect to the skin via an aqueous meniscus. The aqueous meniscus contains dissolved active ingredient and therefore creates a small local spot with a locally high concentration gradient and improved dermal penetration. Therefore, the dermal penetration can be increased with formulations that contain high amounts of particles. This can be achieved by applying formulations with high numbers of particles and/or by applying larger volumes of particle containing formulations to the skin.

Centrifugal pumps are widely employed in the oil refinery industry due to their efficiency and effectiveness in fluid transfer applications. The reliability of pumps plays a pivotal role in ensuring uninterrupted plant productivity and safe operations. Analysis of failure history data shows that bearings have been identified as critical components in oil refinery pump groups. However, traditional reliability estimation theories may not apply when data is limited or subject to right censoring. This paper addresses the complexity of estimating the Weibull distribution parameters using the maximum-likelihood method under the abovementioned conditions. The likelihood equation lacks an explicit analytical solution, necessitating the use of numerical methods for resolution. The proposed approach presented in this article leverages the Expectation-Maximization (EM) algorithm for estimating the Weibull distribution parameters. This method provides more accurate estimates of failure rates and probabilities by accounting for limited and censored data. The findings are demonstrated through a case study, showcasing the practical application of the proposed approach.

The physiological characteristics of liquid digestate retrieved from various biogas plants based in northern Greece are presented. Preliminary photocatalysis experiments on inoculated liquid di-gestate sampled showed that disinfection is possible when pre-treated digestate with a combination of centrifuge-flocculation-μFiltration after 5.5 hours of 0.7g/L suspended TiO2 under UVA illumination for the experimental conditions is used. A novel design photocatalytic nanofiltration reactor was implemented for disinfection experiments on pre-treated liquid digestate, giving promising results. This work sets the basis for the efficient operation and engineering application of technology collaboration with photocatalysis as the final step for liquid digestate sanitation and reusable water recovery.

Clinical and histological similarities between sarcoidosis and tuberculosis have driven repeated investigations looking for a mycobacterial cause of sarcoidosis. Over 50 years ago “anonymous mycobacteria” were suggested to have a role in the etiology of sarcoidosis. Both tuberculosis and sarcoidosis have a predilection for lung involvement though each can be found in any area of the body. A key histopathologic feature of both sarcoidosis and tuberculosis is the granuloma – while the tuberculous caseating granuloma has an area of caseous necrosis with a cheesy consistency; the non-caseating granuloma of sarcoidosis does not have this feature. This article reviews and reiterates the complicity of the infectious agent, Mycobacterium avium ss. paratuberculosis (MAP) as a cause of sarcoidosis. MAP is involved in a parallel story as the putative cause of Crohn’s disease, another disease featuring noncaseating granulomas. MAP is a zoonotic agent infecting ruminant animals and is found in dairy products and in environmental contamination of water and air. Despite increasing evidence tying MAP to several human diseases, there is a continued resistance to embracing its pleiotropic roles. "Who Moved My Cheese" is a simple yet powerful book that explores the ways in which individuals react to change. Extending the metaphor, the “non-cheesy” granuloma of sarcoidosis actually contains the difficult-to-detect “cheese”, MAP; MAP did not move, it was there all along.

The Serra da Estrela Natural Park (NPSEs) in Portugal stands out as a well-preserved region abundant in medicinal plants, particularly known for their pharmaceutical applications in diabetes prevention and treatment. This comprehensive review explores these plants' botanical diversity, traditional uses, pharmacological applications, and chemical composition. The NPSEs boast a rich diversity with 138 medicinal plants across 55 families identified as traditionally and pharmacologically used against diabetes globally. Notably, the Asteraceae and Lamiaceae families are prevalent in anti-diabetic applications. In vitro studies reveal their significant inhibition of carbohydrate-metabolizing enzymes, and certain plant co-products regulate genes involved in carbohydrate metabolism and insulin secretion. In vivo trials demonstrate anti-diabetic effects, including glycaemia regulation, insulin secretion, antioxidant activity, and lipid profile modulation. Medicinal plants in NPSE exhibit various activities beyond anti-diabetic, such as antioxidant, anti-inflammatory, antibacterial, anticancer, and more. Chemical analysis identifies over fifty compounds like phenolic acids, flavonoids, terpenoids, and polysaccharides responsible for their efficacy against diabetes. The findings underscore the potential of NPSE medicinal plants as anti-diabetic candidates, urging further research to develop effective plant-based anti-diabetic drugs, beverages, and supplements.

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common one has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development.

The goal of this work is to develop a sustainable value chain of carbonaceous adsorbents that can be produced from the solid fibrous digestate (SFD) of biogas plants and further applied in integrated desulphurisation-upgrading (CO2/CH4 separation) processes of biogas to yield high purity biomethane. On this purpose, physical and chemical activation of the SFD derived biochar was optimised to afford micro-mesoporous activated carbons (ACs) of high BET surface area (590-2300 m2g−1) and enhanced pore volume (0.57-1.0 cm3g−1). Gas breakthrough experiments from fixed bed columns of the obtained ACs, using real biogas mixture as feedstock, unveiled that the physical and chemical activation conclude to different types of ACs which are sufficient for biogas upgrade and biogas desulphurisation respectively. Performing breakthrough experiments at three temperatures close to ambient it was possible to define the optimum conditions for enhanced H2S/CO2 separation. It was also concluded that the H2S adsorption capacity is significantly affected by restriction to gas diffusion. Hence, the best performance was obtained at 50 oC and the maximum observed in the H2S adsorption capacity vs the temperature is attributed to the counterbalance between adsorption and diffusion processes.

For many years marine algae has been subject of numerous researches and as a source of natural products with antiviral activity, such as terpenes, alkaloids and sulphated polysaccharides. However, the anti-Zika virus (ZIKV) potential of algae has not been studied. In this study we evaluated extracts seven species of the three major classes of seaweeds (Phaeophyceae, Chlorophyceae and Rhodophyceae) against ZIKV. All seaweeds tested are native of the Brazilian coast, except for Kappaphycus alvarezii that can be cultivated. ZIKV a mosquito-borne flavivirus, has become a public health problem. In recent years there has been an increase in the number of cases and a strong association between ZIKV outbreak and the spread of cases of Guillain-Barré Syndrome and microcephaly. All seaweed extracts tested in this work inhibits ZIKV replication in a dose-dependent manner. Caulerpa racemosa, Kappaphycus alvarezii and Osmundaria obtusiloba extracts were able to inhibit viral replication at low concentrations with EC₅₀ values ranging from 1.38 to 1.98 µg/mL. We observed that O. obtusiloba presented a significant virucidal effect. Our results suggest that extracts of C. racemosa, K. alvarezii and O. obtusiloba presented very promising results, being excellent candidates for further studies, demonstrating that marine algae are an interesting source for the development of novel anti-ZIKV agents.

Human endogenous retroviruses (HERV) are remnants of ancient retroviral infections that, over millions of years, became integrated into the human genome. While normally inactive, environmental stimuli such as infections have contributed to the transcriptional reactivation of HERV promoting pathological conditions including the development of autoimmunity, neurodegenerative disease and cancer. What infections then trigger HERV activation? Mycobacterium avium subspecies paratuberculosis (MAP) is a pluripotent driver of human disease. Aside from granulomatous diseases Crohn’s, sarcoidosis and Blau syndrome, MAP is associated with autoimmune disease: type one diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis and autoimmune thyroiditis. MAP is also associated with Parkinson’s disease. Autoimmune diabetes, multiple sclerosis and rheumatoid arthritis are the diseases with the strongest MAP/HERV association. There are several other diseases associated with HERV activation including diseases whose epidemiology and/or pathology would prompt speculation for a causal role of MAP. These include non-solar uveal melanoma, colon cancer, glioblastoma and amyotrophic lateral sclerosis (ALS). This article further points to MAP infection as a contributor to autoimmunity, neurodegenerative disease and cancer via unsilencing of HERV. We examine the link between the ever-increasing number of MAP- associated diseases, the MAP/HERV intersection with these diverse medical conditions and propose treatment opportunities based upon this association.

Native and exotic fruits from the Amazon have varied characteristics, with aroma being a decisive factor in attracting and accepting them and their medicinal use as a nutraceutical supplement. This work aimed to analyze the chemical constituents of the volatile concentrates of some Myrtaceous fruit species sampled in the Brazilian Amazon. The fruit's pulps were subjected to simultaneous distillation-extraction, and gas chromatography-mass spectrometry was used to analyze their volatile chemical composition. In the volatile concentrate of Eugenia stipitata (Araçá-boi) were identified α-pinene (17.5%), citronellyl butanoate (15.6%), and pogostol (13.5%) as primary constituents; in Eugenia uniflora (Ginja) were curzerene (30.5%), germacrone (15.4%), atractylone (13.1%), and (E)-β-ocimene (11.1%); in Myrciaria dubia (Camu-Camu) were α-pinene (55.8%), (E)-β-ocimene (13.1%), and α-terpineol (10.0%); in Psidium guajava (Goiaba) were (2E)-hexenal (21.7%), hexanal (15.4%), caryophylla-4(12),8(13)-dien-5-β-ol (10.5%), caryophyllene oxide (9.2%), and pogostol (8.3%); and in Psidium guineense (Araçá) were limonene (25.2%), ethyl butanoate (12.1%), epi-β-bisabolol (9.8%), and α-pinene (9.2%). The analyzed fruit species' volatile concentrates presented a significant diversity of constituents with a predominance of functional groups, such as monoterpenes, sesquiterpenes, and fatty acid derivatives, originating from the plant's secondary metabolism and representing significant importance regarding their nutritional and medicinal uses.

The addition of biocarriers can improve the biological processes appearing in a bioreactor since their surface allows the immobilization, attachment, protection, and growth of microorganisms. In addition, the development of a biofilm layer allows the colonization of microorganisms in the biocarriers. The structure, composition, and roughness of the biocarriers' surface are crucial factors that affect the development of the biofilm. In the current work, the aluminosilicate zeolites 13X and ZSM-5 were examined as the main building components of the biocarrier scaffolds, using bentonite, montmorillonite, and halloysite nanotubes as inorganic binders in various combinations. 3D printing was utilized to form pastes into monoliths that underwent heat treatment. The 3d-printed biocarriers were subjected to a mechanical analysis, including density, compression, and nanoindentation tests. Furthermore, the 3d-printed biocarriers were morphologically and structurally characterized using nitrogen adsorption at 77 K (LN2), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The stress-strain response of the materials being studied was obtained through nanoindentation tests combined with finite element analysis (FEA). These tests were also utilized to simulate the lattice geometries under compression loading conditions to investigate their deformation and stress distribution in relation to experimental compression testing. The results indicated that the 3d-printed biocarrier 13X/halloysite nanotubes was endowed with high specific surface area and extended mesoporous structure. Due to these assets, its bulk density was one of the lowest observed amongst the biocarriers derived from the various combinations of materials. The biocarriers based on the 13X zeolite exhibited the highest mechanical stability and appropriate morphological features. The 13X/halloysite nanotubes scaffold exhibits a moderate hardness value compared to the rest, while it presents the highest value of modulus of elasticity. In conclusion, Aluminosilicate zeolites and their combination with clays and inorganic nanotubes afford 3d-printed biocarriers of various textural and structural properties, which can be utilized to improve biological processes.

Background: Mycobacterium avium complex includes the commonest nontuberculous mycobacteria associated with human infections. These infections have been associated with the production of biofilms in many cases, but there are only a few studies about biofilms produced by the species included in this group. Methods: Three collection strains (M. avium ATCC25291, M. intracellulare ATCC13950, and M. chimaera DSM756), three clinically significant strains (647, 657, and 655), and three clinically nonsignificant ones (717, 505, and 575) of each species were included. The clinical significance of the clinical isolates was established according to the internationally accepted criteria. Biofilm ultrastructure was studied by Confocal-Laser Scanning Microscopy using Backlight Live dead and Red Nile stains. Viability, covered surface, height and relative autofluorescence were measured in several images/strain. The effect of clarithromycin was studied the technique described by Muñoz-Egea et al with modifications regarding incubation time. The study included clarithromycin in the culture medium at a concentration achievable in the lungs (11.3 mg/L), using one row of wells as the control without antibiotic. Bacterial viability inside the biofilm is expressed as a percentage of viable cells. The differences between the different parameters of biofilm ultrastructure were analysed using Kruskal-Wallis test. The correlation between bacterial viability in the biofilm and treatment time was evaluated using Spearman's rank correlation coefficient (ρ). Results: The strains showed differences between them with all the studied parameters, but neither a species-specific pattern nor a clinical significance-specific patter were detected. For the effect of clarithromycin, the viability of the bacteria contained in the biofilm was inversely proportional to the exposure time of the biofilm (ρ > -0.3; p-value < 0.05), excluding two M. chimaera strains (M. chimaera DSM756 and 575), which showed a weak positive correlation with treatment time (0.2 < ρ < 0.39; p-value < 0.05). Curiously, despite a clarithromycin treatment of 216 hours, the percentage of biofilm viability of the strains evaluated here was not less than 40% at best (M. avium 717). Conclusions: All the M. avium complex studied strains are able to form biofilm in vitro, but ultrastructural characteristics between them suggest that these are strain-specific characteristics unrelated with neither with the species nor with the clinical significance. The clarithromycin effect on MAC species is biofilm age/time of treatment-dependent and appears to be strain-specific while being independent of the clinical significance of the strain.

Few clinical factors can help predict spontaneous culture conversion (SCC) in patients with Mycobacterium avium complex-pulmonary disease (MAC-PD). We retrospectively classified 373 MPD patients who had undergone watchful waiting without antibiotics according to the so-called ‘body mass index (BMI), age, cavity, erythrocyte sedimentation rate (ESR), and sex (BACES)’ severity. We evaluated whether lower severity is associated with a higher rate of SCC. Of 373 patients, 153 (41%) achieved SCC without antibiotics during a median follow-up of 48.1 months. There was a trend toward a higher SCC rate in patients with lower BACES severity: 48% (87/183), 37% (58/157), and 24% (8/33) in the mild, moderate, and severe BACES groups, respectively. In addition, a favorable outcome, defined as maintaining SCC or having two consecutive negative sputum cultures until the last follow-up date, was also more common in patients with lower BACES severity with 53% (97/183), 34% (54/157), and 18% (6/33) in the mild, moderate, and severe BACES groups, respectively. In multivariate analysis, moderate (hazard ratio [HR]=0.63; 95% confidence interval [CI] 0.44 – 0.91; p = 0.013) and severe BACES (HR 0.37; 95% CI 0.16 – 0.90; p = 0.028) had a significantly negative impact on favorable outcomes compared to mild BACES. Lower BACES severity may be associated with SCC in MAC-PD patients.

Background: We assessed the pertinence of yearly updating the International Working Group on the Diabetic Foot (IWGDF) risk classification in people with diabetes by quantifying the changes in the risk group and its accuracy in identifying those developing an ulcer (DFU) in a primary care setting. Methods: In our retrospective cohort study, we included all people with diabetes with a foot as-sessment registry between January 2016 and December 2018 in the Baixo Alentejo Local Health Unit. Foot-related data was collected at baseline after one and two years. DFU and/or death until December 2019 were registered. The proportion of people changing their risk status each year was calculated. Accuracy measures of the IWGDF classification to predict DFU occurrence at one, two, and three years were calculated. Results: A total of 2097 people were followed for three years, during which 0.1% died, and 12.4% developed a DFU. After two years, 3.6% of the participants had progressed to a higher-risk group. The IWGDF classification presented specificity values superior to 90% and negative predictive values superior to 99%. Conclusion: Foot risk status can be safely updated every two years instead of yearly. The IWGDF classification can accurately identify those not at risk of DFU.

Gelatin industry residues are increasingly used as fertilizer and soil conditioner. However, correct residue dosage is critical for grass development and minimizing environmental impacts. This randomized block design study determined adequate dosage of Mixed Gelatin Sludge (MGS) for Marandu grass production in wet/dry seasons in Brazil. Five MGS levels (0%–200% of required nitrogen) were compared to mineral fertilizer. Agronomic/productivity characteristics, bromatological composition, macro/micro-nutrient composition of leaves, and soil chemical attributes were evaluated. Agronomic/productivity characteristics were influenced by MGS dose in both dry/rainy seasons, except for leaf blade pseudostem ratio and percentage of leaves/pseudostem. Bromatological composition was influenced by MGS doses in dry/rainy seasons except for dry/mineral material quantities. Marandu leaf tissue chemical composition was significantly influenced by MGS dose, except for potassium, boron, and iron. Chemical composition of four soil layers between 0–50 cm influenced GMS dose, except for pH, organic matter, magnesium, copper, manganese, and zinc. GMS dose for Marandu production should be 200% of nitrogen (N) requirement. GMS application increased productivity/quality of Marandu grass. Macronutrients (N, phosphorus) and micronutrients (Ca, Mg, S, Cu, and Zn) increased in Marandu grass and in the soil (Ca, S, and Na). Increased sodium (Na) level was not limiting.

The ceramic sherds from approximately 20 samples of lead-glazed tableware, recovered from diverse archaeological sites, including three repurposed storage pits transformed into dumpsters within the medieval city of Santarém (13th-14th century), underwent a meticulous examination. This investigation utilized techniques such as micro-Raman, ground state diffuse reflectance absorption, and X-ray fluorescence spectroscopies, in addition to X-ray diffraction and stereomicroscopy. A parallel study was conducted on contemporaneous European ceramics (glazed sherds) sourced from archaeological sites dating back to the 13th-15th centuries in Saintonge (France), Ardenne, Zomergem, and Bruges (Belgium), as well as Surrey-Hampshire, Kingston, and Cheam (England). The colour of the ceramic bodies is predominantly white or whitish, with a few exhibiting a vivid red hue. Ceramic sherds with a light paste composition were likely crafted using kaolin clays as raw materials. Analyses of fabric, mineralogical, and elemental composition of the sherds suggest that the majority of Santarém's glazed ceramics were locally or regionally produced, potentially derived from a Pliocene kaolin-rich sand formation. However, this conclusion is not supported by the absence of discovered lead glaze kilns or workshops in Santarém for the late Middle Ages. A distinctive feature from the European workshop ceramics lies in the quartz temper content and fabric.

The Herpotrichiellaceae family is an important group of dematiaceous filamentous fungi, associated with a variety of pathogenic fungal species causing Chromoblastomycosis (CBM) and Phaeohyphomycosis (PHM), both with polymorphic clinical manifestations and worldwide incidence. Currently, the identification of this family and determination of the causative agent is challenging due to the subjectivity of morphological identification methods, necessitating the use of molecular techniques to complement diagnosis. In this context, genetic sequencing of the Internal Transcribed Spacer (ITS) has become the norm due to a lack of alternative molecular tools for identifying these agents. Therefore, this study aimed to develop PCR-Multiplex methodologies to address this gap. Sequences from the ITS and Large subunit (LSU) of ribosomal DNA were used, and after manual curation and in vitro analyses, primers were synthesized for the identification of the aforementioned targets. The primers were optimized and validated in vitro, resulting in two PCR-Multiplex methodologies: one for identifying the Herpotrichiellaceae family and the bantiana clade, and another for determining the species Fonsecaea pedrosoi and Fonsecaea monophora. Ultimately, the assays developed in this study aim to complement other identification approaches for these agents, reducing the need for sequencing, improving the management of these infections, and enhancing the accuracy of epidemiological information.

Assessing dietary inadequacies can contribute to understanding the nutritional vulnerabilities of a country. This study aimed to investigate nutrient intakes and micronutrient inadequacies in the Brazilian adult population, with an interest in different age subgroups. Cross-sectional study with 1812 individuals aged 19 to 65 years from a population-based study with a representative sample of Brazilian adults. Dietary intake was assessed by two 24-hour food recalls, and the probabilities of inadequate intake were estimated using the Dietary Reference Intake targets. Adequate macronutrient intake was over 99% for proteins, 84.7% for carbohydrates, and 80.7% for total fats. There was a high probability of inadequacy (above 90%) for vitamins D and E, but vitamin D inadequacy was very similar between sexes. In contrast, vitamin E was more likely to be inadequately consumed among women. A high probability of inadequacy (above 85%) of calcium and magnesium was found in the population, regardless of age group. Except for iron, the probability of inadequacy of other minerals increased with age. The results showed a relevant proportion of nutrient inadequacies, with those most at risk being women and older individuals, helping with better targeting and monitoring of public health policies that address nutritional problems in the population.

Sarcopenia is associated with clinical complications, which increase mortality. Effective sarcopenia risk screening in the elderly should be grounded in a comprehensive assessment, encompassing both nutritional status and medication use. Current screening tools, while emphasizing muscular per-formance, exhibit limited sensitivity in identifying elderly individuals at risk of sarcopenia. This cross-sectional study aimed to develop a sarcopenia risk screening tool, named Sarc-Global, with enhanced sensitivity, incorporating factors pertinent to the overall health of the elderly. Utilizing the criteria established by EWGSOP2 for diagnosing sarcopenia, we evaluated 395 communi-ty-dwelling elderly individuals. This evaluation served as a reference for assessing the efficacy of two validated questionnaires, Sarc-F and Sarc-CalF, and our newly developed Sarc-Global. Con-structed through multiple logistic regression, Sarc-Global integrates variables such as sex, age, medication use, body mass index, arm circumference, and handgrip strength – all significantly associated with sarcopenia (p

We have developed a family of N-alkyl-nitrobenzamides that exhibit promising antitubercular ac-tivities. These compounds are a simplification of the structure of known inhibitors of DprE1, an essential Mtb enzyme and an emergent anti-TB target. The compounds were obtained by a simple synthetic methodology and their antitubercular, cyto-toxicity and stability were studied. The most active compounds were the 3,5-dinitro and the 3-nitro-5-trifluoromethyl derivatives. Lipophilicity was shown to be a main parameter modulating activity within each family. The most active compound presented MIC of 16 ng/mL and had in-termediate lipophilicities. The activity was also studied in a macrophage model of infection. In this model the compounds with chain lengths of 6 and 12 carbon atoms presented the best results, ex-hibiting activity profiles comparable to isoniazid. Docking studies performed suggested that the fit of our highly active compounds to the DprE1 binding pocket was very similar to the literature compounds. Furthermore, the metrics used in the analysis were able to distinguish between the families of the highly and least active compounds. Assessment of susceptibility over multiple species and computational together with the structure of the compounds are complementary information that supports DprE1 as a likely target of action for this type of compounds.

Background: In this investigation, we explored the effects of pharmacological cholinergic stimulation on cardiac function and renal inflammation following acute myocardial infarction (AMI) in spontaneously hypertensive rats (SHRs). Methods: Adult male SHRs were randomized into three experimental groups: sham-operated; AMI+Veh (infarcted, treated with vehicle); and AMI+PY (infarcted, treated with the cholinesterase inhibitor pyridostigmine bromide (PY) - 40 mg/kg, once daily for seven days). Rats were euthanized 7 or 30 days post-surgery. Clinical parameters were assessed on the day before euthanasia. Subsequent to euthanasia, blood samples were collected and renal tissues were harvested for histological and gene expression analyses aimed to evaluate inflammation and injury. Results: Seven days post-surgery, the AMI+PY group demonstrated improvements in left ventricular diastolic function and autonomic regulation, and a reduction in renal macrophage infiltration compared to the AMI+Veh group. Furthermore, there was a notable downregulation of pro-inflammatory gene expression and an upregulation of anti-inflammatory gene expression. Analysis 30 days post-surgery showed that PY treatment had a sustained positive effect on renal gene expression, correlated with a decrease in biomarkers indicative of subclinical kidney injury. Conclusion: Short-term cholinergic stimulation with PY provides both cardiac and renal protection by mitigating the inflammatory response after AMI.

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four week incubation in M63 medium or 24 hour exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in biofilm ultrastructure between the two methods, including variation in appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at &gt;10-fold lower rates than planktonic cells at almost all concentrations. Though these comparisons indicate differences in biofilm responses by M. avium under differing conditions, they also suggest common features of biofilm formation including cellulose production and antimicrobial resistance.

Published data or available literature on sustainable building plan-design, construction, performance, and renovation criteria have covered some stages or some parts of each stage. These data usually have been published partially in many different papers―there have not been any papers that published these data together. Hence, this paper aims to collectively review these data and publish them together. The collection and review of these data were carried out by our twenty-five team members who specialized in sustainable urban, architectural, and civil engineering and construction management. The gathered and reviewed outputs were combined and validated based on a general group consensus. This consensus decision-making proceeded through two major group meetings with several follow-up meetings. The first major meeting was to combine and improve the gathered reviewed sustainable building criteria for Cambodia. The second major meeting was to validate the improved reviewed sustainable building criteria for Cambodia. The several follow-up meetings were to discuss the relevance and importance of the validated data “criteria and their classifications and descriptions” in all stages and more focused on their importance and applicability to Cambodia. The collective reviewed data in this paper would be useful to researchers in the fields. They could also be useful collective knowledge and information for policymakers from governmental agencies and development partners, particularly for sustainable building and construction companies.

Snakebite envenomations (SBE) are a significant global public health threat due to their morbidi-ty and mortality. It is a neglected public health issue in many tropical and subtropical countries. Brazil is in the top ten countries affected by SBE, with 32,160 cases reported in Brazil only in 2020, posing a high burden for this population. We describe the data structure of the Snakebite data on Notifiable Disease Information System (SINAN) made available by the Brazilian Minis-try of Health from 2007 to 2020. In addition, we also provide R scripts that allow a quick and automatic updating of data from the SINAN according to its availability. The data presented in this work is related to clinical and demographic information of SBE cases. Also, data on out-comes, laboratory results, and treatment is available. The database is available and freely acces-sible; however, preprocessing, adjustments, and standardization are necessary due to incom-pleteness and inconsistencies. Regardless of these limitations, it provides a solid basis for as-sessing different aspects and the national burden of envenoming.

The spread of multidrug-resistant mycobacterium strains requires the development of new approaches to combat diseases caused by these pathogens. For that, photodynamic inactivation (PDI) is a promising approach. In this study, a tricarbocyanine (TCC) is used for the first time as a near-infrared (740 nm) activatable PDI photosensitizer to kill mycobacteria with deeper light penetration. For better targeting, a novel tricarbocyanine dye functionalized with two trehalose units (TCC2Tre) is developed. The photodynamic effect of the conjugate against mycobacteria, including Mycobacterium tuberculosis, is evaluated. Under irradiation, TCC2Tre causes more effective killing of mycobacteria compared to the photosensitizer without trehalose conjugation, with 99.99% dead vegetative cells of M. tuberculosis and M. smegmatis. In addition, effective photoinactivation of dormant forms of M. smegmatis is observed after incubation with TCC2Tre. Mycobacteria treated with TCC2Tre are more sensitive to 740 nm light than the Gram-positive Micrococcus luteus and the Gram-negative Escherichia coli. For the first time, this study demonstrates the possibility of in vitro PDI of mycobacteria including the fast-growing M. smegmatis and the slow-growing M. tuberculosis using near-infrared activatable photosensitizers. These findings are useful for the development of new efficient alternatives to antibiotic therapy.

Responses to water stress were measured for sugar maple (Acer saccharum subsp. saccharum Marshall) sources from Oklahoma (Caddo sugar maple), Missouri, Tennessee, Ontario and a black maple (Acer saccharum subsp. nigrum F. Michx.) source from Iowa. Seedling sources were selected for differences in temperature and precipitation of their geographic origins. Seedlings were preconditioned through moist (watered daily) or dry (watered every 4-7 days) cycles and then exposed to prolonged water stress. Dry preconditioned sugar maple seedlings from Oklahoma, Missouri, and Tennessee, sources from warmer and/or drier climates with greater relative evapotranspiration potentials, declined less rapidly in net photosynthesis, stomatal conductance, and water use efficiency (WUE) as water stress increased than dry preconditioned seedlings from Ontario and Iowa having origins in cooler, moister climates. Under imposed water stress the Ontario and Iowa sourced seedlings increased their root to shoot ratios and decreased their specific leaf area, mechanisms for drought avoidance. However, no corresponding changes in these values occurred for Oklahoma, Missouri, and Tennessee sources. Results from this study suggest greater tolerance of water stress in the Oklahoma, Missouri and Tennessee ecotypes from the western and southern range of sugar maple resulted primarily with WUE rather than other water stress coping mechanisms.

Tuberculosis (TB) remains a global health threat, necessitating innovative strategies for control and prevention. This comprehensive review explores the Mycobacterium tuberculosis Lysine Exporter (LysE) gene, unveiling its multifaceted roles and potential uses in controlling and preventing tuberculosis (TB). As a pivotal player in eliminating excess L-lysine and L-arginine, LysE contributes to the survival and virulence of M. tuberculosis. This review synthesizes findings from different electronic databases and includes 13 studies focused on the LysE of M. tuberculosis. The research unveils that LysE can be a potential drug target, a diagnostic marker for TB, and a promising candidate for vaccine development. The absence of LysE in the widely used BCG vaccine underscores its uniqueness and positions it as a novel area for TB prevention. In conclusion, this review underscores the significance of LysE in TB pathogenesis and its potential as a drug target, diagnostic marker, and vaccine candidate. The multifaceted nature of LysE positions it at the forefront of innovative approaches to combat TB, calling for sustained research efforts to harness its full potential in the global fight against this infectious disease.

Mycobacterium tuberculosis is the agent of tuberculosis, one of the most important infectious diseases in the world. This microorganism stands out from other bacteria, not only for its extremely high infection capacity, but also for its cellular characteristics that include an extremely resistant and hydrophobic cellular parade the passage of antibiotics. An incredible ability to adapt to adverse conditions inside the host as well as its vast arsenal of virulence factors that allow its survival within the inhospitable environment within the macrophage can be highlighted. This review aims to discuss several aspects of MTB microbiology, genetics, and physiology. We will address in this review details of the metabolism of MTB that allows it to replicate in the active phase and remain viable during latency, as well as the characteristics of its cell wall that contribute to the blockade of the immune response and its resistance to antibiotics.

Mycobacterium abscessus is a non-tuberculous mycobacteria notoriously known for causing severe, chronic infections. Treatment of these infections is challenging due to either intrinsic or acquired resistance of M. abscessus to multiple antibiotics. Despite prolonged poly-antimicrobial therapy, treatment of M. abscessus infections often fails, leading to progressive morbidity and eventual mortality. Great research efforts are invested in finding new therapeutic options for M. abscessus. Clofazimine and rifabutin are known anti-mycobacterial antibiotics, repurposed for use against M. abscessus. Novel antimicrobials active against M. abscessus include delamanid, pretomanid and PIPD1 and the recently approved beta-lactamase inhibitors avibactam, relebactam and vaborbactam. Previously unused antimicrobial combinations e.g. vancomycin-clarithromycin and dual beta-lactam therapy have been shown to have synergistic effect against M. abscessus in experimental models, suggesting their possible use in multiple-drug regimens. Finally, engineered phage therapy has been reported to be clinically successful in a severe case of disseminated M. abscessus infection. While many of these experimental therapeutics have shown activity against M. abscessus in vitro, as well as intracellular and/or animal models, most have little if any evidence of effect in humans infections. Clinical studies of M. abscesssus treatments are needed in order to reliably determine the value of their incorporation in therapeutic regimens.

Mycobacteria are a large family of over 100 species, most of which do not cause diseases in humans. The majority of the mycobacterial species are referred to as nontuberculous mycobacteria (NTM), meaning they are not the causative agent of tuberculous (TB) or leprosy, i.e. Mycobacterium tuberculous complex and Mycobacterium leprae, respectively. The latter group is undoubtedly the most infamous, with TB infecting an estimated 10 million people and causing over 1.2 million deaths in 2017 alone (1). TB and leprosy also differ from NTM in that they are only transmitted from person to person and have no environmental reservoir, whereas NTM infections are commonly acquired from the environment (2) (3). It took until the 1950's for NTM to be recognised as a potential lung pathogen in people with underlying pulmonary disease and another 3 decades for NTM to be widely recognised by the medical community when NTM, particularly Mycobacterium avium complex (MAC) was recognised as the most common group of opportunistic pathogens in AIDS patients (4). This review focusses on an emerging NTM called Mycobacterium abscessus (M. abs). M. abs is a rapidly growing (RGM) NTM that is responsible for opportunistic pulmonary infections in patients with structural lung disorders such as cystic fibrosis (CF) and bronchiectasis (5), as well as a wide range of skin and soft tissue infections (SSTIs) in humans (6) (7). M. abs is a weakly staining Gram-positive mycobacterium that is neverand is, like other NTM, most often seen in soil and aquatic environments (8). The bacillus-shaped bacterium is 1-6µm long and 0.2-0.5µm in diameter, with curved ends and the presence of cord factor, or trehalose 6-6'-dimycolate, a glycolipid found in the cell wall of virulent species of mycobacteria that results in "serpentine cord" cell morphology is sometimes observed (8) (9). On solid growth medium, M. abs can display either a rough (M. abs-R) or smooth (M. abs-S) morphotype, with the rough morphotype displaying a more virulent phenotype than its smooth variant (10). The rough morphotype is characterised by irregular parallel filaments that form ridges across the colony, whereas a smooth morphology is displays a wet, smooth colony with no filaments or ridges (79). This morphology is driven by cell wall glycopeptidolipid (GPL); a loss of GPL results in the reversion from rough to smooth morphotype (80) (81). Moreover, it has been shown using human tissue culture models of infection that M. abs-R is able to persist and multiply within the host macrophage whereas M. abs-S lacks this capacity, hence its role in virulence (82). Like all other mycobacteria, M. abs are aerobic, non-motile and acid-fast organisms with a characteristically thick, lipid-rich cell wall that is hydrophobic. Due to their unusually impermeable, thick cell wall, mycobacteria are notoriously resistant to many antibiotics, disinfectants and heavy metals (11). When the genome of M. abs became available in 2009, elucidation of the resistance mechanisms of M. abs became an area of focus for scientific research, as the considerable threat it poses to public health became more apparent (12) (13) (14). In this review we will discuss how we came to understand the pathogen, how it is currently treated, as well as a discussion of drug resistance mechanisms and novel treatments currently in development.

Menaquinone is one of the major lipoquinones involved in the respiratory chains of bacteria, which includes ubiquinone. Menaquinone is an essential component in the electron transfer process and ATP synthesis especially in some Gram-positive bacteria including Mycobacterium tuberculosis (M. Tb). However, mammals utilize ubiquinone in the analogous electron transfer processes, and do not synthesize menaquinone. In the biosynthesis of menaquinone, MenA is an important enzyme, which is involved in the conversion of 1,4-dihydroxy-2-napthoate to demethylmenaquinone via prenyl transferase activity. Therefore MenA is chosen as a drug target in determining the inhibitor. While designing and screening MenA inhibitors we observed that benzophenone derivatives showed high coordination with enzymes in transition state and interrupted the growth of M. Tb moderately. In addition, diphenyl ether derivatives also showed moderate MIC and IC50 results. Therefore, to improve the biological activity we synthesized a polymeric compound having both benzophenone and diphenyl ether groups. In addition, a hydrophilic functional group was introduced into the polymeric compound to enhance the biological activity. The synthesized polymeric inhibitor showed improved MIC and IC50 values to inhibit the M. tb infection.

The tetracyclic pyrido[4,3-b]carbazole olivacine and four of its oxygenated derivatives have been synthesized by a late-stage palladium-catalyzed Heck-type cyclization of the pyrrole ring as key step. In a test for inhibition of the growth of Mycobacterium tuberculosis 9-methoxyolivacine showed the most significant inhibiting activity against Mycobacterium tuberculosis with an MIC₉₀ value of 1.5 μM.

Paratuberculosis (PTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects a wide variety of domestic and wild animals. It is considered as one of the diseases with the highest economic impact in the ruminant industry. Despite many efforts and intensive research, PTB control is still controversially discussed and diagnostic and immunoprophylactic tools lack great limitations. Thus, models play a crucial role in understanding the pathogenesis of infection and disease, and in testing novel vaccine candidates. Here, we review the potential and limitations of different experimental approaches currently used in PTB research, focusing on laboratory animals and cell based models. The aim of this review is to offer a vision of the models that have been used and what has been achieved or discovered with each one so that the reader can choose the best model to answer their scientific questions and prove their hypotheses. Also, we bring forward new approaches that we consider worth exploring in the near future.

The emergence of drug-resistant tuberculosis forced the development of new drugs and screnning of more effective or less toxic analogs. Mycolic acids biosynthesis is targeted by several antituberculosis drugs toped by the isoniazid being one of the most important in tuberculosis therapy. Recently, perchlozone, acting on another step in FAS-II cycle, received official approval and was included in the Russian national clinical guidelines. Using the serial dilution method on agar 7H10 plates for perchlozone and Sensititre MYCOTB microdilution plate we analyzed the phenotypic properties of primary M. tuberculosis clinical isolates and analyzed the molecular determinants of resistance to isoniazid, ethionamide and perchlozone. We found a wide variation in the MIC of perchlozone from 2 to 64 mg/L, correlating with the overall resistance profile: the MIC was higher for MDR and pre-XDR isolates. The cross-resistance between the ethionamide and the perchlozone was driven by mutations in ethA gene encoding monooxygenase responsible for activation of both drugs. Presumably susceptible to perchlozone wild-type strains had MICs ranged 2–4 mg/L, and the breakpoint estimated to be at 4 or 8 mg/L. In conclusion, susceptibility to perchlozone retained for a part of MDR strains, as does suceptibility to ethionamide, providing the possibility of therapy for such cases based on phenotypic or molecular analysis.

Mycobacterium tuberculosis variant bovis (MBO) has one of the widest known mammalian host ranges, including humans. Despite characterization of this pathogen in the 1800s, and whole genome sequencing of a UK strain (AF2122) nearly two decades ago, the basis of its host specificity and pathogenicity remains poorly understood. Recent experimental calf infection studies show that MBO strain Ravenel (MBO Ravenel) is attenuated in the cattle host. In the present study, experimental infections were performed to define attenuation and whole genome sequencing completed to identify regions of differences (RD) and single nucleotide polymorphisms (SNPs) to explain the observed attenuation. Comparative genomic analysis of MBO Ravenel against three pathogenic strains of MBO (strains AF2122-97, 10-7428 and 95-1315) was performed. Experimental infection studies on 5 calves each, with either MBO Ravenel or 95-1315, revealed no visible lesions in all 5 animals in the Ravenel group despite robust IFN-γ responses. Out of 486 polymorphisms in the present analysis, 173 were unique to MBO Ravenel among the strains compared. A high-confidence subset of 9 unique SNPs were missense mutations in genes with annotated functions impacting 2 major MBO survival and virulence pathways: 1) Cell wall synthesis & transport [espH (A103T), mmpL8 (V888I), aftB (H484Y), eccC5 (T507M), rpfB (E263G)], and 2) Lipid metabolism & respiration [mycP1(T125I), pks5 (G455S), fadD29 (N231S), fadE29 (V360G)]. These substitutions likely contribute to the observed attenuation. Results from experimental calf infections and the functional attributions of polymorphic loci on the genome of MBO Ravenel provide new insights into the strain’s genotype-disease phenotype associations.

Mycobacterium ulcerans is the causative agent of the Buruli ulcer, also known, in Australia, as Daintree ulcer or Bairnsdale ulcer. This destructive skin disease is characterized by extensive and painless necrosis of the skin and soft tissue with the formation of large ulcers, commonly on the leg or arm. To date, 33 countries with tropical, subtropical and temperate climates in Africa, the Americas, Asia and the Western Pacific have reported cases of Buruli Ulcer. The disease is rarely fatal, although it may lead to permanent disability and/ or disfigurement if not treated appropriately or in time. It is the third most common mycobacterial infection in the world after tuberculosis and leprosy. The precise mode of transmission of M. ulcerans is yet to be elucidated. Nevertheless, it is possible that the mode of transmission varies with different geographical areas and epidemiological settings. The knowledge about the possible route of transmission and potential animal reservoir of M. ulcerans is poorly understood and still remains patchy. We conducted a systematic review with selected key words on PubMed and INFORMIT databases to aggregate available published data on animal reservoirs of M. ulcerans. After certain inclusion and exclusion criteria, a total of 17 studies were included in the review. A variety of animals, e.g rodents, shrews, possums (ringtail and brush tail), horses, dogs, alpacas, koalas and Indian flap-shelled turtles have been recorded as being infected with M. ulcerans around the world. The majority of studies included in this review identified animal reservoirs, either aquatic or terrestrial, as predisposing for the emergence and reemergence of M. ulcerans infection. Taken together, the selected studies in this systematic review and discussed so far, it is clear that exotic wildlife, aquatic animals and native mammals play a significant role as reservoirs for M. ulcerans.

Sweet cherry pomace is a by-product that can be a source of bioactive phenolic compounds. Usually, polyphenols have been extracted using conventional extraction methodologies. However, a significant fraction, called non-extractable polyphenols (NEPs), remains retained in the conventional extraction residues. Therefore, this work is aimed, for the first time, to investigate the release of NEPs from cherry pomace combining pressurized liquid extraction (PLE) and enzyme-assisted extraction (EAE) using Promod enzyme. A response surface methodology was employed to study the influence of temperature, time, and pH on the NEPs extraction. The response variables were the total phenolic content (TPC) measured by Folin-Ciocalteu method, total proanthocyanidin (PA) content evaluated by vanillin, DMAC, and butanol/HCl assays, and total antioxidant capacity determined by Trolox equivalent antioxidant capacity and inhibition of hydroxyl radical assays. The results indicated that PLE-EAE was more suitable and selective to obtain NEPs from sweet cherry pomace than PLE alone. In fact, the extracts obtained by PLE-EAE displayed higher TPC, PA content, and bioactivity than the extracts obtained by PLE under the same extraction conditions, and those obtained by conventional methods. Moreover, size-exclusion chromatography profiles showed that the combination of PLE and EAE enabled the recovery of NEPs with higher molecular weight than PLE without EAE treatment.

Plants have been used in alternative medicine for centuries in the treatment of many diseases with the metabolites they contain. The goal of this work was to investigate the phenolic profile, antioxidant capacity and biological activity of I. helenium subsp. pseudohelenium extracts. Phe-nolic (HPLC) substances in leaf ethanol (L-EtOH), leaf pure water (L-PW), root ethanol (R-EtOH), and root pure water (R-PW) extracts of Inula helenium subsp. pseudohelenium plant were investi-gated. Also, in vitro the antimicrobial (hollow agar), antioxidant (DPPH, ABTS.+, FRAP, CU-PRAC), antiproliferative (MTT assay against HT-29, MCF-7 and L-929), and DNA (Agarose gel electrophoresis) protective activities of the samples were examined. Although gallic acid was not detected in the extracts, the presence of curcumin was detected only in EtOH extracts. L-EtOH exhibited the best antimicrobial activity. According to the antioxidant results, it was found that the L-EtOH extract had an activity profile close to the standard antioxidants in general. It was also found that the L-EtOH extract stabilized the DNA by disrupting the scavenging effect of H2O2+DMSO. According to the antiproliferative activity results, EtOH extracts exhibited the strongest effect, especially against MCF7. When evaluated in general terms, it was found that L-EtOH extract showed better activity than other extracts. It is important to conduct more in vivo and in vitro studies after isolating the active components of extracts e.g. L-EtOH, which have good effects in terms of biological activity.

In a recent effort to mitigate harm from human pathogens, many biosynthetic pathways are extensively evaluated for their ability to inhibit pathogen growth and determine drug targets. Among those, one of the important products/targets of such pathways is isopentenyl diphosphate, the universal precursor of isoprenoids, which are essential for the normal functioning of microorganisms. In general, two biosynthetic pathways lead to the formation of isopentenyl diphosphate: 1) the mevalonate pathway in animals and 2) the non-mevalonate or methylerythritol phosphate (MEP) in many bacteria, some protozoa, and plants. Because the MEP pathway is not found in mammalian cells, it is considered an attractive target for the development of antimicrobials against a variety of human pathogens, including Mycobacterium tuberculosis (M.tb). In the MEP pathway, 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase (IspE) phosphorylates 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME) to form 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDPME2P), followed by cyclization to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECPP) by 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). A virtual high throughput screening was done by docking IspE protein with commercially available compounds and identified an active heterotricyclic compound. Hence, we designed, synthesized, and tested similar new heterotricyclic compounds. This study will provide the critical insight necessary for the ability to develop novel antimicrobials that target the MEP pathways in pathogens.

Peptidoglycan hydrolases are enzymes that cleave the peptidoglycan of the bacterial cell wall, facilitating cell growth, cell division, and peptidoglycan turnover. Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker, encodes an Escherichia coli M23 peptidase EnvC homolog. EnvC is a LytM factor required for septal peptidoglycan splitting and daughter cell separation. In this study we investigate how EnvC contributes to the virulence and cell separation of X. citri. We observed that disruption of the X. citri envC gene (ΔenvC) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime (Citrus limonia Osbeck), the X. citri ΔenvC mutant showed a delay in causing citrus canker symptoms compared with the wild-type X. citri strain. Mutant complementation restored the X. citri wild type phenotype. Subcellular localization confirmed that X. citri EnvC is a periplasmic protein. Moreover, the X. citri ΔenvC mutant exhibited elongated cells, indicative of a cell division defect. These findings support the role of EnvC in regulating virulence, cell wall organization, and cell division in X. citri.

Trachymene incisa subsp. incisa is an Australian endemic taxon that varies greatly in the abundance and length of the leaf trichomes. The essential oil composition of five populations of this subspecies, three corresponding to the typical glabrous form and two of the particularly hairy variant, has been analyzed in an attempt to determinate if that variability is also reflected in their composition. The oils have been extracted by hydrodistillation and analysed by Gas Chromatography (GC) and Gas Chromatography coupled to Mass Spectrometry (GC-MS). The essential oils of T. incisa subsp. incisa were characterized by the high amount of sesquiterpenes that were the major fraction. The sesquiterepene hydrocarbons were significantly higher in the hairy variant in comparison to the glabrous one. According to the main compound three different chemotypes were found: I.- -selinene + bicyclogermacrene and II.- -bisabolene + -pinene for the typical glabrous variant and III.- bicyclogermacrene + -caryophyllene for the hairy variant.

The lungs of tuberculosis (TB) patients contain a spectrum of granulomatous lesions ranging from solid and well vascularized cellular granulomas, to avascular caseous granulomas. In solid granulomas, current therapy kills actively replicating (AR) intracellular bacilli, while in low vascularized caseous granulomas the low oxygen tension stimulates aerobic and microaerophilic AR bacilli to transit into non-replicating (NR), drug-tolerant, extracellular stages. These stages, which do not have genetic mutations and are often referred to as persisters, are difficult to eradicate due to low drug penetration inside caseum and mycobacterial cell walls. The sputum of TB patients contains also viable bacilli called differentially detectable (DD) cells that, unlike persisters, grow in liquid, but not in solid media. This review provides a comprehensive update on drug combinations killing in vitro AR and drug-tolerant bacilli (persisters and DD cells), and sterilizing Mycobacterium tuberculosis-infected BALB/c and caseum-forming C3HeB/FeJ mice. These observations have been important for testing new drug combinations in noninferiority clinical trials, in order to shorten duration of current regimens against TB. In 2022, the World Health Organization, based on one of this trial, supported the use of a 4-month regimen for treatment of drug-susceptible TB as a possible alternative to the current 6-month regimen.

Probiotics are increasingly used by consumers and practitioners to reduce gastrointestinal (GI) distress and improve gut function. Here, we sought to determine whether addition of supplemental bacteriophages (PreforPro) could enhance the effects of a common probiotic, Bifidobacterium animalis subsp. lactis (B. lactis) on GI health. We conducted a 4-week, randomized, parallel-arm, double-blind, placebo-controlled trial where primary outcomes included self-assessments of GI health, a daily stool log, and 16s rRNA analysis of gut microbial populations. We observed within group improvements in GI inflammation (p=0.01) and a trending improvement in colon pain (p=0.08) in individuals consuming B. lactis with PreforPro, but not in the group consuming only the probiotic. There was also a larger increase in Lactobacillus and short chain fatty acid-producing microbial taxa detected in stool of participants taking PreforPro with B. lactis compared to the probiotic alone. Overall, these results suggest the addition of PreforPro as a combination therapy may alter gut ecology to extend the GI benefits of consuming B. lactis or other probiotics.

We have studied Juniperus oxycedrus subsp. badia in the central and southern Iberian Peninsula. Here, the macrobioclimate ranges from Mediterranean-pluviseasonal-oceanic and Mediterranean-pluviseasonal-continental, thermotype from thermomediterranean and supramediterranean. The relevés were taken following the Braun-Blanquet phytosociological methodology. A statistical treatment was applied to establish a separation among Juniperus communities. To understand the presence of Juniperus communities in territories dominated by species of the genus Quercus, we applied the formula of Thornthwaite to calculate potential evapotranspiration. The general cluster analysis clearly separates two groups of plant communities and separates the different associations in each group. All plant communities growing on rocky crests and in steeply extreme sloping areas are significantly influenced by the soil. The Ombroclimatic Index does not explain the presence of plant communities influenced by substrate: so, we propose a new Ombroedaphoxeric Index which explains the presence of the Juniperus communities in territories with a thermotype ranging from the thermo to the supramediterranean belt. The areas of distribution of Juniperus species are expanding due to the spread of rocky areas: this phenomenon causes a rise in edaphoxerophilous areas and a decrease in climatophilous one. We propose four new plant associations and with this update on their structures and floristic compositions, it is possible to make an efficient form of conservation in both territories studied (Spain and Portugal) with specific cross-border-cooperation projects.

For screening excellent lactic acid bacteria (LAB) strains to inhibit Escherichia (E.) coli (ETEC) K88, inhibitory activities of more than 1100 LAB strains isolated from different materials and kept in the lab were evaluated in this study. Nine strains with inhibition zone at least 22.00 mm (including that of hole puncher 10.00 mm) and good physiological and biochemical characteristics identified by 16S DNA gene sequencing and recA gene multiple detection, were assigned to Lactobacillus (L.) plantarum subsp. plantarum (5), L. fermentum (1), L. reuteri (1), W. cibaria (1) and E. faecalis (1), respectively. As investigated for their tolerance abilities and safety, only strain ZA3 possessed high hydrophobicity and auto-aggregation abilities, had high survival rate in low pH, bile salt environment and GI fluids, sensitive to ampicillin, resistant to norfloxacin and amikacin, without hemolytic activity and didn’t carry antibiotic resistance genes, exhibited broad spectrum activity against a wide range of microorganisms, and antibacterial substance may attribute to organic acids, especially lactic acid and acetic acid. The results indicated that the selected strain L. plantarum subsp. plantarum ZA3 could be considered a potential probiotic to inhibit ETEC K88 for further research.

Bovine tuberculosis surveillance in Northern Ireland includes Multiple-Locus Variable number tandem repeat Analysis (MLVA) to determine the Mycobacterium bovis genetic type present in both cattle, and the predominant wildlife host, the European badger (Meles meles). These data are informative for investigating clusters of infection and understanding the scale at which interspecific transmission may occur. We utilised a comprehensive dataset of routinely sampled isolates from infected cattle and from badgers killed in road-traffic accidents to investigate the spatial co-location of MLVA types in, and between, the badger and cattle populations. Furthermore, we investigate the hypothesis that the farming enterprise type might explain some variation in this relationship. MLVA types were spatially co-localised in cattle and RTA badger hosts, indicative of a shared epidemic. Dairy herds were more likely to have at least one MLVA type in common with nearby RTA badgers, compared to non-dairy herd types. Marginally more MLVA spatial clustering was observed in non-dairy herds, which may be a consequence relatively more between-herd movements. For the cattle population, local transmission mechanisms such as infection from contiguous herds, infectious wildlife and short-range between-herd cattle movements appear primarily to drive the epidemic: there appears to be a more limited role for long-range movements. Animal management practices are likely the driving force behind this observation, as beef rearing is associated with elevated numbers of animal movements compared to dairy herds.

The in vitro cultivation of M. leprae has not been possible since it was described as causing leprosy, and the limitation of animal models for clinical aspects makes studies on leprosy and bacteria-human host interaction a challenge. Our aim was to standardize the ex vivo skin model (hOSEC) to maintenance and study of M. leprae as an alternative animal model. Bacillary suspensions were inoculated into human skin explants and sustained in DMEM medium until 60 days. Explants were evaluated by RT-PCR-16SrRNA, and cytokines gene expression. Viability and infectivity of bacilli recovered from explant (D28 and D60) were evaluated using the Shepard’s model. All explants were RT-PCR-16srRNA positive. Viability and infectivity of recovered bacilli from explants, analyzed after 5 months of inoculation in mice showed an average positivity of 31%, being the highest positivity in the D28 groups (80%). Furthermore, our work showed different pattern in cytokines gene expression (TGF-β, IL-10, IL-8 and TNF-α) in the presence of alive or dead bacilli. Although changes can be made for improve future experiments, our results have demonstrated that it is possible to use the hOSEC to maintain M. leprae for 60 days, interacting with the host system, an important step in the development of experimental models for studies on the biology of the bacillus, its interactions, and drug susceptibility.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a predominant cause of mortality, especially in low- and middle-income nations. Recently antimicrobial peptides were discovered that at low concentrations could stimulate the growth of M. tuberculosis (hormetic response). In this study, such a peptide was used to investigate the effects on the time to positivity (TTP). A systematic substitution analysis of peptide 14D was synthesized using Spot synthesis technology, resulting in 171 novel peptides. Our findings revealed a spectrum of interactions, with some peptides accelerating M. tuberculosis growth, potentially aiding in faster diagnostics, while others exhibited inhibitory effects. Notably, peptide NH2-wkivfiwrr-CONH2 significantly reduced the TTP by 25 hours compared to the wild-type peptide 14D, highlighting its potential in improving TB diagnostics by culture. Several peptides demonstrated potent antimycobacterial activity, with a minimum inhibitory concentration (MIC) of 20 µg/mL against H37Rv and a multidrug-resistant M. tuberculosis strain. Additionally, a reduction in the formation of cord-like structures was observed, which is indicative of reduced virulence and transmission potential. This study underscores the multifaceted roles of antimicrobial peptides in TB management, from enhancing diagnostic efficiency to offering therapeutic avenues against M. tuberculosis.

Mycobacterium tuberculosis is a leading cause of human mortality worldwide and the emergence of drug-resistantstrains, demands the discovery of new classes of antimycobacterials that can be employed in the therapeutic pipeline. Previously, a secondary metabolite Chrysomycin A, isolated from Streptomyces sp. OA161 was shown to have potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis and different species of mycobacteria. The antibiotic inhibits the mycobacterial topoisomerase I and DNA gyrase leading to bacterial death, but the mechanisms that could cause resistance are currently unknown. To further understand the resistance mechanism, spontaneous resistance mutants were isolated and subjected to whole-genome sequencing. Mutation in a Tet^R family transcriptional regulator MSMEG_1380 was identified in the resistant isolates and was close to an operon encoding membrane protein MSMEG_1381 and MSMEG_1382. Sequence analysis and modeling studies indicated that they are components of the Mmp family of efflux pumps and over-expression of either the operon or individual genes conferred resistance to chrysomycin A, isoniazid, and ethambutol that are in TB therapy. Our study highlights the role of membrane transporter proteins in conferring multiple drug resistance and the utility of recombinant strains overexpressing membrane transporters in the drug screening pipeline.

Antibiotics have played a crucial role in the reduction of TB incidence globally as evidenced by the fact that before the mid-20th century, the mortality rate within five years of the onset of the disease was 50%. The use of antibiotics has eliminated TB as a devastating disease, but the challenge of resistance to anti-TB drugs, which had already been described at the time of the introduction of streptomycin, has become a major global issue in disease management. Mismanagement of multidrug-resistant tuberculosis (MDR-TB) cases, resulting from intermittent drug use, prescription errors and non-compliance of patients, has been identified as a critical risk factor for the development of extensively drug-resistant tuberculosis (XDR-TB). Antimicrobial resistance (AMR) in TB is a multi-factorial, complex problem of microbes evolving to escape antibiotics, the gradual decline of antibiotic development, and different economic and social conditions. In this review, we summarise recent advances in our understanding of how Mycobacterium tuberculosis evolves drug resistance. We also highlight the importance of developing shorter regimens that rapidly reach bacteria in diverse host environments, eradicating all mycobacterial populations and preventing the evolution of drug resistance. Lastly, we also emphasise that the current burden of this ancient disease is driven by a combination of complex interactions between mycobacterial and host factors, and that only a holistic approach that effectively addresses all the critical issues associated with drug resistance will limit the further spread of drug-resistant strains throught the community.

Mincle, a C-type lectin, is expressed predominantly in macrophages, where it plays a role in the macrophage response to various microorganisms such as mycobacteria. Docking is a computational procedure in which various software packages generate different positions at which ligands bind to their receptors. Discovery Studio docking was used in Molegro Virtual Studios. ChemDraw and ChemDraw3D were used for ligand preparation and construction because the protein preparation performed using docking software was a very lengthy process. Mincle plays a novel and non-redundant role in the induction of inflammatory signaling in response to mycobacteria. One disadvantage of Molegro studio docking is that it does not show the metallic atoms with which it interacts but rather shows hydrogen bonding and amino acid interactions. Hypothetically, trehalose dimycolate (TDM) and trehalose dibenzenate (TDB) detected by macrophages ultimately lead to CARD9 signaling, thereby producing pro-inflammatory cytokines and chemokines that have the potential to be used therapeutically.

Photosynthesis response curves were carried out using different light intensities in 14 olive cultivars subjected to moderate and severe water deficits. For each cultivar, the electronic transfer ratio in response to light was also obtained. The mathematical adjustment of these response curves allowed calculating, with a correlation coefficient above 0.95, parameters such as photosynthetic efficiency, the slope of the curves, the number of electrons required to fix a CO2 molecule, and the number of photons to assimilate a molecule of CO2, among others, as well as their variation with the lack of water. This work represents the first contribution of the response of photosynthesis in olive cultivars to different light intensities in moderate and severe drought conditions for a considerable number of cultivars. The parameters described, and the results provided, pave the road for subsequent work related to plant physiology and other areas of science and technology, and allow the tolerance to water stress to be objectively compared in these fourteen olive cultivars.

Sources of resistance to powdery mildew in wild barley (Hordeum vulgare subsp. spontaneum) collected in Jordan, Lebanon, and Libya Jerzy H. Czembor 1, *, Elżbieta Czembor 1 1 Plant Breeding and Acclimatization Institute – National Research Institute (IHAR-PIB), Radzikow, 05-870 Błonie, Poland; [email protected] (E.C.) *Correspondence: [email protected]) Abstract Barley powdery mildew (BPM) is caused by the pathogen Blumeria graminis f.sp. hordei (Bgh). It is an economically important disease and plant pathologists are looking for new sources of resistance to BPM. Barley genetic resources present in gene banks are often a rich source of disease resistance to be used by breeders. These new sources of resistance to BPM are often used in combination (pyramiding) with those that are already used in modern cultivars. Barley accessions, including the wild subspecies Hordeum vulgare subsp. spontaneum (Hvs), are stored in many gene banks and often are a valuable source of economically important characteristics. This source of biodiversity should be more efficiently used to improve barley in the process of plant breeding. However, their proper characterization and availability are urgently needed. The resistance to BPM in 81 accessions of wild barley (Hvs) collected in Jordan (47), Lebanon (23), and Libya (11) was investigated. The seed samples of these accessions were obtained from the ICARDA gene bank and collected in 10 expeditions from 1981 to 1995. Twenty European differential isolates of BPM were used to select accessions with efficient resistance. Thirty-one resistant single plant lines were selected from 15 accessions from Jordan and Libya based on tests performed with the most avirulent isolate of Bgh available. These resistant single plant lines were tested for the presence of specific resistance genes using a differential set of Bgh isolates. After analysis of obtained results, it was concluded that all tested 31 single plant lines of wild barley have genes for resistance that are not represented in the Pallas isolines differential s. Twenty-six lines of Hvs selected from accessions originated in Jordan and Libya showed resistance reaction to all isolates used. Identified new sources of effective resistance to BPM in single plant lines of Hvs will be further tested and used in barley pre-breeding programs. Keywords: Blumeria graminis; resistance genes; resistance; germplasm; gene bank;

Shea tree (Vitellaria paradoxa) is an important fruit tree crop because of its oil used for cooking and industrial manufacture of cosmetics. Despite its many benefits, quantitative trait loci linked to the economic traits have not yet been studied. In this study, we performed association mapping on a panel of 374 shea tree accessions using 7,530 single-nucleotide polymorphisms (SNPs) markers for oil yield and seed related traits. Twenty three markers that were significantly (–log10 (p) = 4.87) associated to kernel oil content, kernel length; width and weight were identified. The kernel dry matter oil content and kernel width had the most significant Marker Trait Association (MTA) on chromosomes 1 and 8 respectively. Sixteen candidate genes that condition early induction of flower buds and somatic embryos, seed growth and development, substrate binding, transport, lipid biosynthesis, metabolic processes during seed germination and disease resistance and abiotic stress adaptation were identified. The presence of these genes suggest their role in promoting shea bioactive functions that condition high oil synthesis. This study provides insights into the important marker-linked seed traits with genes controlling them, useful for molecular breeding for improving oil yield in the species.

This study investigates the potential antimicrobial utility of the natural substance Amentoflavone against four dangerous bacterial strains—Mycobacterium tuberculosis, Helicobacter pylori, Haemophilus influenzae, and Pseudomonas aeruginosa. Utilizing molecular docking studies, are assessed the binding interactions between Amentoflavone and key protein targets crucial for the viability of these bacteria. The findings suggest promising binding affinities, particularly in the active sites of Aminoglycoside 2'-N-acetyltransferase, Thioredoxin Reductase, N-acetylneuraminate Lyase, and Glucose-1-phosphate Thymidylyltransferase in the respective bacterial strains. While these in silico results provide initial insights, further in vitro and in vivo experiments are essential to validate the biological relevance and efficacy of Amentoflavone as a potential antimicrobial agent against these bacterial pathogens.

Mycobacterium tuberculosis (Mtb) the causative agent of human tuberculosis (TB) is one of the most successfully adapted-human pathogens. Human-to-human transmission occurs at high rates through aerosols containing bacteria, but the pathogen evolved prior to the establishment of crowded populations. Mtb has developed a particular strategy to ensure persistence in the host until an opportunity for transmission arise. It has refined its lifestyle to obviate the need for virulence factors such as capsules, flagella, pilli, or toxins to circumvent mucosal barriers. Instead, the pathogen uses host macrophages, where it establishes intracellular niches, for its migration into the lung parenchyma and other tissues and for the induction of long-lived latency in granulomas. Finally, at the end of the infection cycle Mtb induces necrotic cell death of macrophages to escape to extracellular milieu and instructs a strong inflammatory response required for progression from latency to disease and transmission. Common to all these events is ESAT-6, one of the major virulence factors secreted by the pathogen. This review highlights the recent advances in understanding the role of ESAT-6 in hijacking macrophage function to establish a successful infection and transmission and its use as a target for the development of diagnostic tools, and vaccines.

We studied the link between carbon and water fluxes to understand the response of net ecosystem carbon exchange (NEE) to water availability conditions of three different potato water regimes cropping systems [full irrigation (FI), deficit irrigation (DI) and rainfed (RF)]. Through the eddy covariance technique, we measured CO₂ and water vapor exchanges and determined surface resistances, omega factor, and inherent water use efficiency (IWUE). Additionally, continuous plant growth determinations of leaf area index (LAI) and specific leaf area (SLA) were made over the three cropping systems. The RF potato was a net carbon source (NEE = 187.21 ± 3.84 g C m⁻²), while both, FI (NEE= −311.96 ± 12.82 g C m⁻²) and DI (−17.3 ± 4.6 g C m⁻²) were a net carbon sink. Greater sink activity is due to high fluxes of gross primary productivity (GPP) [where the GPP > ecosystem respiration (R_eco)] and evapotranspiration (ET), and the high efficiency in the exchange of carbon and water. Without water limitations, the larger canopy, with greater photosynthetic activity (GPP/R_eco > 2) as well as with low internal resistance offers a greater area for water and carbon exchange, and the highly coupled and synchronized ET – GPP fluxes are primarily controlled by the radiative environment. The lower sink capacity of the DI potato crop and the carbon source activity from the RF, are consequences of a smaller area for water and carbon exchange due to the smaller canopy, and a low IWUE from decoupled and desynchronized carbon and water exchange caused by unbalanced restrictions on ET and GPP fluxes. Specifically, at DI potato, ET remained at a high rate, while GPP was reduced by means of non-stomatal limitations. In the rainfed potato, vapor pressure deficit (VPD) played a significant role increasing midday canopy resistance (Rc) up to 13 times compared to irrigated sites, when VPD was around 0.8 kPa. In consequence, ET and GPP fluxes decreased together, but GPP decreased more than ET because of stomatal and non-stomatal limitations.

The wild grapevine, Vitis vinifera subsp. sylvestris grows naturally throughout the northern hemisphere, including the Mediterranean region. Wild grapevines were also observed sporadically across the southern Levant and have been considered a non-native feral plant. Nevertheless, no formal characterization was conducted for wild grapevines in this region; thus, its taxonomical assignment remains elusive. Previously we have shown that the wild grapevine populations growing in northern Israel are genetically separated from the feral domesticated forms. The aim of this work was to comprehensively describe the morphological, anatomical, and ecological traits of wild grapevines naturally thriving in two distinct habitats in Israel. The dioicous nature of the wild grapevine, as well as their flower and pollen morphology, their characteristic sylvestris fruit and seed morphology, in addition to the occurrence of natural germination of seeds in close vicinity to the mother plant, all lead to our conclusion that these plants belong to Vitis vinifera subsp. Sylvestris, and should be included in the Flora Palaestina. These findings, combined with the recently-published genetic evidence for these populations significantly advance our understanding of the species' ecology and persistence under climate change.

Proteins are considered to be the main carriers of antigenic properties of Mycobacterium tuberculosis and Mycobacterium bovis, the causative agents of tuberculosis, the most serious infectious disease. Neutrophils, following resident macrophages, are among the first to attack the mycobacteria in defense of the organism. Neutrophils themselves are also the target of mycobacterial products; and their effect on neutrophil functions has been less studied. We have obtained about 70 protein fractions from M. bovis lysate; among them four protein fractions (10, 22, 24, and 40 kDa) had high serological activity against the serum of a rabbit immunized with M. bovis cells. We studied the effect of the serologically active proteins isolated from M. bovis on the generation of reactive oxygen species by bone marrow granulocytes of the Balb/c mouse, evaluated by luminol-dependent chemiluminescence. For comparison, two synthetic peptides (both 3 kDa) carrying immunogenic epitopes of MPB 70 protein (M. bovis) were taken. Proteins of 10, 22, and 24 kDa at concentrations of 83, 80, and 59 ng/mL, respectively, were found to activate ROS generation by mouse granulocytes. The 10- and 40-kDa antigens inhibited the receptor-mediated respiratory responses to opsonized zymosan (OZ) and N-formyl-MLF peptide (fMLF) but significantly enhanced the response to the phorbol ester PMA, which directly activates protein kinase C. Antigens with molecular weights of 22 and 24 kDa enhanced the fMLF-induced response and had no effect on the response activated by PMA. The response to OZ was suppressed in the presence of the 22 kDa protein. Synthetic peptides over a wide range of concentrations showed no activity in any case. All M. bovis proteins examined did not affect both proliferation and cell viability of the A549 and THP-1 cells; therefore, they have no cytotoxic or proliferative effects. Thus, serologically active proteins with molecular weights of 10, 22, 24, and 40 kDa isolated from M. bovis modulated the respiratory responses of mouse granulocytes induced by the formyl peptide, while proteins of 10, 22, and 40 kDa inhibited the responses induced by OZ.

Mycobacterium tuberculosis complex causes tuberculosis (TB), a disease that causes pulmonary inflammation but can also affect other tissues. Despite macrophages having a defined role in TB immunopathogenesis, other innate immune cells, such as neutrophils, are involved in this process. These cells have high phagocytic ability and a microbial-killing machine comprised of enzymes, antimicrobial peptides, and reactive oxygen species. In the last two decades, a new neutrophil immune response, the neutrophil extracellular traps (NETs), has been intensely researched. NETs comprise DNA associated with histones, enzymes, and antimicrobial peptides. These structures are related to antimicrobial immune response and some immuno-pathogenesis mechanisms. This mini review highlights the role of NETs in tuberculosis and how they can be helpful as a diagnostic tool and/or therapeutic target.

In the present study, we aimed to investigate whether an automated molecular diagnostic method based on PCR-reverse blot hybridization assay can discriminate between human Mycobacterium tuberculosis (MTB)-positive and -negative FFPE tissues and to compare the relative mRNA expression levels of various host immune markers between MTB-infected and uninfected human tissues using quantitative reverse transcription (qRT) PCR. A total of 52 human FFPE tissue samples from various regions of the body, including the lungs, lymph nodes, tendons, colon, and appendix, were collected and used for the molecular identification of Mycobacterium species and analysis of cytokine mRNA expression. As a result, IFN-γ, TNF-α, IP-10, CXCL9, CXCL11, and GM-CSF mRNA expression levels in MTB-infected tissues were significantly higher than those in uninfected samples. Additionally, the differences in the mRNA expression levels of IFN-γ, CXCL9, and GM-CSF between MTB-infected and uninfected tissues were statistically significant were statistically significant (p < 0.05). Correlation curve analysis indicated that the mRNA expression of IFN-γ was inversely proportional to that of IP-10 and that the mRNA expression levels of IFN-γ, TNF-α, CXCL9, CXCL11, GM-CSF, and TNFR were proportional and well-correlated. Furthermore, to establish marker profiles for detecting MTB infection, the statistically significant expression levels of three markers were combined. We confirmed that the combined profile of IFN-γ, CXCL9, and GM-CSF expression levels was statistically significant (P < 0.001). Although the mRNA expression patterns of host immune markers may vary according to MTB infection status, these patterns may be highly correlated and can be simultaneously used as an additional indicator for diagnosing TB.

This study aimed to determine the persistence of Salmonella Javiana, Listeria monocytogenes, and Listeria innocua in hydroponic nutrient solution (NS) at 15, 25, 30, and 37°C over a 21-day period to mimic time from seedling to mature lettuce. Bacteria were inoculated in modified Hoagland's NS at 106 CFU/ml and maintained at 15, 25, 30, and 37°C. Samples were collected at various time points and quantified. Data were analyzed using a mixed effect model to compare mean log CFU/ml obtained from each sampling point for all three bacteria at four different temperatures. Least mean squares were calculated to compare mean log CFU/ml. Tukey-Kramer honest significant difference test was used to compare mean values. At all temperatures, S. Javiana persisted in NS throughout the 21-day study period, compared to L. innocua and L. monocytogenes where persistence was limited to d 5 to d 14 and d 1 to d 14, respectively. Similarly, decimal reduction values (D-values) of S. Javiana indicated longer persistence in nutrient solution than L. innocua and L. monocytogenes at most temperatures. For instance, at 15°C and 25°C D-values for S. Javiana were estimated at 82 and 26 d, respectively, compared to D-values of 3.6 and ~3 d for L. monocytogenes. Data indicate that temperature has minimal effect on S. Javiana and thus may pose a greater risk during hydroponic production of leafy greens due to longer survival in NS when compared to Listeria spp. This study furthers the understanding of potential food safety risks associated with hydroponic systems and may aid in developing management strategies to reduce foodborne outbreaks, fresh produce recalls, and economic losses.

Tularemia is a severe infectious disease caused by the Gram-negative bacteria Francisella tularensis. F. tularensis is currently divided into three subspecies, holarctica, tularensis and mediasiatica which differ in their virulence and geographic distribution. Subspecies mediasiatica is the least studied because of its very low documented virulence for humans and limited geographic distribution. It was discovered in sparsely populated regions of Central Asia. Since 2011, a new subsp. mediasiatica lineage was identified in Altai (Russia). In 2021, we isolated one subsp. mediasiatica strain in Krasnoyarsk Territory. In spite of its geographic origin, 500 km east from Altai, this strain belongs to the Altai lineage and contributes surprisingly little genetic diversity to previous knowledge. This improved knowledge of the phylogeography of subsp. mediasiatica led us to propose a scenario in which the two zoonotic lineages, holarctica and tularensis, independently emerged from the mediasiatica lineage in Siberia or Central Asia, and make predictions which will allow to challenge this hypothesis

Pantoea stewartii subsp. indologenes (Psi) is a causative agent of leafspot of foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onion. Our recent host range evaluation study identified two pathovars; P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. that are pathogenic on onion and millets or on millets only, respectively. In the current study we developed a pan-genome using the whole genome sequencing of newly identified/classified Psi strains from both pathovars [pv. cepacicola (n= 4) and pv. setariae (n=13)]. The full spectrum of the pan-genome contained 7,030 genes. Among these, 3,546 (present in genomes of all 17 strains) were the core genes that were a subset of 3,682 soft-core genes (present in ≥16 strains). The accessory genome included 1,308 shell genes and 2,040 cloud genes (present in ≤ 2 strains). The pan-genome showed a clear liner progression with >6,000 genes, suggesting the pan-genome of Psi is open. Comparative phylogenetic analysis showed differences in phylogenetic clustering of Pantoea spp. using PAVs/wgMLST approach in comparison to core genome SNP-based phylogeny. Further, we conducted a horizontal gene transfer (HGT) study including four other Pantoea species namely, P. stewartii subsp. stewartii LMG 2715T, P. ananatis LMG 2665T, P. agglomerans LMG L15, and P. allii LMG 24248T. A total of 317 HGT events among four Pantoea species were identified with most gene transfers observed between Psi pv. cepacicola and Psi pv. setariae. Pan-GWAS analysis predicted a total of 154 genes including seven cluster of genes associated with the pathogenicity phenotype on onion. One of the clusters contain 11 genes with known functions and are found to be chromosomally located.

Multidrug-resistant tuberculosis emerged as a serious challenge to tuberculosis management and control. In the Eastern Cape, the Beijing variants are prevalent and a driving force of multidrug-resistant tuberculosis; hence, we investigated the distribution of gene mutations in Beijing strains compared to non-Beijing strains. Multidrug-resistant tuberculosis and heteroresistant isolates were identified in 412 sputum cultures by drug susceptibility testing. The isolates were analyzed for mutations in three genes associated with resistance to antituberculosis first-line drugs: katG and inhA promoters for isoniazid and rpoB for rifampicin. All isolates were genotyped by spoligotyping. There were more males than females and a more economically active age group in the study. The most prevalent mutations in rpoB resistance were in S531L, katG in S315Tb, and inhA in c-15tb. Heteroresistance was found in 18 isolates. Beijing variants were predominant. Most of the heteroresistant isolates were INH, with heteroresistance occurring more in the inhA gene mutation region c-15tb. Beijing and LAM variants were found more frequently in INH heteroresistant isolates. Mutations in katG S315Tb and rpoB S531L were higher in Beijing variants. The Beijing family is a major contributor to the epidemiological picture and accounts for most of the multidrug-resistant tuberculosis in the study area.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens that cause illness primarily in the elderly, in the immunocompromised or in patients with underlying lung disease. Mycobacterium chimaera is a NTM species belonging to the M. avium Complex (MAC) group of species. Since 2013, a global outbreak of M. chimaera infection related to heater-cooler units (HCU) used in cardio-thoracic surgery has been identified. This outbreak was caused by a single strain of M. chimaera. In order to estimate the prevalence of this outbreak strain in Israel, we sampled M. chimaera from several HCU machines in Israel, as well as from patients, sequenced their genomes and compared them to the outbreak strain. The presence of mixed mycobacteria species in the samples complicated the analysis of obtained sequences. By applying a metagenomic binning strategy, we were able to obtain genomes of single strains from the mixed samples, and characterized them. M. chimaera strains were compared to each other and to previously reported genomes from other countries. The strain causing the outbreak related to the HCU machines was identified in several such machines in Israel but not in any of the clinical samples.

To reveal rare phenotypes in bacterial populations conventional microbiology tools should be advanced to generate rapid, quantitative, accurate and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using Mycobacterium smegmatis cells we applied both conventional assays and a microfluidic-microscopy method to reveal antibiotic-tolerance mechanisms of wild type and the msm2570::Tnmutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the msm2570::Tn mutant was due to the low number of lysed cells during the antibiotic exposure compared with wild-type cells. This is the first study that characterized the antibiotic-tolerance phenotype of the msm2570::Tn mutant that has a transposon insertion in the msm2570 gene encoding a putative xanthine/uracil permease, which enrolls in uptake of nitrogen compound during nitrogen limitation. The experimental results indicate that the msm2570::Tn mutant can be further interrogated to reveal antibiotic killing mechanisms, in particularly, antibiotics those targets cell wall integrity.

Lactococcus lactis subsp. cremoris C60 is a probiotic strain of lactic acid bacteria (LAB) which induces various immune modifications in myeloid lineage cells. These modifications subsequently reg-ulate T cell function, resulting in enhanced immunity both locally and systemically. Here, we report that C60 suppresses tumor growth by enhancing macrophage function via metabolic al-terations, thereby increasing adenosine triphosphate (ATP) production in a murine melanoma model. Intragastric (i.g.) administration of C60 significantly reduced tumor volume compared to saline administration in mice. The anti-tumor function of intratumor (IT) macrophage was up-regulated in mice administered with C60, as evidenced by an increased inflammatory phenotype (M1) rather than an anti-inflammatory/reparative (M2) phenotype, along with enhanced anti-gen-presenting ability, resulting in increased tumor antigen-specific CD8+ T cells. Through this functional modification, we identified that C60 establishes a glycolysis-dominant metabolism, rather than fatty acid oxidation (FAO), in IT macrophages, leading to increased intracellular ATP levels. To address the question of why orally supplemented C60 exhibits functions in distal places, we found a possibility that bacterial cell wall components, which could be distributed throughout the body from the gut, may induce stimulatory signals in peripheral macrophages via Toll-like receptors (TLRs) signaling activation. Thus, C60 strengthens macrophage anti-tumor immunity by promoting a predominant metabolic shift towards glycolysis upon TLR-mediated stimulation, thereby increasing substantial energy production.

The aim of this study was to analyze glucosinolates (GSLs) in germplasms that are currently conserved at the RDA-Genebank. The analysis focused on the glucosinolate diversity among the analyzed germplasms, with the goal of identifying those that would be most useful for future breeding efforts to produce nutritionally rich Choy sum plants..Twenty-three accessions of Choy sum which possessed ample background passport information were selected. By analyzing the glucosinolate content for 17 different glucosinolates, we observed aliphatic GSLs to be the most common. We identified one accession, IT228140 to synthesize high quantities of glucobrassi-canapin and progoitrin which have been reported to contain several therapeutic applications. Overall, this study highlights the importance of conserving and analyzing plant genetic re-sources, such as those held in national genebanks, for the purpose of developing new varieties of crops that are both nutritious and beneficial for human health. The identification of excellent germplasms for breeding material and the confirmation of the anticancer effects of glucosin-olates are significant contributions to the field of agricultural research and have the potential to positively impact public health.

There are several well-studied examples of protective symbiosis between insect-host and symbiotic actinobacteria, producing antimicrobial metabolites to inhibit host pathogens. These mutualistic relationships are best described for some wasps and leaf-cutting ants while a huge variety of insect species still remains poorly explored. For the first time we isolated actinobacteria from harvester ant Messor structor, and evaluated the isolates’ potential as antimicrobial producers. All isolates could be divided into two morphotypes of single and mycelial cells. We found that the highest frequency of occurrence of mycelial morphotype was observed among soldiers, and the lowest among larvae in the studied laboratory colony. The representative of this morphotype was identified as Streptomyces globisporus subsp. globisporus 4-3 by polyphasic approach. It was established using E. coli JW5503 pDualRep2 system, that crude broths of mycelial isolates inhibited a protein synthesis in reporter strains, but it did not disrupt the in vitro synthesis of proteins in cell-free extracts. Active compound was extracted, purified and identified as albomycin δ2. Pronounced ability of albomycin to inhibit the growth of entomopathogens suggests that Streptomyces globisporus subsp. globisporus may be involved in defensive symbiosis with the Messor structor ant against infections.

A fully automated bacteria whole genome sequencing (WGS) assay was evaluated to characterize Mycobacterium tuberculosis (MTB) and non-tuberculosis Mycobacterium (NTM) clinical isolates. Results generated were highly reproducible with 100% concordance in species and sub-lineage classification, and 92% concordance between antimicrobial resistance (AMR) genotypic and phenotypic profiles. These findings demonstrate that a fully automated WGS assay, with a short turnaround time of 24.5 hours, provides timely and valuable insights into MTB outbreak investigation while providing reliable genotypic AMR profiling consistent with traditional antimicrobial susceptibility tests (AST). This study establishes a favorable proposition in the adoption of an end-to-end fully automated WGS solutions for decentralized TB diagnostics, thereby aiding in WHO’s vision of tuberculosis eradication.

Uncontrolled transmission of Mycobacterium tuberculosis (M. tuberculosis, MTB) drug resistant strains is a challenge to control efforts of global tuberculosis programme. Due to increasing multi-drug resistant (MDR) cases in Arunachal Pradesh, a northeastern state of India, the tracking and tracing of these resistant MTB strains is crucial for infection control and spread of drug resistance. This study aims to correlate the phenotypic DST, genomic DST (gDST) and phylogenetic analysis of MDR-MTB strains in the region. Of total 200 suspected MDR-MTB isolates, 125(62.5%) were identified as MTB. MGIT-960 SIRE DST detected 71/125(56.8%) isolates as MDR/RR-MTB of which 22(30.9%) were detected resistant to second line drugs. Whole genome sequencing of 65 isolates and their gDST found Ser315Thr mutation in katG (35/45;77.8%) and Ser531Leu mutation in rpoB (21/41;51.2%) associated with drug resistance. SNP barcoding categorized the dataset with Lineage2 (41;63.1%) being predominant followed by Lineage3 (10;15.4%), Lineage1 (8;12.3%) and Lineage4 (6;9.2%) respectively. Phylogenetic assignment by cgMLST gave insights of two Beijing sub-lineages viz; 2.2.1 (SNP difference &lt; 19) and 2.2.1.2 (SNP difference &lt; 9) associated with recent ongoing transmission in Arunachal Pradesh. This study provides first insight in identifying the ongoing transmission of two virulent Beijing sub-lineages associated with TB drug resistance.

Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Currently, there are no reports on the mycobacterial components that regulate PGE2 production. Previously, we have reported that RpfE-treated dendritic cells (DCs) effectively expanded the Th1 and Th17 cell responses simultaneously; however, the mechanism underlying Th1 and Th17 cell differentiation is unclear. Here, we show that PGE2 produced by RpfE-activated DCs via the MAPK and cyclooxygenase 2 signaling pathways induces Th1 and Th17 cell responses mainly via the EP4 receptor. Furthermore, mice administered intranasally with PGE2 displayed RpfE-induced antigen-specific Th1 and Th17 responses with a significant reduction in bacterial load in the lungs. Furthermore, the addition of optimal PGE2 amount to IL-2-IL-6-IL-23p19-IL-1β was essential for promoting differentiation into Th1/Th17 cells with strong bactericidal activity. These results suggest that RpfE-matured DCs produce PGE2 that induces Th1 and Th17 cell differentiation with potent anti-mycobacterial activity.

Yogurt ice cream (YIC) containing Bifidobacterum longum subsp. longum TISTR 2195 (B. longum) with different fat contents (2 and 10% fat) and prebiotics (galacto-oligosaccharide, GOS and fructo-oligosaccharide, FOS) was investigated for its physicochemical properties, microstructure, and survival of B. longum during storage time and in a simulated gastrointestinal tract. Fat contents affected pH, fat particle size, hardness, melting rate, and solid-like characteristic of ice cream while FOS seemed to enhance the hardness and elasticity of YIC better than GOS due to less solubility and denser microstructure which exhibited more solid-like properties than YIC containing GOS. The interaction effect of fat content at 10% fat and FOS in YIC seemed to promote more sensorial acceptance than the ice cream containing 2% fat and GOS. The fat content, concentration of prebiotics, and type of prebiotics were attributed to the survival of B. longum in YIC. Moreover, the survival number of B. longum in YIC during storage for 180 days and in the simulated gastrointestinal tract was still in 106 CFU g-1, which is considered to benefit human health. Finally, probiotic YIC in this study could be used as a guide for functional ice cream manufacture future.

The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensively resistant (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definition. Sub-lineage 4.3 (52%) was the most frequent genotype and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected 20 novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. Cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug-resistance prediction, presenting the best accuracy values to five of nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) and genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil, genetic and clinical data correlations and demonstrating its utility for surveillance efforts for spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.

Tuberculosis (TB), an infectious airborne disease caused by Mycobacterium tuberculosis (Mtb), is a serious public health threat reported as the leading cause of morbidity and mortality worldwide. South Africa is a high TB burden country with TB being the highest infectious disease killer. The study investigated the distribution and clustering of Mtb mutations and spoligotypes in rural Eastern Cape Province. The Mtb isolates included were 1,157 from DR-TB patients and analysed by LPA followed by spoligotyping of 441 isolates, of these, 36 were whole genome sequenced. Distribution of mutations and spoligotypes was done by spatial analysis and clustering analysis was done by Bayesian model-based clustering of allele frequencies at heterozygous sites, using Mclust package in R. The rpoB gene had highest number of mutations. The distribution of rpoB and katG mutations was more prevalent in four health care facilities, inhA mutations were more prevalent in three healthcare facilities and heteroresistant isolates were more prevalent in five healthcare facilities. The Mtb was genetically diverse with Beijing more prevalent and largely distributed. Spatial analysis and mapping of gene mutations and spoligotypes revealed better picture of distribution. Clustering of isolates indicates that there is transmission of mixed infection in this area.

Precision agriculture including camera-guided row detection and hydraulic steering are often used as an alternative because crop damage can be decreased and driving speed can be increased comparable to herbicide applications.The effects of different approaches as uncontrolled (UC), mechanical weed control (MWC), herbicide weed control (HWC) and mechanical + herbicide control (MWC+HWC) on weed density and yield of sugar beet were tested and evaluated in two trials (2021 and 2022) in South Transylvania at the tested intervals BBCH 19 and 31. Weed control efficacy (WCE) depends on the emergence of the weeds and a good timing of weed controls in all the trials and methods, though the highest yield of sugar beet roots was recorded in the treatment MWC+HWC, with an increase up to 12-15% (56.48 t ha-1) yield from HWC (50.22 t ha-1) and a yield increase of more than 35- 40% than MWC (42.34 t ha-1). Our trials show that is possible to increase yield and have less chemical applications with the introduction of the new precision technologies in agriculture included here the sensor-guided mechanical controls.

Rickettsia is an important pathogenic entity among tick-borne diseases (TBD), which are considered serious emerging public health problems globally. In India, though the widespread distribution of ticks and TBD has been documented, its real burden remains underreported. In a preliminary attempt, rickettsial surveillance was carried out in ticks collected from Sirumalai, Eastern Ghats in Tamil Nadu by using pathogen genome-based phylogenetic inferences generated through multi-locus sequence typing (MLST), targeting the genes 16s rRNA, OmpA, OmpB, and gltA by nested PCR. The laboratory evidence confirms the circulation of Rickettsia in Haemaphysalis intermedia species collected from this area. Analysis of the four gene sequences detected demonstrates their closest identity to the spotted fever group (SFG) available in the GenBank database. Further, multiple sequence alignment with other sequences derived from the GenBank database showed close relatedness to Rickettsia conorii subsp. raoultii (16s rDNA-99.32%, ompA-93.38%, ompB-97.39%, and gltA-98.57%) and Rickettsia felis (ompA-100%, ompB-100% and gltA-99.41%). With this genomic evidence, the circulation of rickettsial pathogens in the pools of H. intermedia ticks infesting livestock in the Sirumalai foothill area has been demonstrated and to complement the microscopic identification of the tick species, DNA barcodes were generated for H. intermedia using the mitochondrial cytochrome c oxidase subunit I gene (COI). Nevertheless, R. raoultii and R. felis were found to be the aetiological agents of tick-borne lymphadenopathy and flea-borne spotted fever in human cases, respectively. Further study on the determination of their diversity, distribution, clinical relevance, and potential risk to the local community in these areas is highly warranted.

Plesiomonas shigelloides is the only species in the genus and has zoonotic importance due to its serious implications resulting from the consumption of contaminated seafood. This is the first report on the genomic features of the whole-genome sequence (WGS) of P. shigelloides strain V-78 recovered from diseased rainbow trout, Oncorhynchus mykiss. The genome of P. shigelloides V-78 consists of 4,478,098 base pairs (bp), which encode 3730 proteins and has a G+C content of 51.1%. The bioinformatics analysis of WGS of V-78 confirmed the presence of 121 tRNA genes and 42 rRNA genes (15 genes for 5S rRNA, 13 genes for 16S rRNA and 14 genes for 23S rRNA). Comprehensive genome analyses revealed that the strain encodes for secondary metabolites, antimicrobial resistance, and virulence genes. The strain V-78 has 31 known antibiotic resistance models, which encode many antimicrobial resistances. Also, strain V-78 has 42 different virulence genes such as adhesion, secretion system, and motility. The digital DNA-DNA hybridization value against P. shigelloides NCTC 10360 was 74.2%, while the average nucleotide identity value was 97.1%. Based on the scrutinized analysis of genomic data, strain V-78 should be considered a novel subspecies of P. shigelloides, for which Plesiomonas shigelloides subsp. oncorhynchi is proposed.

Antibodies are important tools for the study of membrane proteins in biological, biophysics and structural analyses. However, the production of purified membrane proteins is still a formidable challenge due to the amphipathic character and inherent instability of these proteins. In this work, we tested a strategy for producing antibodies against an ATP-Binding Cassette (ABC) transporter without the need to produce the full membrane complex. Instead, just the extra cytoplasmic regions of the ABC-type transporter Rv1819c from Mycobacterium tuberculosis were presented in the RAD-display, a scaffold system based on the engineered Pyrococcus furiosus RadA protein developed for exposition of peptides. The RadA scaffold protein is highly stable, can tolerate long insertions and easy to produce in Escherichia coli. Based on the three-dimensional structure of Rv1891c, we selected two versions of the main extracellular loops of the permease to use as test cases for this approach. Rv1819c-derived peptides displayed on RAD display system were used for immunization of mice and production of polyclonal antibodies. These antibodies were able to recognize the transporter in M. tuberculosis cell extracts, on intact cells by flow cytometry and as purified protein. The results showed the approach can be a useful tool for in vitro analyses, screening, and evaluation of different epitopes in membrane proteins.

Bovine tuberculosis (bTB) outbreaks, caused by Mycobacterium bovis infection, are a costly animal health challenge. Understanding factors associated with the duration of outbreaks, known as breakdowns, could lead to better disease management policy development. We undertook a retrospective observational study (2012-2018) and employed Finite Mixture Models (FMM) to model the outcome parameter, and to investigate how factors were associated with duration for differing subpopulations identified. In addition to traditional risk factors (e.g. herd size, bTB history), we also explored farm geographic area, parcels/farm fragmentation, metrics of intensity via nitrogen loading, and whether herds were designated controlled beef finishing units (CBFU) as potential risk factors for increased duration. The final model fitted log-normal distributions, with two latent classes (k) which partitioned the population into a subpopulation around the central tendency of the distribution, and a second around the tails of the distribution. The latter subpopulation included longer breakdowns of policy interest. Increasing duration was positively associated with recent (<3 yrs) TB history and the number of reactors disclosed, (log) herd size, beef herd-type relative to other herd types, number of land parcels, area, and being designated a controlled finishing unit (“feedlot”), and having high annual inward cattle movements within the “tails” subpopulation. Breakdown length was negatively associated with year of commencement of breakdown (i.e. a decreasing trend) and non-significantly with the organic nitrogen produced on the farm (N kg/hectare), a measure of stocking density. The latter finding may be due to confounding effects with herd size and area. Most variables contributed only moderately to explaining variation in breakdown duration, that is, they had moderate size effects on duration. Herd-size and CBFU had greater effect sizes on the outcome. The findings contribute to evidence-based policy formation in Ireland.

Studies on Protein-Protein interactions (PPI) can be helpful for the annotation of unknown protein function and for the understanding of cellular processes, such as specific virulence mechanisms developed by bacterial pathogens. In that context, several methods have been extensively used in recent years for the characterization of Mycobacterium tuberculosis PPI to further decipher TB pathogenesis. This review aims at compiling the most striking results based on in vivo methods (yeast and bacterial two-hybrid systems, protein complementation assays) for the specific study of PPI in mycobacteria. Moreover, newly developed methods, such as in-cell native mass resonance and proximity-dependent biotinylation identification, will have a deep impact on future mycobacterial research, as they are able to perform dynamic (transient interactions) and integrative (multiprotein complexes) analyses.

Drug-resistant tuberculosis (DR-TB) is still a major public health concern in South Africa. Mutations in M. tuberculosis can cause varying levels of phenotypic resistance to anti-TB medications. There have been no prior studies on gene mutations and the genotyping of DR-TB in the rural Eastern Cape Province; hence we aimed to identify DR-TB mutations, genetic diversity and allocated lineages among patients in this area. Using Xpert® MTB/RIF, we assessed the rifampin-resistance of sputum samples collected from 1157 patients suspected of having tuberculosis. GenoType MTBDR plus VER 2.0 was used for the detection of mutations causing resistance to anti-TB medications. The next step was to spotlight type 441 isolates. The most prevalent rifampin resistance-conferring mutations were in rpoB codon S531L, in INH-resistant strains, katG gene at codon S315TB and the inhA gene at codon C-15TB had highest mutations; 54.5% and 24.7%, respectively. In addition, 24.6% of strains showed mutations in both the rpoB and inhA genes, while 69.9% of strains showed mutations in both the katG and rpoB genes. Heteroresistance was seen in 17.9% of all cases in the study. According to spoligotyping analysis, Beijing families predominated. Investigating the evolutionary lineages of M. tuberculosis isolates can be done using the information provided by the study's diversity of mutations. In locations where these mutations have been discovered, decision-making regarding standardization of treatment regimens or individualized treatment may be aided by the detection frequency of rpoB, katG, and inhA mutations in various study areas.

The study sought to determine the rate of discordant results between genotypic and phe-notypic tests for the diagnosis of drug resistant tuberculosis (DR-TB). Sputum samples and cultured isolates from suspected DR-TB patients were respectively analysed for My-cobacterium tuberculosis by Xpert® MTB/RIF (Cepheid, USA) and Line Probe Assays (LPA) (Hain, Germany). Discrepant Rifampicin (RMP) resistant results were confirmed using BACTEC MGIT960 (BD, USA). Of 224 RMP-resistant by Xpert MTB/RIF, 5.4% were suscep-tible to RMP by LPA. MGIT960 showed 75% agreement with LPA. Discrepancy was at-tributed to either heteroresistance or DNA contamination during LPA testing in 58.3% of cases. In 25% of samples showing agreement in RMP resistance between Xpert MTB/RIF and MGIT960, discrepancy was attributed to laboratory errors causing false RMP suscep-tible results with LPA. Of 16.7% of cases, discrepancy was attributed to false RMP suscep-tible results with Xpert MTB/RIF. Of 224 isolates, susceptibility to isoniazid (INH) by LPA was performed in 73.7% RMP resistant isolates, of which, 80.6% were resistant. All RMP resistant isolates by Xpert MTB/RIF were confirmed in 98.5% by LPA if TB isolates were resistant to INH, but only confirmed in 81.3% if TB isolates were INH susceptible (ρ < 0.001). In conclusion, Laboratory errors should be considered when investigating dis-cordant results.

Tuberculosis (TB) is a contagious disease, caused by Mycobacterium tuberculosis (MTB) that has infected and killed a lot of people in the past. At present treatments against TB are available at a very low cost. Since these chemical drugs have many adverse effects on health, more attention is now given on the plant-derived phytochemicals as potential agents to fight against TB. In this study, 5 phytochemicals, 4-hydroxybenzaldehyde, benzoic acid, bergapten, psoralen, and p-hydroxybenzoic acid, are selected to test their potentiality, safety, and efficacy against two potential targets, the MTB RNA polymerase and enoyl-acyl carrier protein (ACP) reductase, the InhA protein, using various tools of in silico biology. The molecular docking experiment, drug-likeness property test, ADME/T-test, P450 SOM prediction, pharmacophore mapping, and modeling, solubility testing, DFT calculations, and PASS prediction study had confirmed that all the molecules had the good potentiality to inhibit the two targets. However, two agents, 4-hydroxybenzaldehyde and bergapten were considered as the best agents among the five selected agents and they also showed far better results than the two currently used drugs, that function in these pathways, rifampicin (MTB RNA polymerase) and isoniazid (InhA protein). These two agents can be used effectively to treat tuberculosis.

Emergence of multi drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis poses a serious threat to TB control as the available treatment options are less effective in these cases. Therefore, new strategies are required for identification of new drugs and drug targets. The M. tuberculosis dihydrodipicolinate synthase is a putative drug target with no potent inhibitor. Here in this study, we have used a comprehensive computational approach to identify small molecule inhibitors and validated them in vitro. As similar structures tend to have similar functions, therefore 3 approaches, namely, (a) known inhibitor-based approach, (b) substrate analogues, (c) product analogues were used for compound screening from various chemical libraries. The fourth approach we have taken is fragment library screening.In vitro studies revealed that β-hydroxypyruvic acid tartronic acid are inhibitors of Mtb-rDapA with maximum inhibition 48% and 51% at 500µM concentrations respectively. We have also observed that β-hydroxypyruvic acid is a time dependent inhibitor of Mtb-rDapA. Further, thermal shift assays showed that β-hydroxypyruvic acid interacts with Mtb-rDapA and the thermal stability of Mtb-rDapA shifted by 3˚C. Among the product analogues of Mtb-DapA, dipicolinic acid showed to bind to Mtb-rDapB shifting thermal stability of Mtb-rDapB by 0.5˚C in binding. Additionally, we have designed a new approach by combining substrate and product analogues to achieve better inhibitions. In vitro assays validated that this combination approach increased maximum inhibition up to 100%. We envision that these compounds would serve as potent leads for tuberculosis drug discovery.