Abnormalities in sperm nuclei and chromatin can interfere with normal fertilization, embryonic development, implantation, and pregnancy. We aimed in this study to determine the impact of H2BFWT gene variants in sperm DNA on ICSI outcomes in couples undergoing ART treatment. One hundred and nineteen partners were divided into pregnant (G1) and non-pregnant (G2) groups. After semen analysis, complete DNA was extracted from purified sperm samples. The sequence of the H2BFWT gene was amplified by PCR and then subjected to Sanger sequencing. The results showed that there are three mutations in this gene: rs7885967, rs553509 and rs578953. rs553509 and rs578953 showed significant differences in the distribution of alternative and reference alleles between G1 and G2 (P = 0.0004 and P = 0.0020, respectively). However, there was no association between these SNPs and the studied parameters. This study is the first to shed light on the connection between H2BFWT gene variants in sperm DNA and pregnancy after ICSI therapy. This is a pilot study, so further investigations about these gene variants at the transcriptional and translational levels, in a larger cohort of patients, are needed to determine its functional consequences and to understand the mechanism of how the pregnancy is affected by sperm DNA.

Bacterial resistance to antibiotics due to an increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for the efflux analysis are needed. Here we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenyl-alanyl-arginyl-β-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. Results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measure-ments of Et+ interaction with the purified DNA demonstrated that affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably de-creases in presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their in-tricate link to sperm function, embryo development, and overall fertility. Defects in chromatin com-paction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue and chromomy-cin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a Hepes-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species, employing diverse meth-odologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correla-tions with the percentages of sperm stained by aniline blue, methylene blue and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

Cryopreservation is a stressful process for sperm as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon the antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP), and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI), of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14+/PI−) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in PFE than in GFE, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP-OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen-thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP.

The sperm heads of frozen/thawed horse spermatozoa were injected into ovulated mouse oocytes where they decondensed and formed paternal pronuclei in which the DNA damage has been evaluated. The motile spermatozoa exhibit none or minor level of DNA damage and the xenogenic zygotes cleave to the two-cell embryonic stage with regular nuclei and no micronuclei (MNs). On the other hand, the immotile spermatozoa exhibit typically high levels of DNA damage and blastomeres in two-cell stage embryos contain multiple MNs. Moreover, extremely high sperm DNA damage prevents even the cleavage to the two-cell stage.Keywords: keyword 1; keyword 2; keyword 3 (List three to ten pertinent keywords specific to the article yet reasonably common within the subject discipline.)

Advanced paternal age at fertilization has been suggested to be a risk factor for neurodevelopmental, psychiatric and other disorders in offspring. One emerging hypothesis suggests that altered offspring phenotype is linked with age-related accumulation of epigenetic changes in the sperm of fathers. Given that paternal age is increasing in the developed world, understanding aging-related epigenetic changes in sperm is needed as well as environmental factors that modify such changes. In this study, we characterize age-dependent changes in sperm DNA methylation profiles between young pubertal (postnatal day (PNDs) 65) and mature (PND120) Wistar rats. We also analyze these changes in rats exposed perinatally to 0.2 mg/kg of ubiquitous environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). Reduced representation bisulfite sequencing (RRBS) libraries were prepared from caudal epididymal sperm DNA and differentially methylated regions (DMRs; ≥ 10x coverage depth, ≥ 3 CpGs per cluster, ≥ 5% methylation change, q < 0.05) were identified via MethPipe package. In control animals, 5,319 age-dependent DMRs were identified, with 99.3% DMRs hypermethylated in mature animals compared to young pubertal rats. These age-related DMRs were enriched for functional categories essential for embryonic development, such as pattern specification, forebrain and sensory organ development, Hippo and Wnt pathways. Age-related changes in sncRNA, reported in different study, target similar list of genes and biological categories.In BDE-47 exposed rats, sperm DNA methylation was higher in young pubertal and lower in mature animals when compared to controls, which resulted in a significant attenuation in the number of age-dependent DMRs (N = 189) identified in the exposed group. In conclusion, our results indicate that the natural aging process has profound effects on sperm methylation levels and this effect may be modified by environmental exposures. Moreover, our results further support the role of epigenetic mechanisms as a likely link betwen paternal age and offspring health and development.

(1) Background: Double-strand breaks (DSBs) in a single nucleus are usually measured using the sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, and comet assay (CA). We developed mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFGE) and angle-modulated two-dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) to observe DNA fragmentation in separated motile sperm. (2) Methods: We set up comparative standards, calibration curves, the required sensitivity, and eligibility criteria for test sperm to validate their measurement principles. (3) Results: We revealed that the conventional methods overlooked the interference of nucleoproteins in their measurements. In-gel proteolysis improves the measurement accuracies of 1D- and 2D-SCPFGE. We recommend using naked DNA for comparative standards and test specimens. Moreover, we have observed several dysfunctions that may induce DNA damage in the separated motile sperm. Overall, our discussion highlights the need to revisit the conventional univariable analyses based on the SCSA, SCD test, and CA. (4) Conclusions: Human infertility is a complex syndrome, and the aim of quality control in intracytoplasmic sperm injection is to identify the underlying dysfunctions remaining in the separated motile sperm that render them ineligible for injection. Well-designed, multivariable analyses with special consideration to confounding factors are necessary in future cohort studies.

We investigated the development of a simple and sensitive assay for the identification of minute amounts of animal and human biological material. The suggested system uses isothermal amplification of DNA in a rapid assay format, which returns the results in as little as 40 minutes from sampling. The assay used for the proof of concept employs the novel handheld SaLux19 device (Max-Planck Innovation prototype, Germany). This well-established infield detection system could also be utilized for forensic scenarios. The results presented here demonstrated that the assay is sufficiently specific and sensitive and can detect the presence of Sus scrofa nucleic acids.

Many types of banana (Musa spp.)—one of the most economically important horticultural crops—exist, and their ploidy (diploid, triploid, and tetraploid, usually) and genome types (containing A or/and B genome in most cases) differ. At present, observation and genome type detection are commonly used to identify banana germplasm resources. However, the former is tedious, while the latter cannot distinguish categories below genome types. It is therefore urgent that a simple and effective method for identifying banana germplasm resources is established. We sequenced and analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 62 banana germplasm and found that the sequencing peaks, especially the 20 bp region near the 420 bp position (referred to as the “420 bp region”), exhibited relatively recognizable and repeatable polymorphism characteristics. Using the “420 bp region” as a marker, we were able to quickly distinguish bananas belonging to different genome type groups (comprising the AA, AB, AAA, ABB, AAB, ABBB, AAAB, and AAAA groups) or different subgroups in the same genome type group (for example, the Cavendish, Gros Michel, Red, Lakatan, and Ibota Bota subgroups in the AAA group; and the Pisang Raja, Plantain, Pome and Silk subgroups in the AAB group). Moreover, it appeared that Sanger sequencing of ITS could be used for identifying banana hybrid offspring. These results demonstrated that the banana ITS region contains rich genetic information that is useful for genotyping. In general, ITS sequencing simplifies the classification of banana germplasm resources and has potential application in several areas of Musa improvement.

Type 2 diabetes mellitus (T2DM) is a risk factor for male infertility, but the underlying molecular mechanisms remain unclear. Advanced glycation end products (AGEs) are pathogenic molecules for diabetic vascular complications. Here, we investigated the effects of DNA-aptamer raised against AGEs (AGE-Apt) on testicular and sperm abnormalities in a T2DM mouse model. KK-Ay (DM) and wild-type (non-DM) 4- and 7-week-old male mice were sacrificed to collect the testes and spermatozoa for immunofluorescence, RT-PCR, and histological analyses. DM and non-DM 7-week-old mice were subcutaneously infused with AGE-Apt or control aptamer for 6 weeks and then sacrificed. Plasma glucose, testicular AGEs, and RAGE gene expression in 4-week-old DM mice and plasma glucose, testicular AGEs, oxidative stress, and pro-inflammatory gene expressions in 7-week-old DM mice were higher than those in age-matched non-DM mice; the latter of which was associated with seminiferous tubular dilation. AGE-Apt did not affect glycemic parameters, but it inhibited the seminiferous tubular dilation, reduced the number of testicular macrophages and apoptotic cells, and restored the decrease in sperm concentration, motility, and viability of 13-week-old DM mice. Our findings suggest that AGEs-Apt may improve sperm abnormality by suppressing the AGE–RAGE-induced oxidative stress and inflammation in the testes of DM mice.

Dried blood spot (DBS) testing on confetti is one of the most common methods used to collect and extract DNA from field samples for molecular epidemiology of malaria studies. Here, we investigated whether ethanol or other solutions used to clean scissors are a source of sample contamination. DBS were prepared from 3 drops of blood from two Plasmodium falciparum cultures spotted on confetti, and 3 solutions (water, ethanol and DNase) were used to clean scissors between spots during DNA extraction. For each cleaning solution, two blank confetti were used as negative controls. Samples were analyzed by PCR-based genotyping of merozoite surface proteins 1 & 2 (msp-1 and msp-2), and P. falciparum chloroquine resistance transporter (Pfcrt). A total of 15 samples were analyzed. Based on msp-1 and msp-2 amplification, P. falciparum was detected on blank confetti when scissors were washed with ethanol. Based on Pfcrt amplification, DNA of P. falciparum was detected on blank confetti when scissors were cleaned with ethanol or water. No P. falciparum genes were detected on blank confetti when scissors were cleaned with DNase. Scissors cleaned with DNase prevented cross-contamination between samples during processing of dried blood spots, whereas ethanol (which is commonly used) fails to avert cross-contamination.

In the literature, it is well known the correlation between poor semen quality and DNA sperm integrity, which can turn into negative outcomes in terms of embryo development and clinical pregnancy. Sperm selection plays a pivotal role in clinical practice and the most widely used methods are mainly based on sperm motility and morphology. The cumulus oophorus complex (COC) during natural fertilization represents a barrier that spermatozoa must overcome to reach the zona pellucida and fertilize the oocyte. Spermatozoa that can pass through the COC have better structural and metabolic characteristics as well as enhanced acrosome reaction (AR). The present study aimed to evaluate the exposure of sperm to cumulus cell secretome during swim-up treatment (SUC), compared to the routinely used swim-up method (SU). To determine the effectiveness of this method biological factors critical for the ability of sperm to fertilize an oocyte, including capacitation, AR, tyrosine phosphorylation signature, DNA integrity, and mitochondrial functionality were assessed. The SUC selection assures recovery of high-quality spermatozoa, with enhanced mitochondrial functionality and motility compared to both SU-selected or unselected (U) sperm. Furthermore, by this modified swim-up procedure significantly reduced sperm DNA damage (p<0.05) was detected. In conclusion, the SUC approach is a more physiological and integrated method for sperm selection that deserves further investigation for its translation into clinical practice.

Sperm during the freezing and thawing process is damaged by oxidative stress. Thus, its antioxidant scavenger is essential for sperm survival and death in frozen-thawed semen. We used melatonin and silymarin in experiments. Our study aimed to identify the effect of melatonin and silymarin on the motility and viability of sperm, reactive oxygen species (ROS), and nitric oxide (NO) production in frozen-thawed boar semen. Melatonin and silymarin were treated alone and co-treated in the fresh boar semen. Boar semen was collected using the gloved-hand method from ten crossbred pigs, and the age of the experimental ten male pigs was 28.7 ± 3.2 months. We evaluated sperm viability using SYBR-14 and PI kit, and ROS and NO production were detected by DCF-DA and DAF-2, respectively. The sperm motility was not significantly different between non-treatment and treatment. ROS and NO production in frozen-thawed sperm were decreased by melatonin and silymarin (P < 0.05). Moreover, silymarin significantly reduced NO production more than melatonin (P < 0.05). Melatonin (46.37 ± 1.10) and silymarin (46.13 ± 0.80) enhanced the viability of sperm (P < 0.05). We suggest that melatonin and silymarin are essential antioxidants in semen cryopreservation for protecting sperm damage and maintaining sperm viability. Melatonin and silymarin may be useful antioxidants in freezing boar sperm.

Abstract: Congenital cytomegalovirus (cCMV) infection poses significant risks to fetal development, particularly affecting the nervous system. This study reports a fetal autopsy case, examining cCMV infection and focusing on CMV DNA measurements in various fetal organs before formalin fixation, a novel approach for comprehensive CMV DNA evaluations in fetal organs affected by cCMV. A 20-week-old male fetus was diagnosed with cCMV following the detection of CMV DNA in ascites obtained via abdominocentesis in utero. After termination of pregnancy, multiple organs of the fetus, including the cerebrum, thyroid gland, heart, lungs, liver, spleen, kidneys, and adrenal glands, were extracted and examined for CMV DNA loads using real-time polymerase chain reaction. Histopathological examination involved hematoxylin-eosin and CMV-specific immunostaining. A correlation was found between CMV DNA loads and pathology, with higher CMV-infected cell numbers observed in organs positively identified with both staining methods, exhibiting CMV DNA levels of ≥1.0 × 104 copies/mL, compared to those detected solely by CMV-specific immunostaining, where CMV DNA levels ranged from 1.0 x 103 to 1.0 x 104 copies/mL. These results highlight a quantifiable relationship between organ infection extent and CMV DNA concentration, providing insights into cCMV pathogenesis and potentially informing future diagnostic and therapeutic strategies for cCMV infection.

Sperm morphology can predict the reproductive male fertilizing potential. This study aimed to determine the morphological and morphometric spermatozoa characteristics from guinea pigs subjected to different photoperiodic stimulation. Thirty F1 guinea pigs were randomly assigned to three photoperiodic treatments: FT1 (photoperiod with 10L/14D LED light), FT2 (photoperiod with 10L/14D sunlight), and FT0 (room without direct light source). At 107 ± 9.8 days of age, sperm concentration and motility were higher in FT0 and FT1 (p<0.05); furthermore, there were no differences in nucleus length and ellipticity between FT0 and FT1, but FT1 was higher in perimeter and nuclear area, while FT0 was higher in roughness, regularity, midpiece length and tail (p<0.01). Expanding acrosome (Type 2) was more frequent in FT2, but there was variation in head measurements between all morphological categories. Pregnancy rate, calving age and mating age were higher in FT0, meanwhile FT1 initiated successful matings earlier (p<0.01). FT0 had a higher fertility rate, and FT1 age of mating and first calving were earlier than FT0 but no pregnancies were reported for FT2. The photoperiodic stimulation can increase the morphometric dimensions of guinea pig spermatozoa, favoring the reproductive characteristics, but sunlight could reduce their size due to heat stress.

The spread of the infection caused by the new coronavirus SARS-CoV-2 (COVID-19) became pandemic on March 11, 2020. From the time of the first cases (in November 2019, Wuhan, China), to date, a large number of COVID-19 observations have been accumulated in different age groups of patients both in China and abroad. Published scientific data allows us to conclude that children suffer from COVID-19 much less often than adults and tolerate the disease in a milder form, often appear to be asymptomatic. There is currently no final answer why children are less susceptible to this virus; however, scientists are increasingly inclined to consider a complex effect of the immune response and components of the renin-angiotensin system (RAS), which according to recent studies affects not only the cardiovascular system, but is also responsible for the activation of inflammatory reactions. A hypothesis of genetic predisposition to the development of severe forms of COVID-19 has recently been made. We conducted a search for publications in the databases and showed current scientific ideas about COVID-19 pathogenesis and factors influencing the disease development in childhood. Childhood immunity may have several protective features against SARS-CoV-2: immaturity of particular elements of the innate immune response, constitutional lymphocytosis with a shift towards anti-inflammatory Th2-response, as well as "trained" immunity. The influence of renin-angiotensin system reactions in this review is shown from two perspectives: expression of ACE2 receptors and polymorphisms of certain genes of this system. It was established that ACE2 transmembrane protein is not only the entry point for the virus but also plays a regulatory role, turning the pro-inflammatory vasoconstrictor angiotensin II into anti-inflammatory angiotensin (1-7), which has vasodilating properties. Higher ACE2 content in children compared with adults helps maintain balance in the renin-angiotensin system and prevents the development of complications. It was also shown that the presence of certain genetic polymorphisms (AGTR1, AGTR2, ACE2, ACE) could determine the imbalance inside the RAS, leading to more pronounced reactions of alveolocytes, vascular endothelium and smooth muscle fibers in response to SARS-CoV-2 infection due to a shift towards vasoconstrictor, proliferative and profibrotic mechanisms.

We attempted to establish an objective method to accurately evaluate the motility of bull sperm and examined the effects of media for sperm suspensions and frame rates on data of computer-assisted sperm motility analysis (CASA). Sperm incubated in Brackett and Oliphant medium (BO) more clearly showed hyperactivation-like motility than those in synthetic oviductal fluid. Sperm images captured at 150 frames per second (fps) showed a trajectory that was closer to the real pathway than those at other frame rates (30, 50, and 75 fps). We then examined the characteristics of sex-sorted and non-sorted semen using a cluster analysis followed by a discriminant analysis of sperm motility in BO at 150 fps. The results indicated that sex-sorted semen contained sperm with hyperactivation-like motility as the main subpopulation immediately after thawing and this subpopulation decreased after 2-h incubation. The main subpopulation in non-sorted semen had progressive motility that was maintained during incubation. In conclusion, usage of BO for sperm suspensions and capturing sperm motility at 150 fps by CASA were appropriate for evaluating bovine sperm motility. A discriminant analysis using data from a cluster analysis of motile sperm has the ability to accurately describe differences in the structures of sperm motility subpopulations.

A hybrid material, consisting of commercially available nitrocellulose (NC) membrane non-covalently modified with amino-polyethylene glycol functionalized reduced graphene oxide (NH2-PEG-rGO) nanoparticles, was successfully synthesized for oligonucleotide extraction. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the modification of the NC membrane, revealing characteristic peaks of both compounds NC and NH2-PEG-rGO. Scanning Electron Microscopy (SEM) exhibited morphological changes in the NC/NH2-PEG-rGO hybrid membrane, marked by the introduction of NH2-PEG-rGO particles, resulting in a distinctly smothered surface compared to the porous surface of the NC control membrane. Wettability assays revealed hydrophobic behavior for the NC/NH2-PEG-rGO hybrid membrane, with a water contact angle exceeding 90°, contrasting with the hydrophilic behavior characterized by a 16.7° contact angle in the NC membrane. The performance of the NC/NH2-PEG-rGO hybrid membrane was evaluated for the extraction of ssDNA with fewer than 50 nucleotides from solutions containing various ionic species (MnCl2, MgCl2, and MnCl2/MgCl2). The NC/NH2-PEG-rGO hybrid membrane exhibited optimal performance when incubated in MgCl2, presenting the highest fluorescence emission at 525 relative fluorescence units (r.f.u.). This corresponds to the extraction of approximately 610 pg (≈13%) of the total oligo-DNA, underscoring the efficacy of the pristine material, which extracts 286 pg (≈6%) of oligo-DNA in complex solutions.

The cryopreservation of gametes and embryos is an important element of biodiversity conservation. One species in need of conservation is the honey bee Apis mellifera L. Changing environmental factors, especially the anthropogenic factor, have led to a reduction in the numbers of this insect species. In this study, we provide an example of the creation of a biobank of honey bee drone sperm. For sperm cryopreservation, drones of the most common subspecies of honey bees common in Russia were selected. These were the dark forest bee, Apis mellifera mellifera from the Republic of Bashkortostan, with three subspecies (A. m. carnica, A. m. carpatica, and A. m. caucasica) from the southern regions of Russia, as well as two breeding stocks, the Far Eastern bee and Prioksky bee. For subspecies identification, morphometric and genetic methods were used. The subspecies of the studied samples were confirmed by the analysis of the tRNAleu-COII locus of mitochondrial DNA and nine microsatellite markers of nuclear DNA. It was shown that bees of the Prioksky breeding stock belong to the subspecies A. m. caucasica based on phylogenetic analysis, and the Far Eastern breeding stock is a stable hybrid, descending on the maternal line from the evolutionary lineage C or O. The results of the morphometric analysis are consistent with the results of the genetic analysis. For the cryopreservation of sperm, we used a honey diluent. As a result, the viability of frozen–thawed sperm decreased by 20.3% compared to fresh sperm, and overall motility decreased by 25-fold. The measurement of the sperm concentration in the spermatheca of artificially inseminated queens showed that it varied from 0.22 to 4.4 million/μL. Therefore, the use of honey in sperm cryopreservation has great potential.

Sperm cells must undergo a complex maturation process after ejaculation to be able to fertilize an egg. One component of this maturation is hyperpolarization of the membrane potential to a more negative value. The ion channel responsible for this hyperpolarization, SLO3, was first cloned in 1998, and since then much progress has been made to determine how the channel is regulated and how its function intertwines with various signaling pathways involved in sperm maturation. Although SLO3 was originally thought to be present only in the sperm of mammals, recent evidence suggests that a primordial form of the gene is more widely expressed in some fish species. As with many reproductive genes, SLO3 is rapidly evolving with low conservation between closely related species and different regulatory and pharmacological profiles. Despite these differences, SLO3 appears to have a conserved role in regulating sperm membrane potential and driving large changes in response to stimuli. As with the differences in regulation of the channel, the effect of this hyperpolarization of the membrane potential may vary among mammalian species. Recent discoveries have elucidated the role of SLO3 in these processes in human sperm and provided tools to target the channel to affect human fertility.

Assisted reproductive technology has been developed significantly throughout the past few years, particularly diagnosing and treating male infertility. Many studies have been performed showing that Intracytoplasmic Sperm Injection (ICSI) is a successful method to attain clinical pregnancy and live birth through impaired spermatozoa characteristics or low sperm count, such as severe oligospermia. Severe oligospermia indicates low sperm count, which in some cases leads to azoospermia. Severe oligospermia can be caused by several factors such as genetics or medication. In search of efficient treatment for couples with Severe oligospermia, numerous retrospective and prospective researches have reported high pregnancy and live birth rates through testicular sperm for men with severe oligospermia and cryptozoospermia with or without high sperm DNA damage. The research showed that the use of testicular sperm in combination with ICSI yielded a high pregnancy rate and live births over another source of sperm, such as ejaculated sperms. Moreover, the use of ICSI in severe oligospermia has shown successful fertilization and pregnancy.

The zebrafish Danio rerio is suitable to study gametes as a model organism. There were &gt; 70% of zebrafish spermatozoa activated, because they were contaminated with urine or excrement. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation, then damped from the end of the flagellum for 35 s and fully disappear at 61 s after activation. For artificial fertilization, milt must be added to an extender, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 was shown to be the most suitable extender as it allows to store sperm for fertilization for 6 to 12 h at 0-2oC. Sperm motility decreased only to 36% at 12 h post stripping (HPS) for E400 extender and to 19% for Kurokura extender. To achieve an optimal level of fertilization and hatching, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µl of activation solution has proved to be more successful than using a Petri dish. The highest fertilization and hatching rates reached 80% and 40-60%, respectively, with milt stored for 1.5 h in E400 extender at 0-2oC.

The usage of Quantum Similarity through the equation Z = {∀θ ∈ Z → ∃s ∈ S ∧ ∃t ∈ T : θ = (s, t)}, represents a way to analyze the way communication works in our DNA. Being able to create the object set reference for z being (s, t) in our DNA strands, we are able to set logical tags and representations of our DNA in a completely computational form. This will allow us to have a better understanding of the sequences that happen in our DNA. With this approach, we can also utilize mathematical formulas such as the Euler–Mascheroni constant, regression analysis, and computational proofs to answer important questions on Quantum biology, Quantum similarity, and Theoretical Physics.

Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously [1]. In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis.

Lumpy skin disease (LSD), caused by a virus within the Poxviridae family and Capripoxvirus genus, induces nodular skin lesions in cattle, spreading through direct contact and insect vectors, significantly affecting global cattle farming. Despite available vaccines, their effectiveness is limited by poor bioavailability and adverse effects. Our study aimed to identify potential inhibitors targeting LSD-associated DNA polymerase protein, selecting LSDV039 for further investigation through comprehensive analysis and computational methods. Virtual screening revealed rhein and taxifolin as potent binders among 380 phytocompounds, with respective affinities of -8.965 and -7.195 kcal/mol. Canagliflozin and tepotinib exhibited strong affinities (-9.858 and -8.856 kcal/mol) among 718 FDA-approved antiviral drugs. Molecular dynamics simulations of canagliflozin, tepotinib, rhein, and taxifolin highlighted taxifolin's superior stability and binding energy. Rhein displayed compactness in RMSD and RMSF but fluctuations in Rg and SASA, while canagliflozin demonstrated stability compared to tepotinib. The study highlights the promising potential of repurposed drugs and phytocompounds as LSD therapeutics. However, extensive validation through in-vitro, in-vivo, and clinical trials is crucial for their practical application.

The importance of zinc for male fertility only emerged recently, propelled in part by consumer interest in nutritional supplements containing ionic trace minerals. Here, we review the properties, biological roles and cellular mechanisms that are relevant to zinc function in the male reproductive system, survey available peer-reviewed data on nutritional zinc supplementation for fertility improvement in livestock animals and infertility therapy in men, and discuss recently discovered signaling pathways involving zinc in sperm maturation and fertilization. Emphasis is on the zinc-interacting sperm proteome and its involvement in the regulation of sperm structure and function, from spermatogenesis and epididymal sperm maturation to sperm interactions with the female reproductive tract, capacitation, fertilization and embryo development. Merits of dietary zinc supplementation and zinc inclusion into semen processing media are considered with livestock artificial insemination (AI) and human assisted reproductive therapy (ART) in mind. Collectively, the currently available data underline the importance of zinc ions for male fertility, which could be harnessed to improve human reproductive health and reproductive efficiency in agriculturally important livestock species. Further research will advance the field of sperm and fertilization biology, provide new research tools, and ultimately optimize semen processing procedures for human infertility therapy and livestock AI.

Using the nematode C. elegans germline as a model system, we previously reported that PUF-8 (a PUF RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) repress sperm fate at 20°C and the dedifferentiation of spermatocytes into mitotic cells (termed "spermatocyte dedifferentiation") at 25°C. Thus, double mutants lacking both PUF-8 and LIP-1 produce excess sperm at 20°C, and their spermatocytes return to mitotically dividing cells via dedifferentiation at 25°C, resulting in germline tumors. To gain insight into the molecular competence for spermatocyte dedifferentiation, we compared the germline phenotypes of three mutant strains – fem-3(q20gf), puf-8(q725; fem-3(q20gf), and puf-8(q725); lip-1(zh15). Both fem-3(q20gf) and puf-8(q725); fem-3(q20gf) mutants produced excess sperm like puf-8(q725); lip-1(zh15) double mutants. Our results show that spermatocyte dedifferentiation was not observed in fem-3(q20gf) mutants, but it was more aggressive in puf-8(q725); lip-1(zh15) than in puf-8(q725); fem-3(q20gf) mutants. These results suggest that MPK-1 (the C. elegans ERK1/2 MAPK ortholog) activation by removing the function of LIP-1 in the absence of PUF-8 promotes spermatocyte dedifferentiation. This idea was confirmed using Resveratrol (RSV), a potential activator of MPK-1 and ERK1/2 in C. elegans and human cells. Notably, spermatocyte dedifferentiation was significantly enhanced by RSV treatment, and its effect was blocked by mpk-1 RNAi. We, therefore, conclude that PUF-8 and MPK-1 are normally required to inhibit spermatocyte dedifferentiation and tumorigenesis. Since these regulators are broadly conserved, we suggest that similar regulatory circuitry may control cellular dedifferentiation and tumorigenesis in other organisms, including humans.

Much research has been conducted to determine how hair regeneration is regulated, as this could provide therapeutic, cosmetic, and even psychological interventions for hair loss. The current study focused on the hair growth effect and effective utilization of fatty oil obtained from Bryde's whales through high-throughput DNA microarray approach in conjunction with immunohistochemical observations. The research also examined the mechanisms and factors involved in hair growth. In an experiment using female C57BL/6J mice, the vehicle control group (VC: propylene glycol: ethanol: water), the positive control group (MXD: 3% minoxidil), and the experimental group (WO: 20% Whale Oil) were topically applied to the back of the mouse. The results showed that 3% MXD and 20% WO were more effective than VC in promoting hair growth, especially 20% WO. Furthermore, in hematoxylin and eosin-stained skin tissue, an increase in the number of hair follicles and subcutaneous tissue thickness was observed with 20% WO. Whole-genome transcriptomic analysis also showed that 20% WO may have lower stress- and inflammation-related responses than 3% MXD. Therefore, whale oil can be expected to be used as a safe hair growth agent.

This study investigated the relationship of acrosome reactions and fatty acid composition on fertility in boar sperm. The acrosome reaction of sperm was induced via methyl-beta-cyclodextrin (MBCD), and acrosome reaction, plasma membrane integrity, and fertility were analyzed. The fatty acid composition of the excess acrosome reacted sperm was determined via gas chromatography. The results showed that the acrosome reaction in sperm was induced over 85% of the time by 60 mM MBCD treatment, and the plasma membrane integrity was significantly decreased and was dependent on the MBCD level. The acrosome reacted sperm resulted in significantly higher saturated fatty acids (SFAs) and lower unsaturated fatty acids (PUFAs) than the non-acrosome reaction group. Moreover, the acrosome reacted sperm from 60 mM MBCD significantly decreased in vitro fertility and blastocyst formation relative to non-acrosome reacted sperm, and the acrosome reaction was positively correlated with SFAs and negatively correlated with PUFAs. Of these fatty acids, C22:5n-6 (docosapentaenoic acid [DPA]) and C22:6n-3 (docosahexaenoic acid [DHA]) were directly negatively correlated with the acrosome reaction (r = -0.982 and -0.947, respectively). In conclusion, the excessive acrosome reactions may occur by reducing the PUFAs, which may then dramatically decrease sperm fertility in pigs.

Nuclear vacuoles are specific structures present on the head of the human sperm of fertile and non-fertile men. Human sperm head vacuoles have been previously studied using motile sperm organelle morphology examination (MSOME) and their origin related to ab-normal morphology, abnormal chromatin condensation and DNA fragmentation. How-ever, other evidences argued that human sperm vacuoles are physiological structures and consequently, to date, the nature and origin of the nuclear vacuoles remains to be elucidat-ed. Here, we aim to define the incidence, position, morphology, and molecular content of the human sperm vacuoles using transmission electron microscopy (TEM) and immuno-cytochemistry techniques. Results showed that ~50% of the analyzed human sperm cells (n = 1908; 17 normozoospermic human donors) contained vacuoles mainly located (80%) in the anterior head region. A significant positive correlation was also found between the sperm vacuole and nucleus areas. Furthermore, it was confirmed that nuclear vacuoles were invaginations of nuclear envelope containing cytoskeletal proteins and a cytoplas-mic enzyme, discarding a nuclear or acrosomal origin. According to our findings, these human sperm head vacuoles are cellular structures which take the origin from nuclear invaginations and contain perinuclear theca (PT) components, allowing to define a new term of ‘nuclear invaginations’ rather than ‘nuclear vacuoles.

Environmental DNA (eDNA) sampling has emerged as a powerful approach for monitoring biodiversity in the context of diverse terrestrial and aquatic ecosystems, and has broad potential for both environmental surveys and species management. eDNA studies focusing on plant and animal species have capitalized on analytical methodologies developed originally for metagenomic studies of microorganisms, and on technological improvements in sample collection. However, while eDNA studies have proliferated rapidly, efforts to standardize sampling and analytical methods are still nascent. Critically, fundamental data concerning the distribution and persistence of eDNA in different ecological contexts are lacking, complicating both confidence in species detection and comparability between studies. We propose that key deficits could be addressed by using well-designed synthetic DNA standards or tracers, which are widely used in fields ranging from medical diagnostics to hydrology. In-field application of a standardized mixture of barcoded synthetic DNA fragments of varying lengths and concentrations that are recovered concommitantly with eDNA sampling should provide critical yet missing data on environmental DNA distribution and turnover, as well as an internal molecular standard to enhance both species detection and comparability between eDNA studies.

DNA origami has emerged in recent years as a powerful technique for designing and building 2D and 3D nanostructures. While the breadth of structures that have been produced is impressive, one of the remaining challenges, especially for DNA origami structures intended to carry out useful biomedical tasks in vivo, is to endow them with the ability to detect and respond to molecules of interest. Target molecules may be disease indicators or cell surface receptors, and the responses may include conformational changes leading to release of therapeutically relevant cargo. Nucleic acid aptamers are ideally suited to this task and are beginning to be used in DNA origami designs. In this review we consider examples of uses of DNA aptamers in DNA origami structures and summarise what is currently understood regarding aptamer-origami integration. We review three major roles for aptamers in such applications: protein immobilisation, triggering of structural transformation, and cell targeting. Finally, we consider future perspectives for DNA aptamer integration with DNA origami.

Opuntia sp. contain antioxidant phytochemicals resistant to ROS damage, whose excess negatively affect fertilization. We investigate the activity of fruit extracts of O. dillenii and O. ficus indica (cv red and yellow) on sperm quality and cryopreservation. In the first experiment, we exposed the samples to extracts (50 µl) for 1 hour to then evaluate semen parameters (vitality, motility, acrosome reaction, DNA fragmentation and oxidative stress). The results showed a significant increase in the motility (86%±0.19 for OFI cv yellow, 82%±0.15 for OFI cv red and 90%±0.08 for O. dillenii) compared to the control (80%±0.17). Moreover, we noted a reduction of DNA fragmentation on treated (3%±0.03 in OFI cv yellow, 7%±0.09 in OFI cv red and 5%±0.07 in O. dillenii) than the control (40%±0.14). Furthermore, the oxidative stress was reduced after exposure to solutions (3.15mV in the control and 2.94mV in the treated). In the second experiment, 50 µl of solutions were added to the Freezing medium. After thawing, we observed an improvement in vitality and the number of intact acrosomes. Our results suggest that Opuntia sp. fruit extracts improve sperm quality, both before and after cryopreservation, optimizing the potential of fertilization of sperm cells.

The uterine leiomyoma is the most common benign tumor in women of childbearing age. It may lead to problem of conception or complications during gestational period. The methods of treatment can be surgical (myomectomy and hysterectomy, embolization of arteries) and therapeutic treatment (Ulipristal acetate, Leuprolide acetate, Cetrorelix, Goserelin, Mifeprestone). Both approaches are efficient, but are incompatible with pregnancy planning. Therefore, there is a call for medical practice in developing therapeutical means of preventing leiomyoma onset in patients planning on pregnant. Based on the analysis of GWAS data on the search for mononucleotide polymorphisms associated with the risk of leiomyoma, meta-transcriptomic and meta-methylomic studies, target proteins have been proposed. Prospective therapeuticals of leiomyoma may be based on chemical compounds, humanized recombinant antibodies, vaccines based on markers of the uterine leiomyoma cells that are absent in the adult organism, DNA and RNA preparations. Three different nosological forms of the disease associated with driver mutations in the MED12, HMGA2 and FH genes should be considered when developing or prescribing drugs. E.g. synthetic inhibitors and vaccines based on matrix metalloproteinases MMP11 and MMP16 are expected to be effective only for the prevention of the occurrence of MED12-dependent nodules.

After sperm enter the female reproductive tract, the physicochemical and biochemical microenvironment undergoes significant changes. Thus, the response of sperm to environmental factors is vital for successful fertilization. The cation channel of sperm (CatSper) is a sperm-specific, pH-sensitive, and Ca2+-permeable ion channel. It is responsible for the predominant Ca2+ entry in mammalian sperm and is involved in nearly every event of sperm to acquire fertilizing capability. CatSper also serves as a pivotal polymodal chemosensor in mammalian sperm by responding to multiple chemical cues. Physiological ligands (such as progesterone, prostaglandins, β-defensins, and odorants) provoke Ca2+ entry into sperm by activating CatSper and thus triggering sperm functions including hyperactivation, capacitation, the acrosome reaction, and chemotaxis. Additionally, synthetic and natural chemicals (such as medicines, endocrine disrupting chemicals, drugs of abuse, and antioxidants) affect sperm functions by regulating CatSper-dependent Ca2+ signaling. Therefore, understanding the interactions between CatSper and extracellular ligands sheds light on the mechanisms underlying male infertility and offers innovative diagnostic and treatment approaches. This underscores the importance of CatSper as a crucial regulatory target in male reproduction, linking sperm function with the extracellular environment. This review comprehensively summarizes the relevant studies describing the environmental factors that affect CatSper in humans and rodents.

Chromothripsis is a mutational mechanism leading to complex and relatively clustered chromosomal rearrangements resulting in diverse phenotypic outcomes depending on the involved genomic landscapes. It may occur both in the germ and the somatic cells resulting in congenital and developmental disorders and cancer, respectively. Asymptomatic individuals may be carriers of chromotriptic rearrangements and experience recurrent reproductive failures when two or more chromosomes are involved. Several mechanisms are postulated to underly chromothripsis. The most attractive hypothesis involves chromosome pulverization in micronuclei followed by incorrect reassembly of fragments through DNA repair to explain the clustered nature of the observed complex rearrangements. Moreover, exogenous or endogenous DNA damage induction and dicentric bridge formation may be involved. Chromosome instability is commonly observed in the cells of patients with DNA-repair disorders, such as ataxia telangiectasia, Nijmegen breakage syndrome and Bloom syndrome. In addition, germline variations of TP53 have been associated with chromothripsis in Sonic-Hedgehog medulloblastoma and acute myeloid leukemia. In the present review, we focus on the underlying mechanisms of chromothripsis and the involvement of defective DNA-repair genes resulting in chromosome instability and chromothripsis-like rearrangements.

Sperm marking represents a valuable monitoring tool for genetic pest control strategies such as the Sterile Insect Technique, but also provides a key tool for reproductive biology studies. Sperm-marked lines can be generated by introducing transgenes that mediate the expression of fluorescent proteins during spermatogenesis. Homozygous lines established by transgenesis approaches are going through a genetic bottleneck that can lead to reduced fitness. Transgenic SIT approaches have mostly focused on Dipteran and Lepidopteran pests so far. With this study, we provide sperm-marked lines for the Coleopteran pest model organism, the red flour beetle Tribolium castaneum, based on the β2-tubulin promoter/enhancer driving red (DsRed) or green (EGFP) fluorescence. The obtained lines are reasonably competitive and were thus used for studies on reproductive biology confriming the phenomenon of ‘last male sperm precedence’ and that the spermathecae are deployed for long term sperm storage enabling the use of sperm from first matings even after secondary matings for a long period of time. The homozygosity and competiveness of the lines will enable future studies to analyze the controlled process of sperm movement into the long time storage organ as part of a post-mating cryptic female choice mechanism of this extremely promiscuous species.

Na⁺/H⁺ exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility.

Theoretic and empirical studies show that social surroundings experienced by male insects during their larval or adult stage can influence their testicular investment in diverse ways. Although insect pupae do not feed and crawl, they can communicate using sex-specific and/or non-sex specific cues. Yet, it is unknown, in any insect, whether and how male pupae can fine-tune their resource allocation to sperm production and testis size in response to socio-sexual environment. We investigated this question using a moth, Ephestia kuehniella, which produces fertile eupyrene sperm and unfertile apyrene sperm. We held male pupae individually or in groups with different sex ratios, and dissected adults upon eclosion, measured their testis size, and counted both types of sperm. We demonstrated that after exposure to conspecific pupal cues regardless of sex, male pupae increased production of eupyrenes and apyrenes at the same rate but kept testis size unchanged. We suggest that testis size is fixed after pupation because most morphological traits are formed during the larval stage, allowing little room for pupae to adjust testis size. Like adults, male pupae with fully grown testes have sufficient resources to produce more sperm of both types according to the perceived increase of sperm competition risk.

The study titled "Molecular Choreography of DNA: Insights and Innovations" explores the intricate dance of DNA, shedding light on its structure, function, and regulation. This comprehensive review delves into the historical significance of DNA research, from Watson and Crick's double helix discovery to recent innovations in genomics and molecular biology. The review highlights DNA's roles in replication, repair, gene regulation, and chromatin dynamics, emphasizing the precision and complexity of its molecular choreography. Innovations like CRISPR-Cas9 gene editing and advanced sequencing technologies have revolutionized our understanding of DNA, opening new avenues for scientific exploration. This study aims to provide readers with a deeper appreciation of DNA's central role in life's choreographed processes.

Antioxidants have multiple protective roles in cells and can be used as a supplement to protect cells against cryopreservation-induced detrimental effects, including protecting sperm fertility quality. The antioxidant resveratrol (3,5,4-trihydroxy-trans-stilbene; RSV) has been shown to be a protective supplement for the cryopreservation of animal sperm, including human sperm. In this study, we assessed the effect of RSV supplementation on canine sperm cryopreservation. Semen was collected from four dogs and the effect of different concentrations of RSV (0, 100, 200, and 400 µM) on post-thaw quality of sperm was examined. After thawing, sperm motility was assessed using computer aided sperm analysis, and the structural integrity of the plasma membrane, acrosome, and chromatin were examined, as well as mitochondrial activity and gene expression were assessed. Dog sperm cryopreserved with 200 µM RSV showed significant improvement in motility and viability following thawing compared with that of the control group (p &lt; 0.05). Moreover, RSV-supplemented samples showed significantly higher numbers of sperm with an intact plasma membrane, active mitochondria, and structural integrity of acrosomes and chromatin than that of control samples (p &lt; 0.05). Furthermore, gene expression showed that RSV supplemented samples showed lower expression of pro-apoptotic (BAX) oxidative stress-related (ROMO1) and oxidative induced DNA damage repair (OGG1) whereas higher expression levels of anti‐apoptotic (BCL2) protamine-2 (PRM2), protamine-3 (PRM3) and sperm acrosome‐associated (SPACA3) genes than control. Our results suggest that RSV, at its optimum concentration, can be efficiently used as an alternative antioxidant in the cryopreservation of dog sperm.

Sperm capacitation involves biochemical and physiological changes that enable sperm to fertilize the oocyte. It can be induced in vitro under controlled conditions that simulate the environment of the oviduct. While extensively studied in mammals, its approach in lizards remains absent. Understanding the mechanisms that facilitate reproduction is essential for advancing the implementation of assisted reproductive technologies in this group. Fifteen males of Sceloporus torquatus were collected during the early stage of the reproductive season. The sperm were isolated from the seminal plasma and then diluted up to a volume of 150 μl using BWW medium to incubate with 5% CO2 at 30 °C for a maximum duration of 3 hours. A fraction was retrieved hourly for ongoing sperm assessment. We found that total motility is maintained up to 2 hours post-incubation, after which it decreased. However, sperm viability remained constant. From that moment on, we observed a transition to a deeper and less symmetrical flagellar bending in many spermatozoa. The chlortetracycline assay indicated a reduction in the percentage of sperm showing the F pattern and an increase in those exhibiting the capacitated (B) and reactive (RA) patterns, accompanied by an elevation in the percentage of damaged acrosomes as revealed by the lectin binding assay. In mammals, these changes are often associated with sperm capacitation. These observations support the notion that this process may also occur in saurian.

Structural DNA nanotechnology provides an excellent foundation for diverse nanoscale shapes that can be used in various bioapplications and materials research. From all existing DNA assembly techniques, DNA origami has proven to be the most robust one for creating custom nanoshapes. Since its invention in 2006, building from the bottom up using DNA has drastically advanced, and therefore, more and more complex DNA-based systems have become accessible. So far, vast majority of the demonstrated DNA origami frameworks are static by nature, but interestingly, there also exist dynamic DNA origami devices that are increasingly coming into view. In this review, we discuss DNA origami nanostructures that perform controlled translational or rotational movement triggered by predefined DNA strands, various molecular interactions and/or other external stimuli such as light, pH, temperature and electromagnetic fields. The rapid evolution of such dynamic DNA origami tools will undoubtedly have a significant impact on molecular scale precision measurements, targeted drug delivery and diagnostics, but they can also play a role in development of optical/plasmonic sensors, nanophotonic devices and nanorobotics for numerous different tasks.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origin is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases such as DNA polymerase α-primase (Pol α), DNA polymerase δ and ε at replication forks play pivotal roles in the mechanism of initiation reactions including the initiation of signalling unperturbed and stalled replication forks, ‘replication stress’ events, via ATR (ATM-Rad 3-related protein kinase). All these processes are essential for the accurate transfer of the cells’ genetic information to their daughters. Failures and dysfunctions in these processes gives rise to genome instability followed by genetic diseases including cancer. Thus, in their review ‘Hallmarks of Cancer: New Dimensions‘ Hanahan and Weinberg (2022) called genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels which will be discussed in the review.

Tdp1 and Tdp2 are DNA repair enzymes that repair DNA damages caused by various agents, including anticancer drugs. Thus, these enzymes resist anticancer therapy and could be the reasons of resistance to such widely used drugs as topotecan and etoposide. In the present work, we found compounds capable of inhibiting both enzymes among derivatives of (-)-usnic acid. Both (+)- and (-)-enantiomers of compounds act equally effectively against Tdp1, and only (-)-enantiomers inhibited Tdp2. Surprisingly, the compounds protect HEK293FT wild type cells from the cytotoxic effect of etoposide but potentiate it against Tdp2 knockout cells. We assume that the sensitizing effect of the compounds in the absence of Tdp2 is associated with effective inhibition of Tdp1, which could take over the functions of Tdp2.

In E. coli DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

Abstract: Important variables that affect the quality and functionality of sperm and may affect male reproductive health and fertility include telomere length, mitochondrial content, and the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The purpose of this study was to look at the connections between these three sperm characteristics and how they might affect a woman's ability to conceive. Methods: Data were collected from 82 men who underwent IVF/ICSI at the University Hospital of Ioannina's IVF Unit in the Obstetrics and Gynecology Department. Evaluations included sperm morphology, sperm count, sperm motility, and participant history. To address this, males of reproductive age who were categorized into three body mass index (ΒΜΙ) groups—normal, overweight, and obese—had their sperm samples tested. Results: Our findings indicate a negative correlation between relative telomere length and ΒΜΙ for both the normal and overweight groups. Conversely, a positive correlation was observed for the obese group, indicating a possible relationship between mitochondrial health and telomere maintenance. The results were statistically significant only for the obese group. Furthermore, a negative correlation was observed between telomere length and mitochondrial content in both the normal and overweight groups, indicating that a lower mitochondrial content may be linked to longer telomeres. Nonetheless, there was a positive correlation for the group that was obese. For all three groups, the data did not reach statistical significance. Given that mitochondrial con-centration and telomere length are both markers of cellular integrity and health, these correla-tions may have an impact on the quality of sperm. Furthermore, the relative telomere length of the normal and overweight groups had a negative correlation with the ratio of mtDNA to nDNA, though the obese group showed a positive correlation. The findings were not statistical-ly significant in any of the groups under investigation. According to this, male fertility may be negatively impacted by an imbalance in the copy number of the mitochondrial genome com-pared to the nuclear DNA in sperm. Conclusions: Our research, in essence, emphasizes the interaction among human sperm telo-mere length, mitochondrial concentration, and the mtDNA to nDNA ratio. Gaining knowledge of these correlations could help explain the reasons behind male infertility and open up new treatment options for reproductive health problems. The functional importance of these correla-tions and possible uses in therapeutic contexts require more investigation.

Theoretical and experimental approaches have been applied to study the polymer physics under-lying compaction of DNA in the bacterial nucleoid. Knowledge of the compaction mechanism is necessary to obtain a mechanistic understanding of the process of segregation of replicating chromosome arms (replichores) during the cell cycle. In the first part of this review light microscope observations are discussed that have demonstrated that the nucleoid has a lower refractive index and thus a lower density than the cytoplasm. A polymer-physical explanation for this phenomenon was given by the theory of Theo Odijk pro-posed in 1998. By assuming a phase separation between nucleoid and cytoplasm and by imposing equality of osmotic pressure and chemical potential between the two phases, a minimal energy situation is obtained in which soluble proteins are depleted from the nucleoid, thus explaining its lower density. The theory is compared with recent views for DNA compaction that are based on mere exclusion of polyribosomes from the nucleoid or on the transcriptional activity of the cell. These new views raise the question of whether they can still explain the lower refractive index or density of the nucleoid. In the second part of this review the question is discussed how DNA segregation occurs in Esche-richia coli, in the absence of the so-called active ParABS system, present in the majority of bacteria. How is entanglement prevented of nascent chromosome arms, generated at the origin in the pa-rental network of the E. coli nucleoid? The observations of the groups of Sherratt and Hansen in 2006, that the four nascent chromosome arms synthesized in the initial replication bubble, segre-gate to opposite halves of the sister nucleoids implies that extensive intermingling of daughter strands does not occur. Based on the hypothesis that leading and lagging replichores synthesized in the replication bubble, fold into micro-domains that do not intermingle, a passive "four-excluding-arms model" for segregation is proposed. This model implies that the key for seg-regation already lies in the structure of the replication bubble at the very start of DNA replication; it explains the different patterns of chromosome arms as well as the segregation distances between replicated loci as experimentally observed.

Transcription factors (TFs) and microRNAs (miRNAs) are co-actors in genome-scale decoding and regulatory networks, often targeting common genes. In this paper, we describe the algebraic geometry of both TFs and miRNAs thanks to group theory. In TFs, the generator of the group is a DNA-binding domain while, in miRNAs, the generator is the seed of the sequence. For such a generated (infinite) group $\pi$, we compute the $SL(2,\mathbb{C})$ character variety, where $SL(2,\mathbb{C})$ is simultaneously a \lq space-time' (a Lorentz group) and a \lq quantum' (a spin) group. A noteworthy result of our approach is to recognize that optimal regulation occurs when $\pi$ looks like a free group $F_r$ ($r =1$ to $3$) in the cardinality sequence of its subgroups, a result obtained in our previous papers. A non free group structure features a potential disease. A second noteworthy result is about the structure of the Groebner basis $\mathcal{G}$ of the variety. A surface with simple singularities (like the well known Cayley cubic) within $\mathcal{G}$ is a signature of a potential disease even when $G$ looks like a free group $F_r$ in its structure of subgroups. Our methods apply to groups with a generating sequence made of two to four distinct DNA/RNA bases in $\{A,T/U,G,C\}$. Several human TFs and miRNAs are investigated in detail thanks to our approach.

Drug abuse is a major issue worldwide. Methylphenidate (MPH) and alprazolam (ALZ) are commonly prescribed drugs for the treatment of ADHD and anxiety disorders, respectively. The limited studies suggest that abusers primarily use benzodiazepines to counteract adverse effects associated with methylphenidate usage. The main aim of this study was to investigate the interaction of drugs with DNA using spectroscopic methods. Female Wistar rats were administered with MPH (10, 20, 40 mg/kg) and ALZ (5, 10, 20 mg/kg) alone and in combination for a period of 28 days. The FT-IR and UV results reveal some spectral changes in a dose-dependent manner, which indicates interactions of drugs with DNA. Thus, the changes in spectral peaks provide some insight into the mechanism of the interaction of drugs with DNA.

For broadcast spawning invertebrates such as the crown-of-thorns starfish, early life history stages (from spawning to settlement) may be exposed to a wide range of environmental conditions, and could have a major bearing on reproductive success and population replenishment. Arrested development in response to multiple environmental stressors at the earliest stages can be used to define lower and upper limits for normal development. Here, we compared sperm swimming speeds and proportion of motile sperm and rates of fertilization and early development under a range of environmental variables (temperature: 20-36°C, salinity: 20-34 psu, and pH: 7.6-8.2) to identify environmental tipping points and thresholds for reproductive success. We also tested the effects of water-soluble compounds derived from eggs on sperm activity. Our results demonstrate that gametes, fertilization, and early development are robust to a wide range of temperature, salinity, and pH levels that are outside the range found at the geographical limits of adult distribution and can tolerate environmental conditions that exceed expected anomalies as a result of climate change. Water-soluble compounds associated with eggs also enhance sperm activity, particularly in environmental conditions where sperm motility is initially limited. These findings suggest that fertilization and embryonic development of crown-of-thorns starfish are tolerant to a wide range of environmental conditions, though environmental constraints on recruitment success may occur at later ontogenic stages.

Cholangiocarcinoma is an epithelial malignancy arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. The effect of current chemotherapy regimens against cholangiocarcinoma is limited, and the prognosis of patients with cholangiocarcinoma is poor. Aberrant DNA methylation and histone modification induce silencing of tumor suppressor genes and chromosomal instability during carcinogenesis. Studies have shown that the tumor suppressor genes and microRNAs (miRNAs) including MLH1, p14, p16, DAPK, miR-370 and miR-376c are frequently methylated in cholangiocarcinoma. Silencing of these tumor suppressor genes and miRNAs plays critical roles in the initiation and progression of cholangiocarcinoma. In addition, recent studies have demonstrated that DNA methylation inhibitors induce expression of endogenous retroviruses and exert the anti-tumor effect of via an anti-viral immune response. Aberrant DNA methylation of tumor suppressor genes and miRNAs could be a powerful biomarker for diagnosis and treatment of cholangiocarcinoma. Epigenetic therapy with DNA methylation inhibitors hold considerable promise for the treatment of cholangiocarcinoma through re-activation of tumor suppressor genes and miRNAs as well as induction of an anti-viral immune response.

Recently, a rising use of wireless internet technologies has been demonstrated. The devices 18 which use these technologies emit in new spectral regions an electromagnetic radiation (EMFs) 19 which could interact with the male reproductive system. The aim of this study was to investigate in 20 vitro influence of electromagnetic fields at 27 GHz on sperm quality in Mytilus galloprovincialis. 21 Sperm samples, were collected from sexually mature males of M. galloprovincialis and placed in sea- 22 water. Once evaluated the number and quality of spermatozoa, sperm cells were exposed to elec- 23 tromagnetic fields radiated by a pyramidal horn antenna. The effect of exposure was evaluated after 24 10, 20, 30, 40 and 60 minutes with light microscope and using Eosin test. Ten replications were per- 25 formed for each time series, and statistical analysis was carried out byt-test. A significative decrease 26 in sperm motility was observed after 10 minutes of exposure and after 30 minutes most of sperms 27 were immobile and not vital. This study provides useful data on potential ecological impact of the 28 5G high-band on animal fertility, whose effect is currently under investigation.

DNA barcoding has emerged as a powerful tool in the field of taxonomy, transforming the way we identify and classify species. This review article aims to provide an up-to-date overview of the advancements in DNA barcoding techniques, their applications, and their impact on taxonomy and biodiversity studies. We will explore the evolution of DNA barcoding, the development of standardized barcode regions, and the challenges and limitations associated with this methodology. Additionally, we will delve into the diverse range of taxonomic groups for which DNA barcoding has been applied, highlighting key case studies and their contributions to our understanding of biodiversity. Furthermore, we will discuss the integration of DNA barcoding with other molecular and bioinformatic approaches, such as metabarcoding and machine learning, to enhance species identification and biodiversity assessment. This review aims to provide a comprehensive perspective on how DNA barcoding is reshaping the field of taxonomy and its implications for conservation, ecology, and evolutionary biology.

Systematics plays the most crucial role in biodiversity conservation which is at stake due to anthropogenic activities and environmental degradations. The ever-increasing decline of classical taxonomic expertise drives the need to develop molecular marker-based tools for quick, efficient, and reliable detection of organisms, to assess their ecological impacts for deepening our understanding of systematic and evolutionary relationships between organisms which is central to biology. The pace of alpha taxonomy has quickened by its integration with an increasingly fashionable and novel concept called DNA barcoding which utilizes a short genetic marker or barcode to categorize species for enhanced biodiversity assessment. As a supplementary but not complete alternative of systematics research, DNA barcoding, however, not error-free, brings precision in identification by solving existing problems of classical taxonomy and phylogenetics, irrespective of the growth stage of organisms, particularly for known taxa rather than unknown ones. Mitochondrial gene Cytochrome C Oxidase 1 (COI) serves as a universal animal barcode but there is no such universal barcode for plants and developing a suitable one is more challenging. With the recent advancement of Next Generation Sequencing (NGS), DNA metabarcoding technology is advancing rapidly. Still, ambiguity and error prevail with the correct identification of species due to some problems. After extensive analysis of the existing DNA barcoding papers, this review discusses commonly used DNA barcodes in plants and animals, their roles, advantages, limitations to solve existing problems of conservation biology and add the author’s views and recommendations.

DNA methylation, one of the major epigenetic mechanisms, plays critical roles in regulating gene expression, genomic stability and cell lineage commitment. Establishment and maintenance of DNA methylation in mammals is achieved by two groups of DNA methyltransferases: DNMT3A and DNMT3B, which are responsible for installing DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which is essential for propagating DNA methylation patterns during replication. Both groups of DNMTs are multi-modular proteins, containing a large N-terminal regulatory region in addition to the C-terminal methyltransferase domain. Recent structure-function investigations of the individual domains or large fragments of DNMT1 and DNMT3A have revealed the molecular basis for their substrate recognition and specificity, intramolecular domain-domain interactions, as well as their crosstalk with other epigenetic mechanisms. These studies highlight a multifaceted regulation for both DNMT1 and DNMT3A/3B, which is essential for the precise establishment and maintenance of lineage-specific DNA methylation patterns in cells. This review summarizes current understanding of the structure and mechanism of DNMT1- and DNMT3A-mediated DNA methylation, with emphasis on the functional cooperation between the methyltransferase and regulatory domains.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices which have been classified as “possibly carcinogenic”, however their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (RE) may promote genomic instability, here we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of RE, such as long interspersed nuclear element1s (LINE-1), Alu short interspersed nuclear element and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE1 and ribosomal repeats in terms of both average profiles and organization of methylated and un-methylated CpG sites, in different ways in each of the three cell lines studied.

microRNAs can cause male infertility by impacting sperm quality and impaired spermatogenesis. Since the miR-125 family plays an important role in regulating embryo development, but the function of miR-125b-2 in male reproduction remains unknown. In this study, we prepared a model of miR-125b knockout (KO) mice. Among the KO mice, the progeny test showed that litter sizes decreased significantly and the rate of non-parous females increased significantly (p<0.05). At the same time, the testosterone concentration increased significantly (p<0.01), with the remarkable decrease for estradiol (p<0.05). Moreover, sperm count decreased obviously (p<0.05) and the percentage of abnormal sperms increased significantly (p<0.01). Testicular transcriptome sequencing demonstrated that there were 173 up-regulated genes, including Papolb (PAP), and 151 down-regulated genes in KO mice compared with wild type (WT). KEGG and GO analysis showed many of these genes were involved in sperm mitochondrial metabolism and other cellular biological processes. Meanwhile, the sperm mitochondria DNA (mtDNA) copy number was increased significantly (p<0.01) in KO mice, but the integrity of mtDNA and nuclear DNA (nDNA) had no change. In the top 10 up-regulated genes, as a testis specific expressing gene, PAP can affect the process of spermatogenesis. Western blotting and Luciferase Assay validated that PAP was the target of miR-125b-5p. Intriguingly, we also found that both miR-125b and PAP were only highly expressed in germ cells (GC) instead of Leydig cells (LC) and Sertoli cells (SC), and miR-125b-5p could target PAP to regulate TM3 cell secretion of testosterone (p<0.05). Our study firstly demonstrated that miR-125b-2 could regulate testosterone secretion by directly targeting PAP and increase sperm mtDNA copy number to affect semen quality. The study indicated that miR-125b-2 had a positive influence on the reproductive performance of animal and could be a potential therapeutic target for male infertility.

Diffuse midline gliomas are among the deadliest human cancers and have had little progress in treatment in the last 50 years. Cell cultures of these tumors have been developed recently, but it is unknown the degree to which such cultures retain the characteristics of the source tumors. DNA methylation profiling offers a powerful tool to look at genome-wide epigenetic changes that are biologically meaningful and can help assess the similarity of cultured tumor cells to their in vivo progenitors. Paraffinized diagnostic tissue from three diffuse intrinsic pontine gliomas with H3 K27M mutations was compared with subsequent passages of neurosphere cell cultures from those tumors. Each cell line was passaged 3-4 times and analyzed with DNA methylation arrays and standard algorithms that provided comparison of diagnostic classification and cluster analysis. All samples tested maintained high classifier scores and clustered within the reference group of H3 K27M-mutant diffuse midline gliomas. There was a gain of 1q in all cell lines, with two cell lines initially manifesting the gain of 1q only during culture. In vitro cell cultures of H3 K27M-mutant gliomas maintain high degrees of similarity in DNA methylation profiles to their source tumor, confirming their fidelity even with some chromosomal changes.

The DNA Damage Response (DDR) is a complex signaling network that comes into play when cells experience genotoxic stress. Upon DNA damage, cellular signaling pathways are rewired to slow down cell cycle progression and allow recovery. However, when the damage is beyond repair, cells activate complex and still not fully understood mechanisms, leading to a complete proliferative arrest or cell death. A number of conventional and novel anti-neoplastic treatments rely on causing DNA damage or on the inhibition of the DDR in cancer cells. However, the identification of molecular determinants directing cancer cells toward recovery or death upon DNA damage is still far from complete, and it is object of intense investigation. SPRY-COntaining RAN binding Proteins (Scorpins) RANBP9 and RANBP10 are evolutionarily conserved and ubiquitously expressed proteins whose biological functions are still debated. RANBP9 has been previously implicated in cell proliferation, survival, apoptosis and migration. Recent studies also showed that RANBP9 is involved in the Ataxia Telangiectasia Mutated (ATM) signaling upon DNA damage. Accordingly, cells lacking RANBP9 show increased sensitivity to genotoxic treatment. Although there is no published evidence, extensive protein similarities suggest that RANBP10 might have partially overlapping functions with RANBP9. Like RANBP9, RANBP10 bears sites putative target of PIK-kinases and high throughput studies found RANBP10 to be phosphorylated following genotoxic stress. Therefore, this second Scorpin might be another overlooked player of the DDR alone or in combination with RANBP9. This review focuses on the relatively unknown role played by RANBP9 and RANBP10 in responding to genotoxic stress.

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, PDIP46 and PDIP38, both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the splicesosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, Mdm2 alternative splicing and the regulation of the Nox4 NADPH oxidase.

Advanced paternal age at fertilization is a risk factor for multiple disorders in offspring and may be linked with age-related epigenetic changes in fathers sperm. Understanding of aging-related epigenetic changes in sperm and environmental factors that modify such changes is needed. Here we characterize changes in sperm sncRNA between young pubertal and mature rats. We also analyze modification of these changes by exposure to environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). SncRNA libraries prepared from epididymal spermatozoa were sequenced and analyzed using DESeq 2. Distribution of small RNA fractions changed with age, with fractions mapping to rRNA and lncRNA decreasing and fractions mapping to tRNA and miRNA increasing. 249 miRNA, 908 piRNA and 227 tRNA-derived RNA were differentially expressed (2-fold change, FDR p ≤ 0.05) between age groups in control animals. Differentially expressed miRNA and piRNA were enriched for protein-coding targets involved in development and metabolism, piRNA were enriched for LTR targets. BDE-47 accelerated age dependent changes in sncRNA in younger animals, decelerated these changes in older animals and increased the variance in expression of all sncRNA. Our results indicate that the natural aging process has profound effects on sperm sncRNA profiles and this effect may be modified by environmental exposures.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and high-risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. Particularly, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, where many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed association with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Cardiomyopathies are major causes of heart failure. Chagas disease (CD) is caused by the parasite Trypanosoma cruzi, and it is endemic in Central, South America. Thirty percent of the cases evolve into chronic cardiomyopathy (CCC) with worse prognosis as compared with other cardiomyopathies. In vivo bioenergetic analysis and ex vivo proteomic analysis of myocardial tissues highlighted worse mitochondrial dysfunction in CCC, and previous studies identified nuclear-encoded mitochondrial gene variants segregating with CCC. Here, we assessed the role of the mitochondrial genome through mtDNA copy number variations and mtDNA haplotyping and sequencing from heart or blood tissues of severe, moderate CCC and asymptomatic/indeterminate Chagas disease as well as healthy controls as an attempt to help decipher mitochondrial-intrinsic genetic involvement in Chagas disease development. We have found that mtDNA copy number was significantly lower in CCC than in heart tissue from healthy individuals, while blood mtDNA content was similar among asymptomatic Chagas disease, moderate and severe CCC patients. MtDNA haplogrouping study has indicated that African haplogroups were over represented in the Chagas subject groups in comparison with Brazilian healthy individuals. The European lineage is associated to protection against cardiomyopathy and the macro haplogroup H is associated with increased risk towards CCC. By mitochondria DNA sequencing, 84 mtDNA-encoded protein sequence pathogenic variants were associated with CCC. Among them, two variants were associated to left ventricular non-compaction and two to hypertrophic cardiomyopathy. The finding that mitochondrial protein-coding SNPs and mitochondrial haplogroups associate with risk of evolving to CCC is consistent with a key role of mitochondrial DNA in the development of Chronic Chagas disease Cardiomyopathy.

X-ray diffraction (XRD) analysis of complex poly-atomic systems, especially of biomolecules, using ultra-short laser pulses (USP), is currently one of the most important fields of modern physics. The basis for interpreting and "deciphering" experimental data is the well-known theory of X-ray scattering, where the main parameter of USPs - its duration - is not taken into account. In the present work it is shown that for scattering of attosecond USPs on DNA and RNA trinucleotides the pulse length is the most important scattering parameter. In this case the diffraction pattern significantly changes with respect to the previously known scattering theory. The results obtained are extremely important in XRD when using attosecond pulses to study trinucleotides of DNA and RNA because using the previously known scattering theory which does not take into account the duration of USPs one can not correctly interpret and therefore "decode" DNA and RNA structures.

Several clinically useful anticancer drugs selectively kill cancer cells by inducing DNA damage; the genomic instability and DNA repair defects of cancer cells make them more vulnerable than normal cells to the cytotoxicity of DNA-damaging agents. Because epoxide-containing compounds can induce DNA damage, we have used the MTT assay to evaluate the selective cytotoxicity of three epoxyalkyl galactopyranosides against A549 lung cancer cells and MRC-5 lung normal cells. Compound (2S,3S)-2,3-Epoxydecyl 4,6-O-(S)-benzylidene-β-D-galactopyranoside (EDBGP) showed the highest selective anticancer activity and was selected for mechanistic studies. After observing that EDBGP induced cellular DNA damage (comet assay), we found that cells deficient in nucleotide excision repair were hypersensitive to the cytotoxicity of this compound; this suggests that EDBGP may induce bulky DNA adducts. EDBGP did not inhibit glycolysis (glucose consumption and lactate production). Pre-treatment of lung cancer cells with several antioxidants did not reduce the cytotoxicity of EDBGP, thereby indicating that reactive oxygen species do not participate in the anticancer activity of this compound. Finally, EDBGP was screened against a panel of cancer cells and normal cells from several tissues, including three genetically modified skin fibroblasts with increasing degree of malignancy. Our results suggest that epoxyalkyl galactopyranosides are promising lead compounds for the development of new anticancer agents.

Replication protein A (RPA) is a heterotrimeric protein complex and the main single-stranded DNA (ssDNA) binding protein in eukaryotes. RPA has key functions in most of the DNA-associated metabolic pathways and DNA damage signalling. Its high affinity for ssDNA helps to stabilise ssDNA structures and prevents the DNA sequence from nuclease attacks. RPA consists of multiple DNA-binding domains which are oligonucleotide/oligosaccharide-binding (OB)-folds, responsible for DNA binding, and interactions with proteins. These RPA-ssDNA and RPA-protein interactions are crucial for DNA replication, DNA repair, DNA damage signalling, and the conversation of the genetic information of cells. Proteins such as ATR use RPA to locate to regions of DNA damage for DNA damage signalling. Recruitment of nucleases and DNA exchange factors to sites of double strand breaks are also an important RPA function to ensure effective DNA recombination to correct these DNA lesions. Due to its high affinity to ssDNA, RPA’s removal from ssDNA is of central importance to allow these metabolic pathway to proceed and processes to exchange RPA against downstream factors are established in all eukaryotes. These facetted and multi-layered functions of RPA will be discussed in detail in the review. RPA is also a major player in a variety of human diseases, which will be discussed.

Ring necked-pheasant (Phasianus colchicus) is bird of order Galliformes. Ring necked-pheasant is also known as gallinaceous bird and game bird. It looks like chicken bird. It is national bird of South Dakota. It is mostly found in wild areas but in Pakistan it exists as Domestic bird. It has high proteins and low fat in its meat. The present study was conducted to check the effect of ascorbic acid on sperm Motility, sperm cytoplasmic membrane integrity, sperm livability and acrosome integrity of Ring-necked pheasant at different concentrations of ascorbic acid (0mM, 1mM, 2mM, 3mM and 4mM) were used. Semen was collected by abdominal massage technique. Motility of sperm was greater than 70%.Then it was processed further. The cryopreservation of semen was checked at various stages like Post Dilution, Post Cooling, Post Equilibration and Post Thawing. Semen was cooled from post-dilution stage (37⁰ C) to post-cooling (20⁰C).Then it further cooled gradually to get post-equilibration stage (4⁰ C) within 24 hours. After that 10% glycerol was added to the sample. Then it was transferred to liquid nitrogen (LN₂) cylinder for 24 hours. After thawing stage sample was removed from LN₂ cylinders and placed in water bath for 4 hours to achieve post-thawing. Then performed sperm quality assays at each stage of cryopreservation. Sperm motility was assessed by Neubauer Chamber Hemocytometer. Plasma Membrane Integrity was checked by using Hypo Osmotic Swelling Test with the help to 2% Eosin. Sperm livability was assessed by Lake’s glutamate solution and sperm Acrosome Integrity was checked by dual staining technique by using Formal citrate solution and Giemsa stain. The better results were seen at 3mM treatment of ascorbic acid. Sperm motility percentage was significantly different (P < 0.05) on 3mM treatment on all the stages of cryopreservation rather than other treatments. Plasma membrane integrity assay also showed good results at same concentration (P < 0.05) on all the stages of cryopreservation rather than other treatments. At the sperm livability stage 3mM treatment showed significantly difference (P < 0.05) on all the stages of cryopreservation rather than other treatments. The sperm acrosome integrity showed highest percentage on 3mM treatment (P < 0.05) on all stages of cryopreservation rather than other treatments. Effect of ascorbic acid on all the quality parameters (sperm motility, plasma membrane integrity, livability and acrosome integrity) was showed significant difference (P < 0.05) at 3mM treatment of ascorbic acid as compare to control as well as remaining treatments. It was seen that by increasing the concentration of ascorbic acid from 3mM concentration to onward it shows negative results on its cryopreservation. Now this cryopreserved semen can be transfer from one place to another place to obtain good varieties as well as better genetic characteristics of this species.

In the last two decades, plant taxonomy has bloomed, following the development of a novel technique, namely DNA Barcoding. DNA barcodes are standardized sequences, ideally unique, coding or non-coding, either from the genome of the organism or from its organelles, that are used to identify/classify an organismal group; in short the method includes amplification of the DNA barcode, sequencing and comparison with a reference database containing the relevant sequences from different species. In plants, the use a universal DNA barcode, such as COI which is used in animals, has not been achieved so far. Several DNA barcodes from single loci as well as combinations of barcodes have been successfully used in various settings; additionally, as next generation sequencing becomes affordable, very long sequences- super barcodes, such as the chloroplast genome are being used successfully especially to distinguish between close species. In this review we summarize the work done on DNA barcoding in plants and discuss different approaches used so far.

Alzheimer’s disease is associated with prion-like aggregation of the amyloid β (Aβ) peptide and the subsequent accumulation of misfolded neurotoxic aggregates in the brain. Therefore, it is critical to clearly identify the factors that trigger the cascade of Aβ misfolding and aggregation. Numerous studies have pointed out the association between microorganisms and their virulence factors and Alzheimer’s disease; however, their exact mechanisms of action remain unclear. Recently, we discovered a new pathogenic role of bacterial extracellular DNA, triggering the formation of misfolded Tau aggregates. In this study, we investigated the possible role of DNA extracted from different bacterial and eukaryotic cells in triggering Aβ aggregation in vitro. Interestingly, we found that the extracellular DNA of some, but not all, bacteria is an effective trigger of Aβ aggregation. Furthermore, the acceleration of Aβ nucleation and elongation can vary based on the concentration of the bacterial DNA and the bacterial strain from which this DNA had originated. Our findings suggest that bacterial extracellular DNA might play a previously overlooked role in the Aβ protein misfolding associated with Alzheimer’s disease pathogenesis. Moreover, it highlights a new mechanism of how distantly localized bacteria can remotely contribute to protein misfolding and diseases associated with this process. These findings might lead to the use of bacterial DNA as a novel therapeutic target for the prevention and treatment of Alzheimer’s disease.

Proteins and DNA exhibit key physical chemical properties that make them advantageous for building nanostructures with outstanding features. Both DNA and protein nanotechnology have growth notably and proved to be fertile disciplines. The combination of both types of nanotechnologies is helpful to overcome the individual weaknesses and limitations of each one, paving the way for the continuing diversification of the structural nanotechnologies. Recent studies have implemented a synergistic combination of both biomolecules to assemble unique and sophisticate protein-DNA nanostructures. These hybrid nanostructures are highly programmable and display remarkable features that create new opportunities to build in the nanoscale. This review focuses on the strategies deployed to create hybrid protein-DNA nanostructures. Here, we will discuss strategies such as polymerization, spatial directing and organizing, coating, rigidizing or folding DNA into particular shapes or moving parts. The enrichment of structural DNA nanotechnology by incorporating protein nanotechnology has been clearly demonstrated and still shows a large potential to create useful and advanced materials with cell-like properties or dynamic systems. It can be expected that structural protein-DNA nanotechnology will open new avenues in the fabrication of nano-assemblies with unique functional applications and enrich the toolbox of bionanotechnology.

This work demonstrates that a plasmid can function as an efficient biocatalyst in mediating the nitroaldol (Henry) reaction at high temperatures in ultrapure water. Compared with ssDNA, the plasmid displayed a remarkable catalytic efficiency (97% yield within 20 min). Aromatic aldehydes which bear strong or moderate electron-withdrawing groups reacted satisfactorily with nitromethane, which afforded the corresponding β-nitroalcohols in good yields within 2 h (up to 98%). By contrast, aliphatic and aromatic aldehydes with electron-donating groups did not react. Moreover, the yield significantly dropped when the size of the carbanion increased, and the yield was only 10% when nitropropane was used as the donor.

DNA double-strand breaks (DSBs) trigger the Ataxia telangiectasia mutated (ATM)-dependent DNA damage response (DDR), which consists of histone H2AX, MDC1, RNF168, 53BP1, PTIP, RIF1, Rev7, and Shieldin. Early stages of B and T lymphocyte development are dependent on recombination activating gene (RAG)-induced DSBs that form the basis for further V(D)J recombination. Non-homologous end joining (NHEJ) pathway factors recognize, process, and ligate DSBs. Based on numerous loss-of-function studies, DDR factors were thought to be dispensable for the V(D)J recombination. In particular, mice lacking Mediator of DNA Damage Checkpoint Protein 1(MDC1) possessed nearly wild-type levels of mature B and T lymphocytes in the spleen, thymus, and bone marrow. NHEJ factor XRCC4-like factor (XLF)/Cernunnos is functionally redundant with ATM, histone H2AX, and p53-binding protein 1 (53BP1) during the lymphocyte development in mice. Here, we genetically inactivated MDC1, XLF, or both MDC1 and XLF in murine vAbl pro-B cell lines and, using chromosomally integrated substrates, demonstrated that MDC1 stimulates the V(D)J recombination in cells lacking XLF. Moreover, combined inactivation of MDC1 and XLF in mice resulted in synthetic lethality. Together, these findings suggest that MDC1 and XLF are functionally redundant during the mouse development, in general, and the V(D)J recombination, in particular.

In archaea, the interaction of HerA helicase and NurA nuclease is required for 3' end resection during DNA double-stranded break repair, and its disruption results in unfaithful DNA repair. Interactions transpire by the four conserved domains, including the N-terminal HAS domain (HerA-ATP synthase), which serves as a binding site for the exonuclease NurA. As a HerA hexamer between its two subunits, the conserved C-terminal brace extension functions as an adjacent brace. DBD serves as a location for DNA binding, which causes the DNA strands to be propelled into the HerA pocket to begin unwinding. The putative pore entrance is made up of four helix coils, which provide a suitable charged surface for guiding DNA into the pore. The crystal structure of the complex HerA-NurA allows for the identification of key residues for protein-protein interaction. This allows us to predict that most archaeal HerA orthologs will be able to interact with one another. Archaeal HerA is a homo-hexameric assembly featuring a DNA-binding domain that is conserved. It also features distinctive ATP and NurA binding domains. Following the structure and sequence conservation criteria, we find one HerA ortholog in Pyrobaculum calidifontis and multiple HerA paralogs, but the role of none is established yet.Interestingly, orthologous HerA is part of an Mre11-Rad50 gene neighborhood while paralogous HerA is not, which could be misleading. As a result, it sought to describe the criteria that may be used as a possible fingerprint for identifying orthologous genes, as well as the cloning procedures and tactics that would aid in the confirmation of the HerA helicase in P. calidifontis. Their confirmation is based on PCR cloning and characterization following HerA construct creation.

N6-methyladenosine (M6A) modification is the most abundant internal chemical modification on RNA molecules in eukaryotes and can affect mRNA metabolism and regulate RNA transcription, nuclear export, splicing, degradation, translation, ets, and has a significant impact on various aspects of physiology or pathobiology. Radiotherapy is the most common measure of tumor treatment, variety of cellular intrinsic mechanisms affect the responses of cells to ionizing radiation (IR) and effectiveness of cancer radiotherapy. In this review, we summarize and discuss the recent advances about the roles and mechanisms of RNA M6A methylation in cellular responses to radiation-induced DNA damage response, and outcomes of cancer radiotherapy. Insights into the RNA M6A methylation in radiation biology may facilitate the improvement of therapeutic strategies for cancer radiotherapy as well as radioprotection on normal tissues.

Epigenetic modifications, mainly aberrant DNA methylation, have been shown to silence the ex-pression of genes involved in epigenetic diseases, including cancer suppression genes. Almost all conventional cancer therapeutic agents, such as the DNA hypomethylation drug, 5-aza-2-deoxycytidine, have insurmountable side effects. To investigate the role of ectoine in skin cell DNA methylation and cancer cell proliferation, comprehensive methylome sequence analysis, 5-methyl cytosine (5mC) analysis, proliferation and tumorigenicity assays, and DNA methyla-tion-related gene and marker analysis were performed. The results showed that ectoine hypo-methylation in skin cells, especially in the CpG island (CGIs) element and 5mC, was significantly reduced. Moreover, ectoine mildly inhibited skin cell proliferation, but did not induce tumorigenicity in HaCaT cells injected into athymic nude mice. HaCaT cells treated with ectoine for 24 weeks modulated the mRNA expression levels of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3i, Hdac1, Hdac2, Kdm3a, Mettl3, Mettl14, Igf2, Snrpn, and Mest. Overall, ectoine, an excellent DNA protectant, mildly de-methylates DNA in skin cells, modulates the expression of methylation-related genes, and reduces cell proliferation. This evidence suggests that ectoine is a potential anti-aging agent that prevents DNA hypermethylation and subsequently activates cancer-suppressing genes.

Transcription factors (TFs) are proteins that recognize specific DNA fragments in order to decode the genome and ensure its optimal functioning. TFs work at the local and global scales by specifying cell type, cell growth and death, cell migration, organization and timely tasks. We investigate the structure of DNA-binding motifs with the theory of finitely generated groups. The DNA ‘word’ in the binding domain -the motif- may be seen as the generator of a finitely generated group Fdna on four letters, the bases A, T, G and C. It is shown that, most of the time, the DNA-binding motifs have subgroup structure close to free groups of rank three or less, a property that we call ‘syntactical freedom’. Such a property is associated to the aperiodicity of the motif when it is seen as a substitution sequence. Examples are provided for the major families of TFs such as leucine zipper factors, zinc finger factors, homeo-domain factors, etc. We also discuss the exceptions to the existence of such a DNA syntactical rule and their functional role. This includes the TATA box in the promoter region of some genes, the single nucleotide markers (SNP) and the motifs of some genes of ubiquitous role in transcription and regulation.

Pericentromeric regions of chromosomes enriched in tandemly repeated satellite DNA although representing a significant part of eukaryotic genomes are still understudied mainly due to interdisciplinary knowledge gaps. Recent studies suggest their important role in genome regulation, karyotype stability and evolution. Thus, the idea of satellite DNA as a junk part of the genome was refuted. Integration of data about molecular composition, chromosome behaviour and details of in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions of pericentromeric regions non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between cerntomeric regions of X chromosome and autosomes, and associations between centromeric regions of autosomal bivalents forming chromocenters. We conclude that X chromosome form temporary synaptic associations with different autosomes in early meiotic prophase I which normally can be found at pachytene-diplotene without signs of pachytene arrest. These associations are formed between the satellite DNA-enriched centomeric regions of X chromosome and different autosomes but not involve the satellite-poor centromeric region of Y-chromosome. We suggest the mechanism of X chromosome competitive replacement from such associations during synaptic correction. We showed that centromeric region of the X chromosome remains free of γH2Ax-dependent chromatin inactivation, while Y chromosome is completely inactivated. This findings highlights the predominant role of associations between satellite DNA-enriched regions of different chromosomes including X. We assume that X-autosome temporary associations is a manifestation of an additional synaptic disorders checkpoint. These associations are normally corrected before the late diplotene. We revealed that the intense spreading conditions applied to the spermatocytes I nuclei did not lead to destruction of stretched chromatin fibers i.e. elongated chromocenters enriched in satellite DNA. Revealed by us tight associations between pericentromeric regions of different autosomal bivalents and X chromosome may represent the basis for repeat stability maintenance in autosomes an X chromosome. The consequences of our findings are discussed. We obtained the preparations of mouse spermatocytes nuclei in the meiotic prophase I using two approaches: standard and extremely intense surface spread techniques. Using immuno-FISH we visualized tandemly repeated mouse Major and Minor satellite DNA located in the pericentromeric regions of chromosomes and performed a morphological comparison of the standard- and intensely spreaded meiotic nuclei. Based on our results, we assume the remarkable strength of the chromocenter-mediated associations, “chromatin “bridges”, between different bivalents at the pachytene and diplotene stages. We have demonstrated that the chromocenter “bridges” between the centromeric ends of meiotic bivalents are enriched in both tandemly repeated Major and Minor satellite DNA. Association of centromeric regions of autosomal bivalents and X-chromosome but not with Y-chromosome correlates with the absence of Major and Minor satellites on Y-chromosome. We suggest that revealed tight associations between pericentromeric regions of bivalents may represent the network-like system providing dynamic stability of chromosomal territories, as well as add new data for the hypothesis of ectopic recombination in these regions which supports sequence homogeneity between non-homologous chromosomes and does not contradict the meiotic restrictions imposed by the crossing-over interference near centromeres. We conclude that nuclear architecture in meio-sis may play an essential role in contacts between the non-homologous chromosomes providing the specific characteristics of pericentromeric DNA.

The ability to predict physical characteristics from DNA presents significant opportunities for forensic science. Giving scientists an ability to make predictions about the donor of genetic material at a crime scene can then give investigators new intelligence leads for cold cases where DNA evidence has not identified any person of interest. However, the interpretation of this new form of intelligence requires careful analysis. The responses to an online survey, conducted in 2018-19, were used to examine how actors in the criminal justice system assess and interpret different types of DNA evidence and intelligence. The groups of focus for the survey were investigators, legal practitioners and the general public (as potential jurors). Several statistically significant effects were identified based on occupation and whether an individual had prior exposure to new DNA technology. Monitoring how those involved in interpreting reports from different types of DNA evidence and intelligence interpret them helps to ensure that decisions are made based on a sound understanding of their capabilities and limitations and may inform broader training and awareness strategies.

Radical oxygen species formed in human tissue cells by many endogenous and exogenous pathways, cause extensive oxidative damage, which has been linked to various human diseases. This review paper provides an overview of lipid peroxidation and focuses on the free-radicals initiated processes of LDL oxidative modification and DNA oxidative damage, which are widely associated to the initiation and development of atherosclerosis and carcinogenesis, respectively. The article subsequently provides an overview of the recent human trials or even in vitro investigations on the potential of natural antioxidant compounds (such as carotenoids, vitamins C and E) to monitor LDL and DNA oxidative changes.

Abstract Background In tumors, somatic mutagenesis presumably drives DNA damage response (DDR) via altered regulatory pathways increasing genomic instability and proliferative activity. These considerations led to the standard therapeutic strategy against cancer: the disruption of mutation activated DNA repair pathways of tumors. Purpose Justifying that cancer cells are not enemies to be killed, but rather they are ill human cells having the remnants of physiologic regulatory pathways. Results 1. Genomic instability and cancer development may be originated from the flaw of estrogen signal rather than excessive estrogen signaling. 2. Healthy cells with genomic instability exhibit somatic mutations helping DNA restitution. 3. Somatic mutations in tumor cells aim the restoration of DNA damage rather than further genomic derangement. 4. In tumors, estrogen signal drives the pathways of DNA stabilization leading to apoptotic death. 5. In the peritumoral cellular infiltration, the genomic damage of tumor induces inflammatory cytokine secretion and increased estrogen synthesis. In the inflammatory cells, increased growth factor receptor (GFR) signal confers unliganded activation of estrogen receptors (ERs). 6. In tumor cells responsive to genotoxic therapy, constitutive mutations help the upregulation of estrogen signal and consequential apoptosis. In tumors non responsive to genotoxic therapy, the possibilities for ER activation via either liganded or unliganded pathway, are exhausted leading to farther genomic instability and unrestrained proliferation. Conclusion Understanding the real character and behavior of human tumors at molecular level suggests that we should learn the genome repairing methods of tumors and follow them by supportive therapy rather than provoking additional genomic damages.

It has been more than three decades since the discovery of multifunctional factors, Non-POU Domain-Containing Octamer-Binding Protein, NonO and Splicing Factor, Proline-and Glutamine-Rich, SFPQ. Some of their functions, including their participations in transcriptional and posttranscriptional regulation as well as their contribution to paraspeckle subnuclear body organization have been well-documented. In this review, we focus on the roles of NonO and SFPQ in cell cycle with particular attention to their participation in nonhomologous end joining (NHEJ) and homologous recombination (HR) of DNA damage responses induced by irradiation. In these contexts, absence or malfunction of either or both lead to genome instability and ultimately to apoptosis, cellular senescence, and tumor development. This review also will shed light on NonO and SFPQ function in DNA/RNA/protein binding and their association with Ku heterodimer of the NHEJ pathway required for repair of DNA double-strand breaks (DSBs).

Taking a DNA-sequence, a word with letters/bases A, T, G and C, as the relation between the generators of an infinite group $\pi$, one can discriminate two important families: (i) the cardinality structure for conjugacy classes of subgroups of $\pi$ is that of a free group on $1$ to $4$ bases and the DNA word, viewed as a substitution sequence, is aperiodic. (ii) The cardinality structure for conjugacy classes of subgroups of $\pi$ is not that of a free group, the sequence is generally not aperiodic and topological properties of $\pi$ have to be determined differently. The two cases rely on DNA conformations such as A-DNA, B-DNA, Z-DNA, G-quadruplexes, etc. We found a few salient results: Z-DNA, when involved in transcription, replication and regulation in a healthy situation implies (i). The sequence of telomeric repeats comprising $3$ distinct bases, most of the time, satisfies (i). For $2$-base sequences in the free case (i) or non free case (ii), the topology of $\pi$ may be found in terms of the $SL(2,\mathbb{C})$ character variety of $\pi$ and the attached algebraic surfaces. The linking of two unknotted curves --the Hopf link-- may occur in the topology of $\pi$ in cases of biological importance, in telomeres, G-quadruplexes, hairpins and junctions, a feature we already found in the context of models of topological quantum computing. For $3$- and $4$ base sequences, other knotting configurations are noticed and a building block of the topology is the $4$-punctured sphere. Our methods have the potential to discriminate potential diseases associated to the sequences.

Ferric uptake regulation protein is a repressor protein which binds an AT rich region of DNA (the iron box). Fur binds as a dimer in a helix turn helix mode and it is activated by iron(II) and other divalent transition metal ions at elevated concentrations in a process to regulate the ion uptake. Each transition metal ion induces certain conformational changes to aid the Fur binding, both the N-terminal and C-terminal domains take part in binding to DNA in addition to His 88 and His 86 residues. The process is discussed in view of experimental reports. Fe(II), Mn(II) and Co(II) activate Fur to bind DNA experimentally but Zinc plays a structural role and does not activate Fur to bind DNA.

Barcoding of DNA is a species identification technique that utilizes a small DNA fragment from a specific gene or genes known as "sequencing." The idea behind DNA barcoding is that a single sequence can be used to specifically identify an organism to species by comparison with a reference library of these DNA sections. Herbal adulteration can be monitored using DNA barcodes that utilize medicinal plants such as Atropa belladonna. Because it is challenging to distinguish between species solely based on morphology, other plants belonging to the same genus are replacing it in trade. Such replacement with plants not utilized for medical purposes could have negative side effects. Also, Atropa belladonna's positive effects can be completely lost. Taxonomists also have trouble distinguishing the genuine Atropa belladonna plant from cultivated items. As a result, DNA barcoding can now be used to overcome this challenge. The development of DNA barcodes may help with trade control, which protects and conserves endangered species from export outside of the country due to their economic and biological significance. In this review, we discuss the DNA barcoding of Atropa belladonna plant in the medicinal plants field.

This article provides a general overview of the bio-inspired computing method called DNA-Based Computing (DBC), including its theory and applications. The main theme of DBC is the central dogma of molecular biology (once information of DNA/RNA has got into a protein, it can’t get out again), i.e., DNA to RNA (sequences of four types of nucleotides) and DNA/RNA to protein (sequence of twenty types of amino acids) are allowed, not the reverse ones. Thus, DBC transfers few-element information (DNA/RAN-like) to many-element information (protein-like), solving a given cognitive problem. DBC can take many forms; this article elucidates two main forms, denoted as DBC-1 and DBC-2. Using arbitrary numerical examples, it is shown that DBC-1 can solve the following cognitive problems: “similarity indexing between seemingly different but inherently identical objects” and “recognizing regions of an image separated by a complex boundary.” DBC-2 can solve the following cognitive problem: “pattern recognition when the relevant information is insufficient.” Smart manufacturing-based systems (digital twins and big data analytics) must solve the abovementioned problems to make the manufacturing enablers (machine tools and monitoring systems) more self-reliant and autonomous. Consequently, DBC can improve the cognitive problem-solving ability of smart manufacturing-relevant systems and, thereby, can enhance its biologicalization.

Apurinic/apyrimidinic endonuclease 1 (APE1), also known as redox factor-1 (Ref-1), is a multifunctional protein which is widely existing in living organisms. It can specifically recognize and cleave the DNA in apurinic/apyrimidinic (AP) site in the base excision repair (BER) pathway, as well as regulate the expression of genes to activate some transcription factors. The abnormal ex-pression and disruptions in the biological functions of APE1 are linked to a number of diseases, including inflammation, immunodeficiency, and cancer. Hence, it is extremely desired to monitor the activity of APE1, acquiring a thorough understanding of the healing process of damaged DNA and making clinical diagnoses. Thanks to the advent of DNA nanotechnology, some nanodevices are used to image the activity of APE1 with great sensitivity and simplicity. In this review, we will summarize DNA nanotechnology-empowered fluorescence imaging in the past years for APE1 activity according to the types of DNA probe, which are classified into linear DNA probes, composite DNA nanomaterials and three-dimensional (3D) DNA nanostructures. We also highlight the future research directions in the field of APE1 activity imaging.

DNA double-strand breakage is the most lethal damage to chromosomal DNA. It activates a series of cellular DNA damage response pathways, including DNA damage sensing, control of cell cycle arrest and apoptosis, and DNA repair. DNA damage response pathways are regulated by complex signaling machineries. Of the intracellular signaling cascades, diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Because both DG and PA serve as second messengers, DGK activity induces a shift of signaling pathways from DG-mediated to PA-mediated cascades, thereby implicating DGK in the regulation of widely various functions. Reportedly, one member of the DGK family, DGKζ, is intimately involved in the regulation of stress responses through p53 and NF-κB. Stresses such as ischemia and infarction cause DGKζ downregulation. Experimental DGKζ depletion renders cells and mice vulnerable to various stressors such as chemotherapeutic agents and ionizing irradiation. Nevertheless, how DGKζ is involved in DNA repair, a critical event of DNA damage response for survival remains unknown. For this study, we examined how DGKζ depletion affects DNA repair mechanisms. We demonstrated that DGKζ depletion causes attenuation of Akt activation and DNA-PK protein expression upon DNA damage, which might engender downregulated BRCA1 protein synthesis and stability. Results suggest that DGKζ depletion attenuates BRCA1-mediated DNA repair machinery, thereby conferring vulnerability to DNA damage.

The purpose of this study is to provide an accurate formula for calculating entropy for short DNA sequences and to demonstrate how to use it to examine leukemia patient surviving. We used IDIBAPS leukemia patient’s data base with 117 anonymized records. The generalized form of the Robust Entropy Estimator (EnRE) for short DNA sequences was proposed and key EnRE futures was showed. The Survival Analysis has been done using statistical package IBM SPSS. Entropy EnRE were calculated for leukemia patients for two samples: A. 2 groups divided by median EnRE and B. 2 groups of patients were formed according to their belonging to 1st and 4th quartiles of EnRE. The result of survival analysis are statistically significant: A. p &lt; 0.05; B. p &lt; 0.005. The death hazard for a patient with EnRE below median is 1.556 times that of a patient with EnRE over median and that the death hazard for a patient of 1st quartile (lowest EnRE) is 2.143 times that of a patient of 4th quartile (highest EnRE). The transition from median to quartile patients’ groups with more EnRE differentiation confirmed the unique significance of the entropy of DNA sequences for leukemia patients surviving.

We find that the degeneracies and many peculiarities of the DNA genetic code may be described thanks to two closely related (fivefold symmetric) finite groups. The first group has signature $G=\mathbb{Z}_5 \rtimes H$ where $H=\mathbb{Z}_2 . S_4\cong 2O$ is isomorphic to the binary octahedral group $2O$ and $S_4$ is the symmetric group on four letters/bases. The second group has signature $G=\mathbb{Z}_5 \rtimes GL(2,3)$ and points out a threefold symmetry of base pairings. For those groups, the representations for the $22$ conjugacy classes of $G$ are in one-to-one correspondence with the multiplets encoding the proteinogenic amino acids. Additionally, most of the $22$ characters of $G$ attached to those representations are informationally complete. The biological meaning of these coincidences is discussed.

Advancements in DNA methods and biotechnology have enabled forensic scientists to explore the DNA evidence found as part of a criminal investigation on a much more comprehensive and predictive level. This has led to a rise in research into DNA intelligence tools such as phenotypic prediction (i.e., eye and hair colour) and inference of biogeographical ancestry. Both of which can be applied to gain further insights about a scene or sample in question. Although microorganisms have played a role in forensics for decades, investigations were focused on the pathogenicity aspect, mainly to determine the cause and time of death. Recent progress in studying the human microbiome has implicated the potential use of this data in forensics. Since each individual, place, or item has its own microbial pattern, a new suite of tools are now available to be exploited in criminal investigations. Although there is much interest and potential for these emerging metagenomic and microbial forensic tools, best practices and reference ranges need to be established before they are implemented. Here, we discuss existing DNA intelligence tools applied to forensic science, the application of microbial forensics and metagenomics along with the challenges and concerns that future developments entail.

DNA damage causes the mutations that are the principal source of genetic variation. DNA damage detection and repair mechanisms therefore play a determining role in generating the genetic diversity on which natural selection acts. Speciation, it is commonly assumed, occurs at a rate set by the level of standing allelic diversity in a population. The process of speciation is driven by a combination of two evolutionary forces: genetic drift and ecological selection. Genetic drift takes place under conditions of relaxed selection, and results in a balance between rates of mutation and rates of genetic substitution. These two processes, drift and selection, are necessarily mediated by a variety of mechanisms guaranteeing genome stability in any given species. One of the outstanding questions in evolutionary biology concerns the origin of the widely varying phylogenetic distribution of biodiversity across the tree of life, and how the forces of drift and selection contribute to shaping that distribution. The following examines some of the molecular mechanisms underlying genome stability and the adaptive radiations that are associated with biodiversity and the widely varying species richness and evenness in the different eukaryotic lineages.

Advancements in genomics and molecular biology have propelled our understanding of genetic information far beyond the iconic double helix structure of DNA. This comprehensive review delves into the intricate world of DNA and RNA networks, exploring the complexities that extend beyond the classical model. We investigate the dynamic interactions, regulatory mechanisms, and functional implications of the broader genetic landscape, encompassing both coding and non-coding elements. From chromatin architecture to the diverse array of non-coding RNAs, this exploration seeks to provide a holistic perspective on the multifaceted roles played by DNA and RNA in cellular processes. The three-dimensional organization of genetic material within the cell has emerged as a crucial determinant of cellular function. Chromatin architecture, comprising DNA, histones, and other associated proteins, plays a dynamic role in regulating gene expression. We delve into the intricate choreography of chromatin remodeling, deciphering the interplay between transcription factors, epigenetic modifications, and higher-order structures that modulate gene activity. The exploration of chromatin dynamics unveils a nuanced understanding of how the genome responds to environmental cues, cellular signals, and developmental processes. Moreover, the once-dismissed non-coding portion of the genome has emerged as a key regulator of cellular function. Various classes of non-coding RNAs, such as microRNAs, long non-coding RNAs, and circular RNAs, have been implicated in diverse cellular processes, ranging from fine-tuning gene expression to modulating chromatin structure. The review critically examines the functional roles of these non-coding RNAs, shedding light on their involvement in cellular homeostasis, development, and disease. As I traverse this landscape beyond the double helix, our review synthesizes current knowledge on the intricacies of DNA and RNA networks. By exploring the regulatory mechanisms, functional implications, and emerging paradigms, we aim to inspire a deeper appreciation for the molecular choreography that underlies the cellular symphony of life. Furthermore, we discuss the implications of these findings in the context of disease, highlighting potential diagnostic and therapeutic avenues that arise from a comprehensive understanding of DNA and RNA networks. In doing so, this exploration not only broadens our theoretical understanding but also opens new frontiers for translational research in genetics and molecular medicine.

DNA polymerases replicate cell genomes and/or participate in the maintenance of genome integrity. DNA polymerases sharing high sequence homology with E. coli DNA polymerase I (pol I), have been grouped in Family A. Pol I participates in Okazaki fragment maturation and in bacterial genome repair. Since its discovery in 1956, pol I has been extensively studied, primarily to get deeper insights into the mechanism of DNA replication. As research on DNA polymerases advances, many novel functions of this group of polymerases are uncovered. For example, human DNA polymerase θ (a Family A DNA pol) has been shown to synthesize DNA using RNA as a template, a function typically attributed to retroviral reverse transcriptase. A heightened interest in drug discovery against pol θ has emerged due to its roles in cancer. Likewise, Pol I family enzymes also appear attractive drug-development targets against microbial infections. Development of antimalarial compounds targeting apicoplast apPOL, an ortholog of Pol I, further extends the druggability of this family of enzymes. Here, we summarize reported drug-development efforts against Family A polymerases, and future perspective regarding these enzymes as antibiotic targets. Current techniques such artificial intelligence can be used to facilitate the goal of new drugs.

Human papillomavirus testing is increasingly central to screening programs for cervical cancer. Although HPV testing has excellent clinical sensitivity it suffers from modest clinical specificity. Cytology has taken an important role as a triage test, while more recently HPV16/18 genotyping has provided modest incremental benefits. Despite such progress issues remain to be addressed, some of which are how to: 1) improve access to the best methods, 2) decrease costs so more women can be screened, 3) simplify management algorithms, 4) Recognize the evolving diagnostic landscape created by widespread HPV vaccination. Part of the solution lies in improving the positive predictive value of the initial screen and reflex triage. An aspirational goal should be that most women attending colposcopy have true precancer; DNA methylation testing has a role to play here. Cervical cancers and CIN2/3 generally have high levels of methylation in target human and HPV genes. In contrast normal and healthy women with no intraepithelial lesions or malignancy usually have low methylation. Furthermore, there is an interesting and useful secular gradient of increasing cervical DNA methylation as women progress toward precancer and cancer. Recent results from clinical studies using commercially available routine qPCR-based DNA methylation tests have been encouraging, demonstrating the potential for higher sensitivity and specificity than current triage methods.

Assessment of the genetic distinctiveness of a cultivar through morphological descriptors is an important tool for both the registration and the protection. New mango genotypes have been improved using valuable diverse germplasm resources to ensure food security. DNA fingerprinting based simple sequence repeats (SSR)-markers have been the most broadly used, effective and accurate in evaluation of genetic characterization of a cultivar. Molecular breeding is an effective source of genetic gain after improvement of fruit trees using marker assisted genomic selection. Mango (Mangifera indica L.) is an allotetraploid (2n = 4X= 40) drupe fruit and has high nutritional value belongs to genus Mangifera and family Anacardiaceae. Mango cultivars are used with worldwide acceptance to pharmacological, ethnomedical, and phytochemical industries. This study investigated the molecular evaluation of a new mango cultivar ‘Azeem Chaunsa’ using a set of the most effective 50 hyper-variable polymorphic SSR markers. Highly specific DNA fingerprints were identified in the genome of this mango cultivar, ‘Azeem Chaunsa’ compared with three standard cultivars such as Sindhri, Samar Bahisht (S.B) Chaunsa and Sufaid Chaunsa. Our results showed that SSR markers could efficiently assess genetic diversity in mango. An agglomerative hierarchical clustering method was used to construct dendrogram based on the ‘Unweighted Pair- Group Method with Arithmetic Mean’ (UPGMA). The genetic similarity coefficients were recorded between the mangos cultivars ranged from 0.49 to 0.67. Cultivar identification (CID) evaluates association among standard cultivars and Azeem Chaunsa and further concludes significant variations. CID results concluded that cultivar ‘Azeem Chaunsa’ varied significantly from the check cultivar, Sindhri (46.2%), S.B Chaunsa (45%) and Sufaid Chaunsa (46.7%). The results obtained in this study will orient cultivar identification strategies for a successful future mango breeding programmes in the context of climate change.

Background: Despite recent advances in epithelial ovarian carcinoma (EOC) treatment, recur-rence and mortality rates have not improved significantly. DNA hypermethylation has generally been associated with ominous prognosis and chemotherapy resistance, but the role of DNMTs in EOC remains to be investigated. Methods: In the current study, we systematically retrieved gene expression data from patients with EOC and studied the immunohistochemical expression of DNMTs in 108 primary and 26 relapsed tumors. Results: Our results showed that DNMT1, DNMT3A, DNMT3B and DNMT3L RNA levels were higher and DNMT2 lower in tumors compared to non-neoplastic tissue, and DNMT3A and DNMT2 expression decreased from Stage II to Stage IV carcinomas. Proteomic data also suggested that DNMT1 and DNMT3A levels were increased in tumors. Similarly, DNMT1, DNMT3A and DNMT3L protein levels were overex-pressed and DNMT2 expression was reduced in high grade carcinomas compared to non-neoplastic tissue and low-grade tumors. Moreover, DNMT1 and DNMT3L were increased in relapsed tumors compared to their primaries. DNMT3A, DNMT1 and DNMT3B mRNA lev-els were correlated with overall survival. Conclusions: Our study demonstrates that DNMT1 and DNMT3L are upregulated in primary high grade EOC and further increase in relapses, whereas DNMT3A is upregulated only in the earlier stages of cancer progression. DNMT2 downregulation highlights a presumptive tumor-suppressor activity of this gene in ovarian car-cinoma.

Currently available DNA detection techniques frequently require compromises between simplicity, speed, accuracy, and cost. Here, we propose a simple, label-free and cost-effective DNA detection platform developed at screen printed carbon electrodes (SPCEs) modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs). The preparation of the detection platform involved a two-step electrochemical procedure based on GO reduction onto SPCEs followed by the electrochemical reduction of HAuCl4 to facilitate the post-grafting reaction with AuNPs. The final sensor was fabricated by the simple physical adsorption of a single-stranded DNA (ssDNA) probe onto AuNPs-RGO/SPCE electrode. Each preparation step was confirmed by morphological and structural characterization using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy, respectively. Furthermore, the electrochemical properties of the modified electrodes have been investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that the introduction of AuNPs onto RGO/SPCEs led to an enhancement in surface conductivity, characteristic that favored an increased sensitivity in detection. The detection process relied on the change in the electrochemical signal induced by the binding of target DNA to the bioreceptor, and was monitored particularly by the change in the charge transfer resistance of a [Fe(CN)6]4−/3− redox couple added in the test solution.

Inflammatory bowel disease (IBD), including Crohn’s Disease (CD) and Ulcerative Colitis (UC) are chronic multifactorial disorders which affect the gastrointestinal tract with variable extent. Despite extensive research, their etiology and exact pathogenesis are still unknown. Cell-free DNAs (cfDNAs) are defined as any DNA fragments which are free from origin cell and able to circulate into bloodstream with or without microvescicles. CfDNAs are now being increasingly studied in different human diseases, like cancer or inflammatory diseases. However, to date it is unclear how IBD etiology is linked to cfDNAs in plasma. Extrachromosomal circular DNA (eccDNA) are non-plasmidic, nuclear, circular and closed DNA molecules found in all eukaryotes tested. CfDNAs appear to play an important role in autoimmune diseases, inflammatory processes, and cancer; recently, interest has also grown in IBD, and their role in the pathogenesis of IBD has been suggested. We now suggest that eccDNAs also plays a role in IBD. In this review we have comprehensively collected available knowledge in literature regarding cfDNA, eccDNA, and structures involving them such as neutrophil extracellular traps and exosomes, and their role in IBD. Finally, we focused on old and novel potential molecular therapies and drug delivery systems, such as nanoparticles, for IBD treatment.

Mature B cells notably diversify immunoglobulin (Ig) production through class switch recombination (CSR), allowing the junction of distant “switch” (S) regions. CSR is initiated by activation-induced deaminase (AID) which targets cytosines adequately exposed within single-stranded DNA of transcribed targeted S regions, with a specific affinity for WRCY motifs. In mammals, G-rich sequences are additionally present in S regions, forming canonical G-quadruplexes (G4s) DNA structures which favor CSR. Small molecules interacting with G4-DNA (G4 ligands), proved able to regulate CSR in B lymphocytes, either positively (such as for nucleoside diphosphate kinase isoforms) or negatively (such as for RHPS4). G4-DNA is also implicated in the control of transcription, and due to their impact on both CSR and transcriptional regulation, G4-rich sequences likely play a role in the natural history of B cell malignancies. Since G4-DNA stands at multiple locations in the genome, and notably within oncogene promoters, it remains to be clarified how it can more specifically promote legitimate CSR in physiology, rather than pathogenic translocation. The specific regulatory role of G4 structures in transcribed DNA and/or in corresponding transcripts and recombination hereby appears as a major issue for understanding immune responses and lymphomagenesis.