Detection Of DNA Fragmentation Research Articles

A universal fluorescence biosensor based on rolling circle amplification and locking probe for DNA detection.

Astable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.

A Review on Toxicity and Degradation of Ethidium Bromide (EtBr)

Ethidium bromide (EtBr) serves as an interpolating agent utilized for its fluorescent properties in gel electrophoresis, aiding in the detection of various DNA fragments. However, its usage has been associated with DNA mutations, inhibition of mitochondrial protein synthesis and other alterations, particularly at high concentrations. Due to its remarkable stability in the environment and propensity to degrade into mutagenic compounds, especially when exposed to bleaching agents, there are significant concerns regarding its widespread use. Given the potential toxic and mutagenic effects of EtBr and the possibility of its entry into the food chain, it becomes crucial to monitor its concentration at emission sources across different ecosystems, including aquatic, terrestrial, and plant environments. Improper disposal of EtBr poses substantial risks, including the induction of mutations and cancer. Various disposal methods have been explored, such as phytoremediation and employing bacteria capable of degrading this chemical. Proper management of EtBr disposal is paramount to mitigate its harmful impact on the environment and living organisms.

Exploring Gut Microbiome Composition and Circulating Microbial DNA Fragments in Patients with Stage II/III Colorectal Cancer: A Comprehensive Analysis.

Colorectal cancer (CRC) significantly contributes to cancer-related mortality, necessitating the exploration of prognostic factors beyond TNM staging. This study investigates the composition of the gut microbiome and microbial DNA fragments in stage II/III CRC. A cohort of 142 patients with stage II/III CRC and 91 healthy controls underwent comprehensive microbiome analysis. Fecal samples were collected for 16S rRNA sequencing, and blood samples were tested for the presence of microbial DNA fragments. De novo clustering analysis categorized individuals based on their microbial profiles. Alpha and beta diversity metrics were calculated, and taxonomic profiling was conducted. Patients with CRC exhibited distinct microbial composition compared to controls. Beta diversity analysis confirmed CRC-specific microbial profiles. Taxonomic profiling revealed unique taxonomies in the patient cohort. De novo clustering separated individuals into distinct groups, with specific microbial DNA fragment detection associated with certain patient clusters. The gut microbiota can differentiate patients with CRC from healthy individuals. Detecting microbial DNA fragments in the bloodstream may be linked to CRC prognosis. These findings suggest that the gut microbiome could serve as a prognostic factor in stage II/III CRC. Identifying specific microbial markers associated with CRC prognosis has potential clinical implications, including personalized treatment strategies and reduced healthcare costs. Further research is needed to validate these findings and uncover underlying mechanisms.

Assessing DNA Degradation through Differential Amplification Efficiency of Total Human and Human Male DNA in a Forensic qPCR Assay.

The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of degradation. The possibility of deriving information on DNA degradation was evaluated in a forensic qPCR assay not specifically designed to detect DNA fragmentation, the Plexor HY (Promega), by calculating the ratio between the estimated concentrations of autosomal (99 bp) and Y-chromosomal (133 bp) targets ("[Auto]/[Y]"). The [Auto]/[Y] ratio measured in 57 formalin-fixed, paraffin-embedded samples was compared to a quality score (QS) calculated for corresponding STR profiles using quantitative data (allele peak height). A statistically significant inverse correlation was observed between [Auto]/[Y] and QS (R = -0.65, p < 0.001). The [Auto]/[Y] values were highly correlated (R = 0.75, p < 0.001) with the "[Auto]/[D]" values obtained using the PowerQuant (Promega) assay, expressly designed to detect DNA degradation through simultaneous quantification of a short (Auto) and a long (D) autosomal target. These results indicate that it is possible to estimate DNA degradation in male samples through Plexor HY data and suggest an alternative strategy for laboratories lacking the equipment required for the assessment of DNA integrity through dedicated qPCR assays.

Real-Time Monitoring of a Nucleic Acid Amplification Reaction Using a Mass Sensor Based on a Quartz-Crystal Microbalance.

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids.

Protective Effects of Hesperetin on Cardiomyocyte Integrity and Cytoskeletal Stability in a Murine Model of Epirubicin-Induced Cardiotoxicity: A Histopathological Study

Anthracyclines, including epirubicin (Epi), are effective chemotherapeutics but are known for their cardiotoxic side effects, primarily inducing cardiomyocyte apoptosis. This study investigates the protective role of hesperetin (HSP) against cardiomyopathy triggered by Epi in a murine model. Male CD1 mice were divided into four groups, with the Epi group receiving a cumulative dose of 12 mg/kg intraperitoneally, reflecting a clinically relevant dosage. The co-treatment group received 100 mg/kg of HSP daily for 13 days. After the treatment period, mice were euthanized, and heart tissues were collected for histopathological, immunofluorescence/immunohistochemistry, and transmission electron microscopy (TEM) analyses. Histologically, Epi treatment led to cytoplasmic vacuolization, myofibril loss, and fiber disarray, while co-treatment with HSP preserved cardiac structure. Immunofluorescent analysis of Bcl-2 family proteins revealed Epi-induced upregulation of the pro-apoptotic protein Bax and a decrease in anti-apoptotic Bcl-2, which HSP treatment reversed. TEM observations confirmed the preservation of mitochondrial ultrastructure with HSP treatment. Moreover, in situ detection of DNA fragmentation highlighted a decrease in apoptotic nuclei with HSP treatment. In conclusion, HSP demonstrates a protective effect against Epi-induced cardiac injury and apoptosis, suggesting its potential as an adjunctive therapy in anthracycline-induced cardiomyopathy. Further studies, including chronic cardiotoxicity models and clinical trials, are warranted to optimize its therapeutic application in Epi-related cardiac dysfunction.

AI-based Apoptosis Cell Classification Using Phase-contrast Images of K562 Cells.

This study aimed to automate the classification of cells, particularly in identifying apoptosis, using artificial intelligence (AI) in conjunction with phase-contrast microscopy. The objective was to reduce reliance on manual observation, which is often time-consuming and subject to human error. K562 cells were used as a model system and apoptosis was induced following administration of gamma-secretase inhibitors. Fluorescence staining was applied to detect DNA fragmentation and caspase activity. Cell images were obtained using both phase-contrast and fluorescence microscopy. Two AI models, Lobe(R) and a server-based ResNet50, were trained using these images and evaluated using F-values through five-fold cross-validation. Both AI models demonstrated effectively categorized individual cells into three groups: caspase-negative/no DNA fragmentation, caspase-positive/no DNA fragmentation, and caspase-positive/DNA fragmentation. Notably, the AI models' ability to differentiate cells relied on subtle variations in phase-contrast images, potentially linked to changes in refractive indices during apoptosis progression. Both AI models exhibited high accuracy, with the server-based ResNet50 model showing improved performance through repeated training. This study demonstrates the potential of AI-assisted phase-contrast microscopy as a powerful tool for automating cell classification, especially in the context of apoptosis research and the discovery of anticancer substances. By reducing the need for manual labor and enhancing classification accuracy, this approach holds promise for expediting high-throughput cell screening, significantly contributing to advancements in medical diagnostics and drug development.

Studying the effects of secondary metabolites isolated from Cycas thouarsii R.Br. leaves on MDA-MB-231 breast cancer cells

The various therapeutic drugs that are currently utilized for the management of cancer, especially breast cancer, are greatly challenged by the augmented resistance that is either acquired or de novo by the cancer cells owing to the long treatment periods. So, this study aimed at elucidating the possible anticancer potential of four compounds 7, 4′, 7′′, 4′′′-tetra-O-methyl amentoflavone, hesperidin, ferulic acid, and chlorogenic acid that are isolated from Cycas thouarsii leaves n-butanol fraction for the first time. The MTT assay evaluated the cytotoxic action of four isolated compounds against MDA-MB-231 breast cancer cells and oral epithelial cells. Interestingly, ferulic acid revealed the lowest IC50 of 12.52 µg/mL against MDA-MB-231 cells and a high IC50 of 80.2 µg/mL against oral epithelial cells. Also, using an inverted microscope, the influence of ferulic acid was studied on the MDA-MB-231, which revealed the appearance of apoptosis characteristics like shrinkage of the cells and blebbing of the cell membrane. In addition, the flow cytometric analysis showed that the MDA-MB-231 cells stained with Annexin V/PI had a rise in the count of the cells in the early and late apoptosis stages. Moreover, gel electrophoresis detected DNA fragmentation in the ferulic acid-treated cells. Finally, the effect of the compound was tested at the molecular level by qRT-PCR. An upregulation of the pro-apoptotic genes (BAX and P53) and a downregulation of the anti-apoptotic gene (BCL-2) were observed. Consequently, our study demonstrated that these isolated compounds, especially ferulic acid, may be vital anticancer agents, particularly for breast cancer, through its induction of apoptosis through the P53-dependent pathway.

Salvia officinalis restores semen quality and testicular functionality in cadmium-intoxicated male rats

The present study investigated the potential ability of Salvia officinalis, one of the oldest medicinal plants, to protect male rats against cadmium reproductive toxicity. Twenty-eight healthy male rats were randomly allocated into four groups (n = 7); control, Salvia-extract treated group, cadmium treated group and a group treated with both Cd and Salvia. Administration of cadmium reduced the relative testis to body weight and significantly affected sperm parameters by decreasing motility, viability, count and increasing morphological aberrations. Comet assay was used to detect DNA fragmentation in sperms of the rats exposed to Cd. Serum levels of testosterone T, follicle stimulating hormone FSH, and luteinizing hormone LH were significantly decreased. The biochemical analysis of testicular tissue showed a significant rise in Malondialdehyde MDA level coupled with a decrease in the activity of antioxidant enzymes (superoxide dismutase SOD, glutathione peroxidase GPx and catalase CAT). The histological examination of testis sections after Cd administration revealed severe degeneration of spermatogenic cells. Seminiferous tubules were filled with homogenous eosinophilic fluid associated with atrophy of other seminiferous tubules. Co-treatment with the Salvia officinalis extract restored the oxidative enzymes activities and decreased the formation of lipid peroxidation byproduct, which in turn ameliorated the effect of Cd on sperm parameters, DNA damage and testis histopathology. Taken together, it can be concluded that the synergistic antioxidant and radical savaging activities of Salvia officinalis prevented the effect of Cd on semen quality, sperm DNA damage, along with the oxidative stress and histological abnormalities in the testis tissues.

Processing biological samples from simulated radiological terrorist events using Rapid DNA instruments

Two commercially available portable Rapid DNA instruments were evaluated for their ability to process 1 µL and 10 µL saliva samples deposited on metal and plastic surfaces and contaminated with surrogates of cesium (Cs)-137, strontium (Sr)-90 and cobalt (Co)-60; radioactive materials potentially released during a nuclear weapon accident or a radiological dispersal device detonation. A comparable success rate was noted for both Rapid DNA instruments when considering the number of complete and balanced DNA profiles, the number of profiles with a minimum of 10 autosomal STR loci (out of 23 [FlexPlex™ 27] or 21 [GlobalFiler™ Express]), and the possibility to search a national DNA database in Canada and the United States. Cobalt had an adverse impact on the quality of the megaplex short tandem repeat (STR) DNA profiles derived on each instrument for two of the three contamination levels tested in this study, i.e., 0.05 M and 0.1 M as reflected by a reduced number of detected alleles and decreased profile peak heights. Strontium exhibited some adverse effect on the Rapid DNA results when used at the highest contamination level (0.1 M) whereas cesium had none. No new artifacts were observed in the Rapid DNA profiles of samples spiked with the non-radiogenic surrogates. Importantly, in the context of a radiological/nuclear (RN) event, the ANDE™ 6C offers the possibility to dispose of all radioactive materials associated with contaminated samples quickly using a chip on which all steps of the Rapid DNA process are performed whereas the RapidHIT™ ID accumulates radioactive materials for many days before disposal. An individual handling 25 samples in a week (5 per day) on the RapidHIT™ ID at a 30.5 cm distance with a 5 min exposure to the radioactive source estimated at every run would exceed the 0.042 µSv/5 min limit with gamma dose rates for Cs at 0.13 mSv and for Co at 3.8 mSv. Beta dose rates calculated for the surrogate isotopes at the three concentrations tested were also above the recommended radiation exposure limit of 1 mSv/yr (0.042 µSv/5 min). Various potential mechanisms of action behind the interference noted for Sr and Co at high concentrations are presented. These elements may play a role in the steps prior to PCR (at the DNA molecule by binding to bases or to phosphate groups), during PCR (at the DNA polymerase as cofactors for catalytic sites), or even during amplified DNA fragment detection (as fluorescence quenchers).

Highly Sensitive Detection of DNA Fragments by an Inexpensive and Simple Method Fabricated In-House Blue LED Transilluminator

Agarose gel electrophoresis is a routinely employed experimental technique for visualizing nucleic acids, typically combined with ethidium bromide or some other molecular dye staining and a UV transilluminator. However, the utilization of a UV transilluminator is restricted due to high cost and safety considerations. In this study, an inexpensive and simple in-house blue Light Emitting Diode (LED) transilluminator was developed using a high-brightness blue LED, carbon resistor, and smartphone charging cable. Its sensitivity for DNA detection was compared with conventional methods. A detection limit of 4 ng/band was confirmed by observing a 100 bp DNA fragment, which is comparable to ethidium bromide staining and UV transilluminator. Additionally, the in-house blue LED transilluminator can be attached to existing electrophoresis chambers, allowing real-time observation of electrophoresis and offering a superior alternative to conventional methods. In conclusion, the developed in-house blue LED transilluminator demonstrates high sensitivity in detecting DNA fragments and holds potential for advancing low-cost research facilities.

Internal validation of the GA118-24B Genetic Analyzer, a stable capillary electrophoresis system for forensic DNA identification.

The GA118-24B Genetic Analyzer (hereafter, "GA118-24B") is an independently developed capillary electrophoresis instrument. In the present research, we designed a series of validation experiments to test its performance at detecting DNA fragments compared to the Applied Biosystems 3500 Genetic Analyzer (hereafter, "3500"). Three commercially available autosomal short tandem repeat multiplex kits were used in this validation. The results showed that GA118-24B had acceptable spectral calibration for three kits. The results of accuracy and concordance studies were also satisfactory. GA118-24B showed excellent precision, with a standard deviation of less than 0.1 bp. Sensitivity and mixture studies indicated that GA118-24B could detect low-template DNA and complex mixtures as well as the results generated by 3500 in parallel experiments. Based on the experimental results, we set specific analytical and stochastic thresholds. Besides, GA118-24B showed superiority than 3500 within certain size ranges in the resolution study. Instead of conventional commercial multiplex kits, GA118-24B performed stably on a self-developed eight-dye multiplex system, which were not performed on 3500 Genetic Analyzer. We compared our validation results with those of previous research and found our results to be convincing. Overall, we conclude that GA118-24B is a stable and reliable genetic analyzer for forensic DNA identification.

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method.

Detection of heavy metal contamination in Batlama Stream (Turkiye) and the potential toxicity profile

In this study, heavy metal pollution in Batlama stream flowing into the Black Sea from Giresun (Turkiye) province and the toxicity induced by this pollution were investigated by Allium test. Heavy metal concentrations in stream water were analyzed by using ICP-MS. Germination percentage, weight gain, root length, micronucleus (MN), mitotic index (MI), chromosomal abnormalities (CAs), proline, chlorophyll, malondialdehyde (MDA), antioxidant enzyme activities were used as indicators of physiological, cytogenetic and biochemical toxicity. In addition, Comet assay was performed for detecting DNA fragmentation. Anatomical changes caused by heavy metals in the root meristem cells were observed under the microscope. A. cepa bulbs are divided into two groups as control and treatment. The bulbs in the control group were germinated with tap water and the bulbs in the treatment group were germinated with stream water. As a result, heavy metals such as Al, Ti and Co and radioactive heavy metals such as Rb, Sr, Sb and Ba were detected in the stream water above the acceptable parametric values. Heavy metals in the water caused a decrease in germination, root elongation, weight gain, MI and chlorophyll values, and an increase in MDA, proline, SOD, CAT, MN and CAs values. Comet assays indicated the presence of severe DNA damage. In addition, heavy metals in stream water caused different types of CAs and anatomical damage in root meristem cells. As a result, it was determined that there is intense heavy metal pollution in the stream water and this pollution promotes multi-dimensional toxicity in A. cepa, which is an indicator organism. For this reason, the first priority should be to prevent pollution of water resources in order to prevent heavy metal-induced toxicity in water.

Development and validation of an environmental DNA assay to detect federally threatened groundwater salamanders in central Texas.

The molecular detection of DNA fragments that are shed into the environment (eDNA) has become an increasingly applied tool used to inventory biological communities and to perform targeted species surveys. This method is particularly useful in habitats where it is difficult or not practical to visually detect or trap the target organisms. Central Texas Eurycea salamanders inhabit both surface and subterranean aquatic environments. Subterranean surveys are challenging or infeasible, and the detection of salamander eDNA in water samples is an appealing survey technique for these situations. Here, we develop and validate an eDNA assay using quantitative PCR for E. chisholmensis, E. naufragia, and E. tonkawae. These three species are federally threatened and constitute the Septentriomolge clade that occurs in the northern segment of the Edwards Aquifer. First, we validated the specificity of the assay in silico and with DNA extracted from tissue samples of both target Septentriomolge and non-target amphibians that overlap in distribution. Then, we evaluated the sensitivity of the assay in two controls, one with salamander-positive water and one at field sites known to be occupied by Septentriomolge. For the salamander-positive control, the estimated probability of eDNA occurrence (ψ) was 0.981 (SE = 0.019), and the estimated probability of detecting eDNA in a qPCR replicate (p) was 0.981 (SE = 0.011). For the field control, the estimated probability of eDNA occurring at a site (ψ) was 0.938 (95% CRI: 0.714-0.998). The estimated probability of collecting eDNA in a water sample (θ) was positively correlated with salamander relative density and ranged from 0.371 (95% CRI: 0.201-0.561) to 0.999 (95% CRI: 0.850- > 0.999) among sampled sites. Therefore, sites with low salamander density require more water samples for eDNA evaluation, and we determined that our site with the lowest estimated θ would require seven water samples for the cumulative collection probability to exceed 0.95. The estimated probability of detecting eDNA in a qPCR replicate (p) was 0.882 (95% CRI: 0.807-0.936), and our assay required two qPCR replicates for the cumulative detection probability to exceed 0.95. In complementary visual encounter surveys, the estimated probability of salamanders occurring at a known-occupied site was 0.905 (SE = 0.096), and the estimated probability of detecting salamanders in a visual encounter survey was 0.925 (SE = 0.052). We additionally discuss future research needed to refine this method and understand its limitations before practical application and incorporation into formal survey protocols for these taxa.

Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM).

Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population's health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.

Enzalutamide combination with Arsenic trioxide suppresses the progression of castration-resistant prostate cancer

The study aimed to investigate the anti-tumor effects and underlying mechanisms of Enzalutamide (ENZ) and Arsenic trioxide (ATO) co-treatment on castration-resistant prostate cancer (CRPC). The effects on C4-2B cells were initially evaluated by colony formation assay, FACS analysis, and DNA fragmentation detection. Bioinformatics methods including mRNA-sequencing and gene enrichment analysis were used to screen the underlying target genes and pathways related to their actions. Western blot was employed to assess the expression levels of protein-related angiogenesis, apoptosis, DNA repair, and the screened genes. Finally, the effects were further verified in subcutaneous tumor models and tissue sections from the xenografts. It was found that not only could ENZ combination with ATO significantly inhibit cell proliferation and angiogenesis, but also induce cell arrest and apoptosis in C4-2B cells. In addition, interruption of the DNA damage repair-related pathways also occurred as a result of their combined effects. Western blot analysis further suggested that proteins involved in these pathways, especially P-ATR and P-CHEK1 were significantly reduced. In addition, their combination also inhibited the tumor growth of xenografts. Altogether, ENZ combination with ATO synergistically improved the therapeutic effects and suppressed CRPC progression through regulation of the ATR-CHEK1-CDC25C pathway.

Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS

BackgroundSpermatozoa have the task of delivering an intact paternal genome to the oocyte and supporting successful embryo development. The detection of sperm DNA fragmentation (SDF) has been emerging as a complementary test to conventional semen analysis for male infertility evaluation, but the mechanism leading to SDF and its impact on assisted reproduction remain unclear. Therefore, the study identified and analyzed the differentially expressed proteins of sperm with high and low SDF.MethodsSemen samples from men attended the infertility clinic during June 2020 and August 2020 were analyzed, and sperm DNA fragmentation index (DFI) was detected by the sperm chromatin structure assay. Semen samples with low DFI (< 30%, control group) and high DFI (≥ 30%, experimental group) were optimized by density gradient centrifugation (DGC), and the differentially expressed proteins of obtained sperm were identified by the Sequential Window Acquisition of All Theoretical Mass Spectra Mass Spectrometry (SWATH-MS) and performed GO and KEGG analysis.ResultsA total of 2186 proteins were identified and 1591 proteins were quantified, of which 252 proteins were identified as differentially expressed proteins, including 124 upregulated and 128 downregulated. These differentially expressed proteins were involved in metabolic pathways, replication/recombination/repair, acrosomal vesicles, kinase regulators, fertilization, tyrosine metabolism, etc. Western blotting results showed that the expression levels of RAD23B and DFFA proteins and the levels of posttranslational ubiquitination and acetylation modifications in the experimental group were significantly higher than those in the control group, which was consistent with the results of proteomics analysis.ConclusionsProteomic markers of sperm with high DNA fragmentation can be identified by the SWATH-MS and bioinformatic analysis, and new protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. Our findings may improve our understanding of the basic molecular mechanism of sperm DNA damage.

Varicocelectomy improves sperm parameters, sperm DNA integrity as well as the other critical semen features.

Varicocele is a major entity defined within male infertility. In this report we have studied the influence of laparoscopic varicocelectomy on semen quality, biochemical parameters of seminal plasma and sperm DNA fragmentation. In this study, the semen samples from patients with left-side varicocele of grade II-III before and after laparoscopic varicocelectomy were compared to healthy individuals and separated into three groups. The volume of semen, sperm concentration (106/ml), motility (%), viability (%) and normal morphology (%) were assessed. Total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) together with other biochemical substances in seminal plasma as alpha-glucosidase (α-Glu), fructose (Fr) and citric acid (CA) were determined by ELISA method. The spermatozoa activity including ion-transports through sodium, potassium ATPase (Na+, K+-ATPase) and calcium, magnesium ATPase (Ca2+, Mg2+-ATPase) were determined by using spectrophotometry. In addition, flow cytometry method for detection of sperm DNA fragmentation was used. The results showed, that three months after varicocelectomy such intervention led to significant postoperative improvement in volume of semen (p<0.001), total sperm count (p<0.001), sperm motility (p<0.001) and spermatozoa with normal morphology (p<0.001). We found decreased α-Glu levels due to varicocelectomy (p<0.05). There has been shown a high positive correlation between Na+, K+-ATPase and Ca2+, Mg2+-ATPase activity with total number of spermatozoa (p<0.05). The TAC levels and DNA fragmentation values after varicocelectomy can be considered as significant indicators of good prognosis after surgical intervention. It has to be emphasized that α-Glu levels and total sperm count expressed statistically significant both positive and negative predictive values for semen assessment. Varicocelectomy may lead to significant improvement of semen quality although the observations must be correlated with clinical pregnancies observed thereafter.

Agarose Gel Electrophoresis-Based RAPD-PCR-An Optimization of the Conditions to Rapidly Detect Similarity of the Alert Pathogens for the Purpose of Epidemiological Studies.

Agarose gel electrophoresis is a well-known tool to detect DNA fragments amplified in polymerase chain reaction (PCR). Its usefulness has also been confirmed for epidemiological studies based on restriction fragments length polymorphism (RFLP), usually performed using pulsed-field gel electrophoresis (PFGE). Little is known on the effectiveness for alert-pathogen epidemiological studies of another less time-consuming and costly technique called randomly amplified polymorphic DNA-PCR (RAPD-PCR). Meanwhile, its usefulness is believed to be comparable to RFLP-PFGE. Therefore, the aim of the study was to establish and optimize the conditions of agarose gel electrophoresis following RAPD-PCR for 19 Enterococcus faecium strains derived from epidemic outbreaks at intensive care units. An application of different PCR primers, primer combinations, and, in particular, agarose gel concentrations and electrophoresis conditions revealed the usefulness of this relatively fast and inexpensive method based on RAPD-PCR for epidemiological studies without a compulsion to use the specialized equipment necessary for RFLP-PFGE.