Polymerase Chain Research Articles

Persistence evaluation of fecal pollution indicators in dewatered sludge and dewatering filtrate of municipal sewage sludge: The impacts of ambient temperature and conditioning treatments

Sludge resource utilization is one of the important routines for transmitting fecal pollution to water and soil, and sludge dewatering is a crucial step for sludge resource utilization. However, it remains unclear the decay characteristics and persistence of fecal pollution indicators after sludge dewatering. In this study, the persistence of six fecal pollution indicators, namely E. coli (EC), human-specific HF183 Bacteroides (HF183), human adenovirus (HAdV), human JC and BK polyomavirus (JCPyV and BKPyV), and crAssphage, in dewatered sludge cake and dewatering filtrate deriving from raw sewage sludge, as well as three types of sludge conditioned with polyacrylamide (PAM), Fenton's reagent, or Fe[III] and CaO were analyzed. The quantitative polymerase chain reaction (qPCR) and viability-qPCR methods were used to analyze the variation in abundances and infectivity of fecal pollution indicators in dewatered sludge cake or dewatering filtrate over the storage time, respectively. Decay predications of fecal pollution indicators over time were modeled using either the first-order or the biphasic decay model. The qPCR results revealed that fecal pollution indicators in dewatered sludge cake persisted longer than those in dewatering filtrate at the same temperature. Increasing temperature can accelerate the decay of fecal pollution indicators in both dewatered sludge cake and dewatering filtrate. Notably, sludge conditioning treatment may prolong the persistence of fecal pollution indicators in both dewatered sludge cake and dewatering filtrate. Viability-qPCR results indicated that the fecal pollution indicators (except HAdV) in dewatered sludge cakes deriving from both raw sewage sludge and conditioned sludges remained infectious for up to 30 days. After a storage period of 40 days, the abundances of fecal pollution indicators (except for EC) in sludge conditioned with Fenton's reagent were effectively decreased and meanwhile the infectivity of EC was reduced, exhibiting the lowest levels of fecal pollution. Therefore, both ambient temperature and conditioning treatment greatly impacted the decay characteristics and persistence of fecal pollution indicators in dewatered sludge cake and dewatering filtrate, and selecting suitable conditioning method can minimize environmental risks associated with fecal pollution in sewage sludge.

Identification of Reliable Reference Genes for Use in Gene Expression Studies in Rat Febrile Seizure Model.

The study of the pathogenesis of febrile seizures and their consequences frequently necessitates gene expression analysis. The primary methodology employed for such analysis is reverse transcription with quantitative polymerase chain reaction (RT-qPCR). To ensure the accuracy of data obtained by RT-qPCR, it is crucial to utilize stably expressed reference genes. The objective of this study was to identify the most suitable reference genes for use in the analysis of mRNA production in various brain regions of rats following prolonged neonatal febrile seizures. The expression stability of eight housekeeping genes was evaluated using the online tool RefFinder in the dorsal and ventral hippocampal regions and in the temporal and medial prefrontal cortex of the brain. The Ppia gene exhibited the greatest stability of expression. Conversely, the genes with the least stable expression levels were Actb and Ywhaz; thus, it is not recommended to use them for normalization in a febrile seizure model. Additionally, the majority of housekeeping genes demonstrate age-related, region-specific fluctuations. Therefore, it is crucial to employ the appropriate housekeeping genes for each brain structure under investigation when examining the expression dynamics of genes of interest in a febrile seizure model.

Differential Expression of Mitogen-Activated Protein Kinase Kinase 3 (MAP2K3) and Interleukin12B (IL12B) in the Bursa of Fabricius of chicken infected with Infectious Bursal Disease Virus

Infectious Bursal Disease (IBD) caused by Infectious Bursal Disease virus (IBDV), poses a significant threat to the poultry industry, causing immunosuppression and economic losses since its discovery in 1957. To understand the host response to IBDV infection, this study examined the modulation of MAP2K3 and IL12B in chickens infected with IBDV. A local IBDV isolate propagated in embryonated chicken eggs was inoculated into 18-day-old chicken. The mRNA transcription levels of MAP2K3 and IL12B in bursal tissue were quantified by real-time quantitative polymerase chain reaction on day two post-infection and compared with those of uninfected, age-matched control birds (sham inoculated). The study revealed increased expression of the MAP2K3 and IL12B genes, indicating their role in inflammatory response during IBDV infection. Thus, the present investigation sheds light on the involvement of MAP2K3 and IL12B in the inflammatory response to IBDV infection in chickens. Keywords: Infectious bursal disease virus, immunosuppression, MAP2K3, IL12B, pro-inflammatory cytokine gene expression

Association between human telomerase reverse transcriptase (hTERT) MNS16A polymorphism and risk of breast cancer.

It is well known that telomerase activity is suppressed in normal human tissues and reactivated in tumors, suggesting that the human telomerase reverse transcriptase (hTERT, MIM: 187270) gene may be involved in carcinogenesis. A polymorphic tandem repeat minisatellite located downstream of exon 16 of hTERT and upstream in the putative promoter region of an antisense hTERT transcript, termed MNS16A, results in a functional polymorphism. Because the association between the MNS16A genetic polymorphism and breast cancer (BC) risk remains an open question, the present case-control study was conducted in Shiraz (Fars Province, Southern Iran). A total of 711 samples were collected, including 362 BC patients and 349 healthy individuals. Genotyping was performed by polymerase chain reaction method. Alleles were determined by classifying DNA amplicons of less than and greater than 300bp as short (S) and long (L) alleles, respectively. Different inheritance models (codominant, dominant, recessive, overdominant genotype models and the allele model) were used to evaluate the association between the MNS16A polymorphism and the risk of BC. No significant association was observed in any of the analyses. It should be noted that the statistical power of the comparisons was low. The present study did not support the association between hTERT MNS16A polymorphism and breast cancer risk. Similar studies in other populations with larger sample sizes are needed to determine the association between the hTERT MNS16A polymorphism and susceptibility to breast cancer.

Oral Bacteria Counter Using Dielectrophoretic Impedance Measurement: Usefulness and Usage Considerations.

The oral cavity hosts numerous bacteria that are associated with various systemic diseases. The Oral Bacteria Counter (PHC Corporation, Tokyo, Japan), a microorganism quantitative analyzer that utilizes dielectrophoretic impedance measurements, enables rapid bacterial counting and is widely used in dental practice in Japan. However, it may also detect nonviable bacteria. This study aimed to assess the impact of disinfectants, electrolytes, and viscosity on the accuracy of the Oral Bacteria Counter and to determine whether it measures non-viable bacteria similarly to viable bacteria. To evaluate the effect of the disinfectants, samples of 7% povidone-iodine (PV-I), 0.2% benzethonium chloride, 5% chlorhexidine (CHX), 0.2% CHX, 0.05% CHX, sterile water, and saline were measured using the Oral Bacteria Counter. The effect of viscosity was assessed by mixing sterile water with glycerol in various ratios and measuring the dielectrophoretic impedance of the bacterial counts at different viscosities. For the electrolyte effects, samples of Staphylococcus aureus diluted in sterile water or saline were measured using the Oral Bacteria Counter. Additionally, samples of 7% PV-I or 5% CHX diluted in sterile water or saline were measured. Bacterial counts were then measured and compared using the Oral Bacteria Counter, our developed delayed real-time polymerase chain reaction (DR-PCR) method (which quantifies only viable bacteria), and culture methods. Disinfectants such as 5% CHX and 7% PV-I produced high readings on the Oral Bacteria Counter, even when no viable bacteria were present. Higher glycerol concentrations, which increased the viscosity, resulted in lower bacterial counts. The presence of electrolytes, particularly saline, led to higher readings on the Oral Bacteria Counter, which detected both viable and non-viable bacteria, whereas DR-PCR and culture methods did not detect non-viable bacteria. The Oral Bacteria Counter may be influenced by disinfectants, viscosity, and electrolytes, leading to potential inaccuracies in bacterial quantification. For accurate bacterial measurements, it is essential to consider these factors and ideally combine the results from the Oral Bacteria Counter with methods such as DR-PCR for more reliable assessment.

Supplementing exogenous xylanase improves the liver antioxidant capacity and immune response of Tibetan sheep fed wheat-based diets

Xylanase is a heteropolysaccharide found in the plant cell wall, which aids in the absorption and utilisation of nutrients. When embedded in animal feed, it enhances nutrient availability, leading to improved animal performance. The aim of this study was to explore the effect of xylanase supplementation in wheat-based feeds on the hepatic development, antioxidant capacity, and immune response in Tibetan sheep. Sixty healthy, weaned, non-neutered male lambs (19.35 ± 2.18 kg) were randomly assigned to two equal groups (n = 30 per group). Dietary treatments were: basal diet (H group), and basal diet with 0.2% xylanase supplementation (E group). After a 10–day adaptation period, the feeding trial was carried out for 90 days. The live weight, morphology, antioxidant capacity and immune response were measured. The differential genes in liver were identified by RNA-sequencing. Our results showed that the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were significantly higher in E group than in H group (p < 0.05). Enzyme-linked immunosorbent assay (ELISA) revealed that immunoglobulin A (IgA), IgM, and IgG were significantly higher in E group than in H group (p < 0.05). The hepatocytes of sheep fed xylanase diet showed arranged radially into hepatic plates cantered around the central vein, and the plates linked with each other to form a lost-like structure. A total of 4,509 differential genes (DEGs) were identified, among which 4,427 were up-regulated and 82 down-regulated. Additionally, the expression of antioxidant-related genes in cluding catalase (CAT), superoxide dismutase 1 (SOD1), oxidation resistance 1 (OXR1) and glutathione peroxidase 2 (GPX2), and immunity-related gene including mitogen-activated protein kinase 1 (MAPK1), signal transducer and activator of transcription 5B (STAT5B), janus kinase 3 (JAK3) and jun proto-oncogene (JUN) were verified using the quantitative reverse transcription polymerase chain reaction (qRT-PCR). Conclusion: In conclusion, dietary 0.2% xylanase supplementation promoted hepatic antioxidant capacity and immune response via modulating the expression of functional genes in Tibetan sheep.

Bladder Cancer detection by urinary methylation markers GHSR/MAL: a validation study

PurposeAlthough cystoscopy is a reliable tool for detecting bladder cancer, it poses a high burden on patients and entails high costs. This highlights the need for non-invasive and cost-effective alternatives. This study aimed to validate a previously developed urinary methylation marker panel containing GHSR and MAL.MethodsWe enrolled 134 patients who underwent cystoscopy because of hematuria, including 63 individuals with primary bladder cancer and 71 with non-malignant findings. Urine samples were self-collected at home and sent via regular mail. Subsequently, DNA was extracted and the hypermethylation of GHSR and MAL was evaluated using quantitative methylation-specific polymerase chain reaction. The performance of methylation markers was assessed using area-under-the-curve (AUC) analysis and sensitivity and specificity based on pre-established cut-off values.ResultsValidation of the marker panel GHSR/MAL resulted in an AUC of 0.87 at 79% sensitivity and 80% specificity. Sensitivity was comparable to the previous investigation (P > 0.9), though specificity was significantly lower (P = 0.026). Sensitivity was higher for high-grade tumors compared to low-grade tumors (94% vs. 60%, P = 0.002).ConclusionValidation of the GHSR/MAL methylation marker panel on at home collected urine samples confirms its robust performance for bladder cancer detection in a hematuria population, and underscores the diagnostic potential for future clinical application.

The effectiveness of liver function tests in the prediction of mortality in patients with COVID-19

Objective: To evaluate the impact of liver function test abnormalities on mortality in critically ill coronavirus disease-2019 patients. Method: The single-centre, retrospective study was conducted at the Department of Anaesthesiology and Reanimation, Mersin City Training and Research Hospital, Turkiye, and comprised data from March 2020 to January 2022 of patients with positive real-time reverse transcription polymerase chain reaction test on nasopharyngeal swab sample for coronavirus disease-2019. Comorbidities, demographic data, admission to and discharge from the intensive care unit, Acute Physiology and Chronic Health Evaluation II scores, complete blood count on the first and second day of hospitalisation, coagulation parameters, biochemical analysis, 7-day, 28-day and total mortality of the patients in the intensive care unit were recorded. The patients were then divided into groups, with Group 1 having patients with normal liver enzymes, Group 2 having patients with abnormal liver enzymes, and Group 3 having patients with liver damage. Data was analysed using SPSS 20. Results: Of the 940 subjects, 587(62.4) were males and 353(37.6) were females. The overall mean age of the patients was 67.11±16.65 years. There were 395(42%) patients in Group 1, 534(56.8%) in Group 2 and 11(1.2%) in Group 3. Gamma-glutamyl transferase-1s, aspartate aminotransferase-2nd and albumin-2nd were significant predictors of mortality (p&lt;0.05). Conclusion: High gamma-glutamyl transferase, aspartate aminotransferase and low albumin levels in critically ill coronavirus disease-2019 patients could be considered indicators of high mortality rate. Key Words: Coronavirus, Critical illness, Liver function test, Pandemic.

Effects of Cannabidiol on Intestinal Myofibroblast Fibrotic PathwaysPATHWAYS

Inflammatory bowel disease (IBD) includes two immune-mediated disorders, Crohn’s disease (CD) and ulcerative colitis (UC). IBD is characterized by recurrent and chronic inflammation of the gastrointestinal tract. IBD is lifelong, and there is no cure. There has been an interest in new advanced medications to decrease inflammation and manage symptoms. Many of these new medications are expensive and depress the immune system, leaving many patients looking for alternatives to current medical therapies. Some IBD patients use medical marijuana to relieve the symptoms of this disease, and cannabidiol (CBD) in particular shows potential for reducing inflammation. This study investigates the potential therapeutic effects of CBD on IBD by examining its impact on fibrotic pathways in human intestinal myofibroblast (InMyoFib) cells. Cultured InMyoFibs were pre-treated with CBD, followed by transforming growth factor-beta 1 (TGF-β1) to stimulate a pro-fibrotic phenotype and mimic human IBD in the lab. Expression of genes associated with fibrosis, inflammation, and cannabinoid signaling was measured by quantitative polymerase chain reaction (qPCR). Fibrotic protein expression in the culture media was analyzed using enzyme-linked immunosorbent assays (ELISA). CBD pre-treatment before TGF-β1 stimulation significantly reduced the expression of pro-fibrotic genes ACTA2, CCN2, COL1A1, and SERPINH1 compared to cells stimulated with TGF-β1 alone. Additionally, CBD increased the expression of inflammation-related genes IL6 and PTGS2 and the lipid metabolism gene PPARG, known to have anti-fibrotic properties. These findings suggest that CBD may modulate fibrotic and inflammatory signaling pathways, providing a potential therapeutic avenue for IBD treatment.

Transcriptome Analysis of Porphyromonas gingivalis Lipopolysaccharide-Induced Early Gene Expression in Human Gingival Keratinocytes.

Porphyromonas gingivalis lipopolysaccharide (PgLPS) is a significant virulence factor and a driver of early innate immune responses in epithelial cells. The presence of PgLPS in immediate proximity to gingival epithelium induces significant inflammatory responses. In primary human gingival keratinocytes (HGK), we utilized transcriptome analysis to elucidate the change in early gene expression induced by PgLPS. HGK cell cultures were treated with PgLPS (4 h), and RNA was extracted and prepared for RNA sequence (RNAseq) analysis. Differentially expressed genes (DEGs) were identified, and potential interactions between these genes were subsequently examined using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analytic approaches to identify significantly enriched pathways. Expression of genes associated with relevant pathways was evaluated using real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR). RNAseq analysis identified 25 DEGs, and GO and KEGG analytic approaches showed related genes expressed in two general pathways. First, pathways broadly related to urokinase and coagulation included the genes PLAU, PLAUR, and SerpinB2. In RT-qPCR analysis, these genes were induced by PgLPS over time (4-24 h), and these data were consistent with PgLPS induction of cell migration. Second, interleukin-1 (IL-1) receptor binding and cytokine-activity pathways were also enriched. Genes associated with these pathways included IL36G, IL1B, IL1RN, and CXCL14. RT-qPCR analysis confirmed PgLPS induction of genes associated with the IL-1family. When expression of IL1B and IL36G genes was examined in relation to their respective antagonists, only IL36G gene expression was increased. CXCL14 gene expression was reduced over time, and this was consistent with RNAseq analysis. Genes associated with significantly enriched GO and KEGG pathways are relevant to aspects of periodontal disease (PDD) pathogenesis. First, PgLPS induced expression of PLAU, PLAUR, and SerpinB2, and these changes were consistent with an increase in cell migration that was found. Second, both IL36G and IL1B gene expression was significantly induced, but only IL36G in relation to its selective antagonist (IL36RN) was increased. These data support that early upregulation of IL36G may serve as an alarmin that can drive early innate immune inflammatory responses in HGK. Further invivo testing of these findings is ongoing.

The Distribution of Acme Gene and MRSA Related Virulence Genes in Staphylococcus Aureus Strains from Nigeria

Background: Arginine Catabolic Mobile Element (acme) is a Staphylococcal genomic island that enhances fitness and ability of bacteria cells to colonize on mucous membrane and skin. It is stongly associated with the epidemic and virulent S. aureus USA 300. This study determined the distribution of acme and Methicillin Resistant Staphyllococus Aureus (MRSA)-related genes in S. aureus isolated from patients in five tertiary hospitals in Nigeria. Methods: A total of 51 S. aureus isolates from the clinical specimens submitted to laboratories in five tertiary hospitals in Nigeria were used in this study. Phenotypic and genotypic identifications of the S. aureus were performed. Antibiotic Susceptibility Test (AST) was carried out to determine the susceptibility pattern of the isolates using various antibiotics discs. Minimum Inhibitory Concentration (MIC) was used to determine the degree of resistance of the isolates to methicillin and vancomycin. Polymerase Chain Reaction (PCR) was used to screen for the presence of mecA, acme sae, sarA, PVL, α-psm and norB genes using specific primers. The SCCmec type was determined for all the MRSA isolates using polymerase chain reaction. Results: The MIC for mecA negative strains was ≤4 µg/ml, while the MIC for mecA positive was 8 µg/ml. mecA gene was detected in 35 (68.6%) of 51 strains of S. aureus. The prevalence of sae, sarA, mecA, acme, PVL, α-psm and norB gene were estimated to be 70.6%, 68.6%, 68.6%, 29.4%, 5.1%, 39.2% and 33.3% respectively. There was an association between the distribution of mecA+ and norB+ strains (P = 0.034) and the hospitals (P =0.008) where the isolates were obtained from, whereas there was no association between acme positive strains and the hospitals (P = 0.669) from which the isolates were obtained from. Also, there was no association between mecA, norB, acme with the sex, age and hospital admission status (P&gt;0.05). Furthermore, there was an association between PVL gene and the two geographical regions (South-Western and North-Western, Nigeria) (χ2 = 7.77; p &lt; 0.05). The SCCmec typing showed that 21 (60.0%) out of the 35 MRSA used in this study carried the SCCmec type elements such as type I, II, III and VIII which were all the characteristic of HA-MRSA while the remaining 14 (40%) carried the SCCmec type elements such as type IV, V, VI and VII which were all characteristic of CA-MRSA. Thus the overall prevalence of HA-MRSA and CA-MRSA in this study was 60.0% and 40.0%, respectively. Conclusion: The prevalence of acme and α-psm genes in S. aureus are high and this is a novel discovery in Nigeria which has opened a new era in the transmission and fitness of circulating MRSA in causing infection in the community at large. The study concluded that there is high prevalence of HA-MRSA in South-Western, Nigeria and high prevalence of CA-MRSA in the North-Western, Nigeria.

CURRENT STATUS OF DIAGNOSTIC INFECTIONS CAUSED BY HELICOBACTER PYLORI

The article is devoted to the review of the latest scientific research related to methods of diagnosis of infections caused by Helicobacter pylori (H. pylori) bacteria. Based on the review of research in the field of diagnosis of infections caused by Helicobacter pylori, modern approaches used in global medical practice were analysed. In modern times, diagnosing H. pylori is a significant problem given the variety of methods and their effectiveness, and there is no single universal approach for detecting this pathogenic bacterium. Infection caused by the bacterium Helicobacter pylori is one of the world's most common stomach and intestinal infections. More than half of the world's population is infected with this bacterium, which often leads to severe diseases such as stomach and duodenal ulcers, as well as stomach cancer. Modern medicine develops every day, providing new methods of diagnosis and treatment. Significant scientific and technological changes are taking place in Helicobacter pylori diagnostics, which open up new perspectives and opportunities. One of the leading prospects is the search for more accurate, fast and convenient diagnostic methods that would be less invasive and more accessible to patients. The development of molecular and genetic methods, in particular polymerase chain reaction (PCR), opens up opportunities for accurate and rapid detection of H. pylori and the determination of its virulent properties. Another perspective is the development of accurate biomarkers and prognostic methods that will predict the risk of developing complications due to H. pylori infection. This will help plan and individualise treatment strategies for patients with this infection. The search for new drugs to eradicate H. pylori is also a critical prospect, especially considering the growing problem of antibiotic resistance. The article compares invasive and non-invasive methods of disease research, highlighting their advantages and limitations. Therefore, leading scientists and practitioners in the field of gastroenterology are recommended to use two or more methods, in particular, to combine invasive and non-invasive approaches, to use a serological method for population screening, to control and eradicate H. pylori mainly by non-invasive methods, using PCR as the most accurate method for diagnosing H. pylori, as well as to determine the molecular and genetic characteristics of the microorganism and assess its virulence to form an idea of the further course and prognosis of the disease. The purpose of the work is to systematically review modern methods of diagnosing infections caused by the bacterium Helicobacter pylori (H. pylori) to evaluate their effectiveness, advantages and limitations. An overview of the state of the art in diagnosing Helicobacter pylori infections is presented, focusing on the various detection methods, their advantages and disadvantages, and their role in the course of the disease and the treatment of this infection. A variety of diagnostic methods are reviewed, including both invasive and non-invasive approaches, including endoscopy, histology, urea breath test (UBT), polymerase chain reaction (PCR)-based methods, next-generation sequencing (NGS), biosensors, and MALDI-TOF MS . The advantages and limitations of each of the methods, as well as their effectiveness in the diagnosis of H. pylori infections, are discussed. Studies have shown the importance of developing new approaches and their application in practice to improve the diagnosis and treatment of this infection.

Lost in the Ds: Unexplained Loss of Rh(D) Antigen Expression By Serological Testing In Two Patients

Abstract Introduction/Objective While the loss of ABOH antigens has been well documented, reduced-to-absent expression of the RHD antigen is comparatively rare. Here, we describe two cases with D antigen loss presenting associated clinical settings, smear results, and red blood cell (RBC) genotyping findings to elucidate this unusual phenomenon. Methods/Case Report Cases (n=2) were retrospectively reviewed based on discovery during routine serological testing in our Blood Bank. RhD blood typing was performed via gel methods [Grifols, Los Angeles, CA]. Clinical information and laboratory data were extracted via the electronic medical record. Case 1 is a 79-year-old female with metastatic breast cancer. She initially typed as D positive (4+) in at least two separate analyses, but her D front type progressively became weaker until turning entirely negative after several weeks. Spherocytes and stomatocytes were present on her peripheral blood smear. This patient received 10 D+ red blood cell (RBC) units without anti-D formation. RBC genotyping revealed traditional “dce” by standard polymerase chain reaction (PCR). Case 2 is a 61-year-old male with a history of pre-B acute lymphocytic leukemia post-stem cell transplant, myelodysplastic syndrome, and progressive loss of D antigen expression (4+ to 0). Standard RBC genotyping revealed “Dce” by PCR. The peripheral blood smear was significant for RBC anisopoikilocytosis with increased stomatocytes, spherocytes, and ovalocytes. Results (if a Case Study enter NA) NA Conclusion The disappearance of the D antigen is rare, and our experience suggests an association between malignancies (both hematopoietic and non-hematopoietic) and D antigenic loss. Beyond these disease associations, however, loss of RhD is not otherwise readily explained in either case. Further analysis to test for other RHAG and RHCE mutational variants is an ongoing area of investigation in these cases, as more sensitive testing may reconcile the traditional PCR results with our initial detection of the D antigen.

DNA fragmentation of sperm and microdeletions in the long arm of the Y chromosome in male infertility patients from private fertilization units of Barquisiemto, Lara State

Abstract Introduction/Objective Figures for infertility area increasing worldwide, with a large share of causes related to the male gender. The objective of this study is to relate the frequency of abnormal sperm DNA fragmentation and the frecuency of microdeletions in the long arm of Y chromosome (Yq) in patients with male infertility from private fertilitation units of Barquisimeto, Lara, during the 2010-2012 period. Methods/Case Report We analyzed 38 samples of semen from infertile male patients to measure DNA fragmentation index (FI) by TUNEL technique, and 36 semen samples from patients with male infertility to measure FI employing SCD method. To dertermine the frecuency of Yq microdeletions by polymerase chain reaction (PCR), samples of peripheral blood from 33 patients with male infertility were evaluated. Among the 38 patients in whom the FI was measured by TUNEL technique, eight(8) of them were also tested for detection of Yq microdeletions, and one (1) of these patients performed SCD test for medical reasons. Results (if a Case Study enter NA) The frecuency of abnormal fragmentation index of sperm DNA, determined by the TUNEL technique in patients with male infertility was 47.37%. The frecuency of abnormal fragmentation index of sperm DNA determoined by SCD method in infertile male patients was 22.22%. And the frecuency of microdeletions of the long arm of the Y chromosome in patients with male infertility was 9.09%. All microdeletions were found at DAZSY208 in AZFc region and in one case there was also detected a microdeletion at DAZSY255. Conclusion It was also showed that, sperm DNA fragmentation is not depending on the existence of Yq microdeletions.

Production of anti-rM14 and anti-rM42 antibodies and their application in the stidy of expression of metalloproteases M14 and M42 of keratinolytic strain of Bacillus paralicheniformis

In the present study, two metalloproteases M14 and M42 from a Kazakh strain of Bacillus paralicheniformis T7 with high proteolytic activity were successfully cloned, expressed and purified. Protease genes were amplified by polymerase chain reaction (PCR) using specific primers selected on the basis of full genome sequencing data of B. paralicheniformis strain N7 (GenBank accession #CP124861). The amplified fragments were cloned in the expression vector pET-28c(+). Recombinant analogs of the metalloproteases M14 and M42 were obtained by plasmid expression in Escherichia coli cells of the strain ArcticExpressRP(DE3). Using metal affinity chromatography, rM14 and rM42 proteins were purified and polyclonal antibodies were produced by immunization of rabbits. Using anti-rM14 and anti-rM42 antibodies, the expression level of native metalloproteases M14 and M42 in cell lysate and culture fluid of B. paralicheniformis T7 strain was examined after culturing the strain on keratin-containing media: chicken feather, horn, hoof, hide and wool of cattle. Substrate-dependent expression of metalloprotease genes of B. paralicheniformis strain T7 was established. The obtained results indicate the prospective use B. paralicheniformis strain T7 as a producer of metalloproteases M14 and M42.

Human Schistosomiasis due to Schistosoma bovis in Nigeria.

Schistosomiasis is a global public health challenge, especially in sub-Saharan Africa, where Nigeria has the highest burden of disease. Schistosoma hybrids have been discovered in various countries, including Nigeria, where livestock and human beings share common water resources. This study, carried out in three communities, two of which are endemic for urinary schistosomiasis, aimed to identify the urinary schistosomiasis causative agent by using a species-specific molecular technique and their evolutionary relationship. Polymerase chain reaction using primers specific for a partial DNA sequence of the mitochondrial cox1 gene of Schistosoma haematobium was carried out on pooled urine sediments preserved in 70% ethanol. DNA from the high-intensity pooled samples from Onye-Uku camp amplified, and a BLAST search identified the pooled samples as Schistosoma bovis, whereas S. haematobium DNA did not amplify. The phylogenetic relationship of the sequence showed that it clustered with an S. bovis hybrid obtained from a human host in Côte d'Ivoire and had close ancestry with isolates from cattle in Cameroon. This finding revealed the prevalence of S. bovis among some inhabitants in a zone in Nigeria where schistosomiasis is endemic.

Characterizing the Impact of Dysregulated Micrornas on CRISP3 Isoforms in Male Infertility.

microRNAs (miRNAs) have a serious and dynamic function in spermatogenesis. These molecules have been recognized as crucial parts of the control of gene activity, and their involvement in the regulation of target genes has been extensively studied. This research aimed to determine the expression of CRISP3 and miR-493-5p, miR-204-5p, and miR-182-5p in the seminal plasma fluid and spermatozoa and to examine the relationship between CRISP3 and the mentioned miRNAs in 57 infertile men with Asthenozoospermia (AZ) (n = 19), Teratoasthenozoospermia (TAZ) (n = 19), and Normozoospermia (NZ) (n = 19).The selection of these three miRNAs, miR-493-5p, miR-204-5p, and miR-182-5p, was conducted using computational prediction algorithms. These miRNAs were nominated as CRISP3-associated miRNAs that can target CRISP3. We performed the quantitative real-time polymerase chain reaction (qRT-PCR) method to determine the levels of the studied miRNA expression. In the following stage, the expression of two protein isoforms of CRISP3, targeted by these miRNAs, was quantified using western blotting. The results demonstrate significant differences in the levels of miR-182-5p, miR-204-5p, miR-493-5p, and CRISP3 isoforms among the patient groups. In TAZ individuals, miR-182-5p and miR-204-5p expression decreased, while miR-493-5p expression increased compared to the control samples. Additionally, significant differences were observed in the expression levels of unglycosylated and glycosylated CRISP3 isoforms between the AZ and NZ groups. Correlation analysis revealed associations between miRNA expression and the expression of CRISP3 isoforms in the patient groups. Additionally, there were correlations between the expression of CRISP3 isoforms and sperm motility and morphology. These results offer valuable insights into the underlying molecular processes associated with male infertility.

PSAT1 is upregulated by METTL3 to attenuate high glucose-induced retinal pigment epithelial cell apoptosis and oxidative stress

BackgroundDiabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported.MethodsHigh-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay.ResultsPSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation.ConclusionMETTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR.

AML typical mutations (CEBPA, FLT3, NPM1) identify a high-risk chronic myelomonocytic leukemia independent of CPSS molecular

AbstractMutations commonly associated with acute myeloid leukemia (AML), such as CEBPA, FLT3, IDH1/2, and NPM1, are rarely found in chronic myelomonocytic leukemia (CMML), and their prognostic significance in CMML has not been clearly identified. In 127 patients with CMML, we have retrospectively analyzed next-generation sequencing and polymerase chain reaction data from bone marrow samples collected at the time of CMML diagnosis. Seven patients harbored CEBPA mutations, 8 FLT3 mutations, 12 IDH1 mutations, 26 IDH2 mutations, and 11 NPM1 mutations. Patients with CMML harboring CEBPA, FLT3, and/or NPM1 mutations (mutCFN) more frequently had the myeloproliferative subtype, a high prevalence of severe cytopenia, and elevated blast counts. Regardless of their CMML Prognostic Scoring System molecular classification, mutCFN patients with CMML had a poor prognosis, and the multivariate analysis identified mutCFN as an independent marker of overall survival. The genetic profile of these mutCFN patients with CMML closely resembled that of patients with AML, with higher-risk clinical characteristics. Our findings lead us to suggest including the assessment of these mutations in CMML prognostic models and treating these patients with AML-type therapies, including intensive chemotherapy and allogeneic stem cell transplantation, whenever feasible. Furthermore, certain targeted therapies approved for use in AML should be considered.

Functional and transcriptional regulation of the anthocyanidin acyl modifier gene Gs5AT of Gentiana sino-ornata.

The Chinese gentian, Gentiana sino-ornata produces brilliant blue flowers. To investigate the biological function and transcriptional regulation mechanism of the anthocyanin 5-O-acyltransferase gene (Gs5AT ) in the corolla, it is beneficial to analyse the mechanism of blue flower colour presentation. In this investigation, we obtained the CDS and promoter sequences of the gene Gs5AT . Yeast one-hybrid experiments were used to identify the transcription factor GsbHLH7 that activates the gene Gs5AT . According to quantitive reverse transcription polymerase chain reaction analysis, the expression of the gene Gs5AT was significantly and positively correlated with the gene GsbHLH7 . The colour phenotype of the flowers was significantly altered by the virus-induced gene silencing transduction of Gs5AT and GsbHLH7 , with GsbHLH7 silencing producing more pronounced changes in the corolla colour than Gs5AT . The expression of GsF3'5'H , GsDFR , GsANS , Gs3GT , and Gs5GT all fell to varying degrees after GsbHLH7 silencing, indicating that GsbHLH7 may regulate transcription of these genes as well as Gs5AT . The results of this study indicate that Gs5AT was positively regulated by the GsbHLH7 , and thus affects the colour presentation of the blue corolla.