Polymerase Chain Reaction Method Research Articles

Development and validation of a multiplex real-time PCR method for screening genetically modified plants

The diversity of genetically modified plants used for food and feed is increasing worldwide. For the detection and control of these products, efficient and reliable analytical tools are a prerequisite. This can be done by screening for specific DNA-elements and constructs characteristic of transgenic plants. In the past, numerous methods have already been published. However, several genetically modified plants are not covered by common screening methods. Here, a new qualitative triplex real-time polymerase chain reaction (PCR) method is presented, detecting two transgene flanking sequences and the transition between the Cassava Vein Mosaic Virus Promotor (P-CsVMV) and the phosphinothricin-N-acetyltransferase (pat) gene. These sequences are present in several transgenic plants and therefore, the described triplex method can be used as a screening tool to guide further analysis and increase the efficiency of the analysis strategy for GMO detection. The method is characterized by high specificity, sensitivity and robustness and is provided as a ring-trial validated method in the Official Collection of Methods according to the German Food and Feed Act.

Comparison of gut microbiota profiles between patients suffering from elderly frailty syndrome and non-frail elderly individuals.

Frailty syndrome is a state of increased vulnerability to stressors, marked by lowered physical strength and increased dependence on others. The well-established changes in gut microbiota associated with old age suggest a probable relationship between gut microbiota and frailty. This study was aimed at finding the relationship between gut microbiota and frailty syndrome, by comparing the sociodemographic data and the gut microbiota profiles of 23 non-frail and 14 frail elderly individuals. We used the quantitative polymerase chain reaction method (qPCR) to determine the bacterial loads of Bifidobacteria, Lactobacillus, Bacteroidetes, Prevotella, and Escherichia coli in stool samples from test subjects. We discovered a significant increase in the bacterial load of Prevotella in frail elderly individuals aged 70 or above. Other bacterial loads and ratios were not significantly different between the two groups. More comprehensive studies with larger sample sizes and encompassing a wider range of inflammation-related bacteria need to be performed to discover the existence and exact nature of these relations.

The Slaughter House and the Re-emerging Foodborne Illness with Special Reference to Bovine Tuberculosis

The Slaughter house is the place in which the animals are slaughtered for human consumption. The Slaughter house plays important role in prevention of zoonotic diseases between animals and humans like Mycobacterium tuberculosis as reemerging foodborne illness and also prevent infectious diseases between animals. Bovine Mycobacterium tuberculosis is caused by a species of pathogenic Gram positive, acid fast stain bacteria in the Mycobacteriaceae family. the causative agent bacteria of Bovine tuberculosis as reemerging foodborne illness tuberculosis bacteria has an waxy cover on its surface primarily due to the presence of acid called mycolic which refers the cells impervious to Gram staining, and as a result, the causative agent bacteria of Bovine tuberculosis as reemerging foodborne illness may appear weakly Gram-positive. Acid-fast bacilli by using certain stains called Ziehl Nielsen, or through using stain called fluorescent such as aura mine are used to identify the cause of Bovine tuberculosis as reemerging foodborne illness with a microscope. The Bacteria cause Bovine tuberculosis as a reemerging foodborne illness is aerobic bacteria and needs high concentrations of oxygen. Mainly this bacteria is pathogenic to human and mammal's respiratory system, it infects the lungs. The most diagnostic means for Bovine tuberculosis as a reemerging foodborne illness are the tuberculin skin examination, stain of acid-fast, laboratory culture, and through using polymerase chain reaction method.

Study based on the acetaldehyde dehydrogenase 2 gene polymorphism and acetaminophen-induced liver injury

Objective: To explore the association between aldehyde dehydrogenase 2 (ALDH2) gene polymorphisms and abnormal liver function-induced by acetaminophen (APAP) drugs. Methods: An ALDH2 gene knockout mouse model was constructed using CRISPR/Cas9 gene editing technology. The obtained heterozygous mice were mated with opposite sex of heterozygotes. Genomic DNA was extracted from the tail of the offspring mouse. The polymerase chain reaction (PCR) method was used to determine the ALDH2 genotype. APAP was further used to induce acute drug-induced liver injury models in wild-type and ALDH2 knockout mice. Blood and liver tissues of mice were collected for liver function index, HE staining, F4/80 immunohistochemistry, and other detections. The intergroup mean was compared using a one-way ANOVA. The LSD- t test was used for pairwise comparison. Results: ALDH2 knockout mice were bred successfully. The genotyping of the offspring was segregated into the wild-type (ALDH2(+/+)), heterozygous mutant (ALDH2(+/-)), and homozygous mutant (ALDH2(-/-)), respectively. Biochemical and histological results after APAP modeling showed that the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil) was not significantly increased in the blank control group (P < 0.05), while the ALT, AST,ALP, and TBil were all elevated in the APAP experimental group. The levels of ALT (P = 0.004), AST (P = 0.002), and TBil (P = 0.012) were significantly elevated among the mutant group compared to those in the wild-type group, and the expression levels of these indicators were also significantly elevated among the homozygous mutant group compared to those in the heterozygous mutant group (P = 0.003, 0 and 0.006). In addition, the ALP levels were higher in the heterozygous mutation group than those in the homozygous mutant group (P = 0.085) and wild-type group mice, but the difference was only statistically significant compared to wild-type mice (P = 0.002). HE staining results showed that mice in the APAP experimental group had hepatocyte degeneration, necrosis, and increased inflammatory cell infiltration, which was mostly evident in mutant mice. Simultaneously, the F4/80 immunohistochemical staining results showed that brown granules were visible in the liver tissue of APAP experimental group mice, and its expression levels were significantly enhanced compared to the blank control group. Conclusion: APAP-induced liver function abnormalities were associated with the ALDH2 gene polymorphism. The liver injury symptoms were increased in ALDH2 mutant mice following APAP modeling, and the ALDH2 gene defect may alleviate, to some extent, APAP-induced liver function abnormalities.

Evaluation of Mir-1290 as a Possible Diagnostic Factor in the Serum of Oral Squamous Cell Carcinoma Patients with Qualitative Real-Time Polymerase Chain Reaction.

This study aimed to determine the incidence of microRNA (miRNA; miR-1290) in the serum of oral squamous cell carcinoma (OSCC) patients compared to a control group using the qualitative real-time polymerase chain reaction (PCR) method. Blood serum samples were obtained from patients diagnosed with OSCC and confirmed through biopsy. The samples were collected from patients referred to the Mashhad Dental Faculty and Ghaem Hospital. The OSCC group consisted of 17 patients, while the healthy group included 15 individuals. RNA was extracted from the patient samples, and samples with an A260/280 ratio between 1.8 and 2.0 (indicating acceptable RNA quality) were immediately converted into complementary DNA (cDNA) using albumin and cDNA reference genes. The SYBR green real-time reverse transcriptase PCR method was used to measure the presence of miR-1290 in the blood samples. A total of 32 patients were examined in this study, including 17 women (53.1%) and 15 men (46.9%). The mean age was 46.7 years in the healthy group and 54.6 years in the SCC group, indicating a significant difference (P<0.05). The expression level of the miR-1290 gene was higher in patients with SCC compared to the healthy group (P=0.000). While the expression level of miR-1290 was higher in grade 3 and advanced stage than in grades 2 and 1 and early stage, the differences were not statistically significant (P=0.173 and P=0.564 for grade and stage, respectively). The expression level of miR-1290 may increase in SCC patients compared to healthy individuals, making it a potential circulating biomarker. Further investigations for diagnostic utility would be warranted.

Early detection of oral bacteria causing gum infections and dental caries in children.

Periodontal diseases are resulted from gum infections and dental plaques, which are mainly caused by the bacterial agents. Since dental monitoring includes important prognostic roles, the aim of this study was to detect the most common periodontal pathogenic bacteria in children. A total of 200 clinical samples were collected from dental plaques and gingival grooves. Target-specific primers were designed for hbpA in Aggregatibacter actinomycetemcomitans, fimA in Porphyromonas gingivalis and 16S rRNA in Prevotella intermedia, Tannerella forsythia and Treponema denticola. Then, a multiplex polymerase chain reaction method was optimized for the highlighted bacterial agents. In general, the highest and the lowest bacterial prevalence rates belonged to Tannerella forsythia (88%) and Porphyromonas gingivalis (13%), respectively. Furthermore, prevalence rates of Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Treponema denticola were 25, 21 and 45% in samples, respectively. There were significant associations between dental/oral health and microbial community. Metabolism of the oral bacteria, including biofilm formation, can affect gums and develop dental plaques and hence dental caries, especially in children. Early diagnosis of dental caries in children via rapid, accurate molecular methods can increase the diagnostic capacity in clinical cases and therefore prevent periodontal infections in adulthood.

Analytical validation and clinical utilization of K-4CARE™: a comprehensive genomic profiling assay with personalized MRD detection.

Background: Biomarker testing has gradually become standard of care in precision oncology to help physicians select optimal treatment for patients. Compared to single-gene or small gene panel testing, comprehensive genomic profiling (CGP) has emerged as a more time- and tissue-efficient method. This study demonstrated in-depth analytical validation of K-4CARE, a CGP assay that integrates circulating tumor DNA (ctDNA) tracking for residual cancer surveillance. Methods: The assay utilized a panel of 473 cancer-relevant genes with a total length of 1.7Mb. Reference standards were used to evaluate limit of detection (LOD), concordance, sensitivity, specificity and precision of the assay to detect single nucleotide variants (SNVs), small insertion/deletions (Indels), gene amplification and fusion, microsatellite instability (MSI) and tumor mutational burden (TMB). The assay was then benchmarked against orthogonal methods using 155 clinical samples from 10 cancer types. In selected cancers, top tumor-derived somatic mutations, as ranked by our proprietary algorithm, were used to detect ctDNA in the plasma. Results: For detection of somatic SNVs and Indels, gene fusion and amplification, the assay had sensitivity of >99%, 94% and >99% respectively, and specificity of >99%. Detection of germline variants also achieved sensitivity and specificity of >99%. For TMB measurement, the correlation coefficient between whole-exome sequencing and our targeted panel was 97%. MSI analysis when benchmarked against polymerase chain reaction method showed sensitivity of 94% and specificity of >99%. The concordance between our assay and the TruSight Oncology 500 assay for detection of somatic variants, TMB and MSI measurement was 100%, 89%, and 98% respectively. When CGP-informed mutations were used to personalize ctDNA tracking, the detection rate of ctDNA in liquid biopsy was 79%, and clinical utility in cancer surveillance was demonstrated in 2 case studies. Conclusion: K-4CARE™ assay provides comprehensive and reliable genomic information that fulfills all guideline-based biomarker testing for both targeted therapy and immunotherapy. Integration of ctDNA tracking helps clinicians to further monitor treatment response and ultimately provide well-rounded care to cancer patients.

Occurrence of Eimeria spp. and Intestinal Helminths in Free-Range Chickens from Northwest and Central Romania.

Chickens raised in backyard free-range systems are confronted with a significant threat of parasitic infections. Among the parasitic agents, protozoa belonging to the genus Eimeria and helminths, including Ascaridia galli, Capillaria spp., Heterakis gallinarum, and Strongyloides avium, stand out as the most prevalent. The sampling protocol included sixteen localities in four counties within the Transylvania region of Romania. Fecal samples were collected from chickens reared in a backyard system. Fecal samples were screened for oocysts (O) and eggs (E) by flotation method, and their number per gram of feces (OPG/EPG) was calculated after counting them by McMaster method. Positive samples for Eimeria spp. were further analyzed by PCR (polymerase chain reaction) method to identify the Eimeria species. A total of 145 flocks were tested and the overall prevalence of infection was 53.1%. The most prevalent infections were with A. galli/H. gallinarum (25.5%), and Eimeria spp. (24.8%), followed by Capillaria spp. and strongyles. The mean OPG/EPG values were as follows: 63,577 for Eimeria spp., 157 for Ascaridia/Heterakis, 362 for Capillaria spp., and 1671 for Strongyle eggs. Identified Eimeria species were E. acervulina (41.7%), E. tenella (27.8%), E. praecox (16.7%), E. brunetti (16.7%), OTUy (operational taxonomic unit y) (8.3%), OTUz (operational taxonomic unit z) (8.3%) and E. mitis (5.6%). Intestinal parasites exhibit a high prevalence among chickens in backyard poultry flocks, and the presence of significant parasite burdens can adversely affect both productive and economic aspects. To the best of our knowledge, this is the first comprehensive study that aimed to analyze the prevalence of gastrointestinal parasites in chickens raised in a backyard free-range system in Romania, and the first report of OTUy species in Europe.

PREVALENCE OF BOVINE MASTITIS AND THE FIRST DETECTION OF MYCOPLASMA BOVIS IN VIETNAM’S DAIRY FARMS

Mastitis still remains an economic disease in dairy farms all over the world. This study aimed to report the prevalence of this disease in large-scale farms in Vietnam, the pathogens profile, and first report the detection of Mycoplasma bovis (M. bovis) by applying conventional culture and real-time PCR (polymerase chain reaction) methods. We examined 64,802 enrolled dairy cows from six farms in Vietnam, yielding 1,874 (2.9%) total cases of clinical mastitis. The majority of the 14 pathogens were identified. Klebsiella spp. accounted for 21.0% of the total isolates. Streptococcus uberis (S. uberis), Escherichia coli (E. coli), other Streptococcus spp, and Coagulase-Negative Staphylococci (CNS), followed at 12.6, 7.6, 6.0, and 5.5 percent, respectively. The rest pathogens include Pseudomonas spp., Enterococcus faecalis, Enterobacter spp., Bacillus spp., Citrobacter spp., Pasteurella spp., Proteus spp., and Staphylococcus aureus were isolated with less than 1% each. To the best of our knowledge, this is the first report for detecting M. bovis in mastitis milk samples in Vietnam, where 35 out of 1,422 samples submitted in 2022 were M. bovis positive. Moreover, of the 35 positive cases, 11 cases had only M. bovis, while 24 cases had M. bovis and other pathogens, including 13 Enterobacteriaceae, 7 Streptococcus spp., and 4 CNS.

The Supression of Migration and Metastasis via Inhibition of Vascular Endothelial Growth Factor in Pancreatic Adenocarcinoma Cells Applied Danusertib.

Pancreatic ductal adenocarcinoma is an extremely deadly type of cancer with a high metastatic potential. Genetic factors in cellular events play an important role in the emergence of this situation. One of these factors is Aurora kinase family members, which play a role in migration, invasion, and cell cycle. In this study, the expression of vascular endothelial growth factor gene, which plays a role in migration, metastasis, and angiogenesis, on cystic fibrosis human pancreatic ductal adenocarcinoma 1 cells of danusertib, a pan-Aurora kinase inhibitor, was examined. The half maximal inhibitory concentration (IC50) value (400 nM) of danusertib in cystic fibrosis human pancreatic ductal adenocarcinoma 1 cells was determined by the wound-healing test depending on the dose and time and migration with CIM-Plate 16 in the xCELLingence system. In addition, the effect of danusertib on migration was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) method and vascular endothelial growth factor gene expression. When the dose- and time-dependent danusertib-applied cystic fibrosis human pancreatic ductal adenocarcinoma 1 cells were compared with the control group, it was observed that the wound formed did not close. In the xCELLigence system CIM-Plate 16 migration analysis, it was observed that migration was inhibited in the group administered danusertib in parallel with the wound dehiscence experiment. The gene expressions of vascular endothelial growth factor decreased 0.5-fold at the 24th hour and 0.3-fold at the 48th hour in the Danusertib-administered groups. Danusertib, a pan-Aurora kinase inhibitor, is predicted to be used as a potential agent in pancreatic cancers due to its antitumor and anti-metastatic effect.

Special Real-time PCR and Conventional PCR for Detection of Rhino-Orbital-Cerebral Mucormycosis

Background: Mucormycosis is an invasive fungal infection that occurs in immunodeficiency patients and belongs to infections caused by mucoral fungi, such as Rhizopus and Rhizomucor. Objectives: This study investigated the identification and detection of the mucor fungus in tissue samples and other samples by the real-time polymerase chain reaction (PCR) method. Methods: This was a cross-sectional-descriptive study. A total of 80 tissue samples were collected from referring patients to diagnose fungi with the opinion of a specialist doctor. After extracting deoxyribonucleic acid (DNA) from the samples, PCR and real-time PCR tests were performed using specific primers for mucor and universal-pan and compared to culture. The results were confirmed by sequencing. Results: In this study, 80 samples were examined. In the PCR method, 74 and 75 cases of fungi were confirmed from the DNA obtained from the colonies and tissues, respectively. By using a specific primer for mucor, 12 patients were reported as positive for mucor. Additionally, 76 and 77 cases of fungi were confirmed in the real-time PCR method from the DNA obtained from the colonies and tissues, respectively, and positive cases were reported. By using specific primers and the real-time PCR test for mucor, 16 cases of mucor were reported from colonies, and 15 cases of mucor were reported positive from tissues. Conclusions: This study showed that real-time PCR and PCR are high-speed and more accurate methods than the culture test in identifying its species and have acceptable results in clinical cases.

Features of &lt;i&gt;ESCHERICHIA COLI&lt;/i&gt; samples from patients with diarrheal syndrome in the Republic of Guinea

Introduction. Diarrheal diseases are a global public health issue and cause 15% of deaths in children under 5 years old, of which about 80% occur in the regions of Africa and Southeast Asia. According to the Global Enteric Multicentre Study (GEMS) conducted in a number of African countries, one of the leading pathogens of high risk of death in infants and young children is diarrheagenic E. coli (DEC). In recent decades, antimicrobial resistance (AMR) has become globally ubiquitous. The Republic of Guinea urgently needs large-scale studies devoted to assessing DEC distribution and antibiotic resistance. The purpose of the study is to assess the pattern of E. coli infections and to test the susceptibility to antibiotics in strains of diarrheagenic E. coli sampled from individuals residing in the Republic of Guinea.&#x0D; Materials and methods. From 2019 to 2022, we studied 724 samples of faeces of patients with acute diarrhea, among them 72 (9.9%) children aged 1–5 years, 128 (17.7%) children aged 6–17 years, and 524 (72.4%) people aged 18 years and older; a method of polymerase chain reaction (PCR) was applied with the use of the AmpliSense® Escherichioses-FL reagent kit to identify the genetic determinants of DEC: EPEC, EHEC, ETEC, EIEC, and EAgEC (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia). Susceptibility to 15 antimicrobial agents was found by the disc-diffusion method using Mueller–Hinton agar (Russia) and Oxoid discs (UK). Results were interpreted according EUCAST criteria, versions 2019–2022 (https://www.eucast.org/ast_of_bacteria/previous_versions_of_documents).&#x0D; Results. For the period from 2019 to 2022, the percentage of E. coli infections in the etiological pattern of acute intestinal infections amounted to 51.7%. In the age-related manner, DEC was significantly more common in young children aged 0–5 (96.9%, p 0.05) as compared to school age children aged 6–17 (53.9%) and adults (45.6%). In all years of observation, EAgEC strains prevailed, accounting for 38.4%. Other DEC pathotypes, EPEC, ETEC, EIEC and STEC, accounted for 27.2%, 17.5%, 11.8%, and 5.1%, respectively. DEC strains are susceptible to meropenem, amikacin, and nitrofurantoin. The activity of other antibiotics ranged from 11.3% for ampicillin, 28.3% for trimethoprim-sulfamethoxazole, and 34.0% for tetracycline to 73.6% for cephalosporins, 84.0% for aminoglycosides, and 98.1% for fluorinated quinolones.&#x0D; Conclusion. To reduce the burden of diarrheal diseases in the Republic of Guinea, it may be necessary to conduct targeted epidemiological and microbiological studies to identify DEC and monitor the development of antimicrobial resistance of E. coli infection pathogens in the population.

Bifidobacterium species viability in dairy-based probiotic foods: challenges and innovative approaches for accurate viability determination and monitoring of probiotic functionality.

Bifidobacterium species are essential members of a healthy human gut microbiota. Their presence in the gut is associated with numerous health outcomes such as protection against gastrointestinal tract infections, inflammation, and metabolic diseases. Regular intake of Bifidobacterium in foods is a sustainable way of maintaining the health benefits associated with its use as a probiotic. Owing to their global acceptance, fermented dairy products (particularly yogurt) are considered the ideal probiotic carrier foods. As envisioned in the definition of probiotics as "live organisms," the therapeutic functionalities of Bifidobacterium spp. depend on maintaining their viability in the foods up to the point of consumption. However, sustaining Bifidobacterium spp. viability during the manufacture and shelf-life of fermented dairy products remains challenging. Hence, this paper discusses the significance of viability as a prerequisite for Bifidobacterium spp. probiotic functionality. The paper focuses on the stress factors that influence Bifidobacterium spp. viability during the manufacture and shelf life of yogurt as an archetypical fermented dairy product that is widely accepted as a delivery vehicle for probiotics. It further expounds the Bifidobacterium spp. physiological and genetic stress response mechanisms as well as the methods for viability retention in yogurt, such as microencapsulation, use of oxygen scavenging lactic acid bacterial strains, and stress-protective agents. The report also explores the topic of viability determination as a critical factor in probiotic quality assurance, wherein, the limitations of culture-based enumeration methods, the challenges of species and strain resolution in the presence of lactic acid bacterial starter and probiotic species are discussed. Finally, new developments and potential applications of next-generation viability determination methods such as flow cytometry, propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), next-generation sequencing, and single-cell Raman spectroscopy (SCRS) methods are examined.

Association between Genetic Variants Linked to Premature Ovarian Insufficiency and Inflammatory Markers: A Cross-Sectional Study.

Premature menopause (PM) is the cessation of ovarian function before age 40. PM women are more likely to have cardiovascular diseases (CVDs), diabetes, and mental disorders. This is the first study that assessed the association of single nucleotide polymorphisms (SNPs) with anti-heat shock protein 27 (Hsp27), High-sensitivity C-reactive protein (hs- CRP), and PM and serum pro-oxidant-antioxidant balance (PAB), as putative risk factors for CVDs. We aimed to explore the association of oxidative stress markers with eight different SNPs shown to be related to premature menopause. In this cross-sectional research, we included 183 healthy women and 117 premature menopausal women. We determined baseline characteristics for all participants and measured serum hs-CRP, anti-HSP-27 antibody titer, and PAB levels using the established methods. Genotyping for eight SNPs was done using the tetra amplification refractory mutation system polymerase chain reaction (Tetra-ARMS PCR) and allele-specific oligonucleotide PCR (ASO-PCR) methods. We found a significant difference between mean serum PAB levels and the genetic variant of rs16991615 (P=0.03). ANCOVA showed a significant effect of the genotypes rs4806660 and rs10183486 on hs-CRP serum levels in the case and control groups, respectively (P=0.04 and P=0.007). ANCOVA also showed an association between rs244715 genotypes and anti-hsp27 serum levels in the case group (P=0.02). There was a significant effect of the genotypes of rs451417 on the serum hs-CRP level in the control group (P=0.03). There was a significant association of the genetic variants related to PM with oxidative stress and inflammatory markers (serum PAB, anti-hsp27 antibody, and hs-CRP). Accordingly, this seems to be an effective approach to predicting susceptible subjects for cardiovascular and mental disorders as well as various cancers.

Detecting and genotyping high-risk human papillomavirus among male patients during 2015–2023 in Beijing, China

ABSTRACT Few studies focused on human papillomavirus (HPV) in male patients. This study aimed to explore the detection rate and genotyping of HPV among male patients in Beijing to provide a reference for formulating prevention strategies for HPV infection. The cross-sectional study was conducted in Beijing Chaoyang Hospital from November 2015 to March 2023. It covered male patients from the urology and dermatology departments. Fifteen high-risk HPV genotypes were detected by the multiplex real-time polymerase chain reaction method. The overall detection rate of HPV was 25.19% (1288/5114, 95% confidence interval [CI] 24.00%−26.38%), of which the single infection rate was 16.99% (869/5114, 95% CI 15.97%−18.05%) and the co-infection rate was 8.19% (419/5114, 95% CI 7.46%−8.98%). The detection rate of HPV was 40.77% (521/1278), 35.58% (58/163), 32.69% (101/309), 31.91% (60/188), 12.63% (299/2367), and 32.35% (131/405) among male patients with balanitis, warts, rash, urethritis, prostatitis, and other urinary inflammation, respectively (P < 0.001). The top five HPV genotypes were HPV-52, HPV-58, HPV-16, HPV-51, and HPV-66. After the first positive HPV test, the proportion of male patients who turned negative was 22.47% within 3 months, 26.40% within 3−6 months, 24.72% within 6−12 months, 17.98% within 12−24 months, and 8.43% more than 24 months. The detection rate of HPV was high among male patients from the urology and dermatology departments in Beijing, which should be considered to develop HPV vaccines with better prevention effects.

Mitigation of arsenic poisoning induced oxidative stress and genotoxicity by Ocimum gratissimum L.

Arsenic toxicity is a major problem across the world due to geogenic activity and has been supposed to generate free radicals and genotoxicity among the arsenic-poisoned population. There is a need to find suitable free radical quenching compounds for the arsenic-induced free radical-affected population. In the present study, Na3AsO3- induced oxidative stress and genotoxicity were evaluated in Oryctolagus cuniculus L, and quenching competency of Ocimum species was examined by applying enzymatic and non-enzymatic in vitro tests, comet assay, and Random Amplified Polymorphic Deoxyribonucleic acid – Polymerase Chain Reaction (RAPD-PCR) methods. In the present study, oxidative damage due to Na3AsO3 intoxication in O. cuniculus L has been confirmed followed by substantive genotoxicity, and in a further study, it has also been reported that the extract of O. gratissimum L lowers the oxidative stress in experimental animals confirmed by a decrease in Malondialdehyde (MDA) 4.78 ± 0.05 (nmol/mg protein), and an increase in Glutathione (GSH) 2.87 ± 0.50 (μmoles/mg proteins), Superoxide Dismutase (SOD) 1.78 ± 0.03(Units/mg protein), Catalase (CAT) 2.72 ± 0.02 (μmoles of H2O2 consumed/min/mg proteins) and Glutathione peroxidase (GPX) 7.43 ± 0.01 (μg of glutathione utilized/min/mg protein). A positive impact of extract of O. gratissimum L on protection of genotoxicity has been also confirmed by Random Amplified Polymorphic DNA (RAPD) based reduction in polymorphic bands of Deoxyribonucleic acid (DNA) from 6.5 to 3.16 and comet assay-based increase in head DNA % (87.86 ± 1.58), tail moment (1.07 ± 0.27) and decrease in tail DNA % (12.13 ± 1.58) & tail length (8.2 ± 1.46) at 5% P in lymphocytes. A significant level reduction in free radicals and reduction in DNA polymorphism has proved the competency of test material for the development of suitable antidotes against arsenicosis.

KARYOLOGICAL CHANGES OF BUCCAL EPITHELIOCYTES IN CHILDREN WITH ACUTE RESPIRATORY PATHOLOGY CAUSED BY SARS-COV-2

Pneumonia is one of the leading causes of death in children under 5 years of age in lowincome countries worldwide. This problem became especially acute with the onset ofthe new coronaviral infection caused by SARS-CoV-2. Community- acquired pneumoniais also one of the most common diseases in children, which requires extensive use ofantibiotics and admission to pediatric clinics. That is why diagnosis of the infectiousinflammatory process activity and prediction of the disease severity is the main factorin optimizing the treatment of children who suffer from infectious respiratory pathology.Objective – to improve the prediction of the severe course of acute respiratory pathologycaused by the SARS-CoV-2 virus by analyzing the results of kariological indicators ofexfoliative cells of the buccal epithelium in children of different ages. Materials and methods. 63 children with acute respiratory pathology caused by theSARS-CoV-2 virus (average age – 10.6±0.5 years) were comprehensively examinedby the method of simple random sampling using the «experiment- control» method.The verification of the etiological factor of inflammation of the respiratory tract wascarried out by the method of polymerase chain reaction with reverse transcriptase(a set of COVID-19 Multiplex RT-PCR in real time from Labsystems Diagnostics Oy) ina nasopharyngeal swab and/or lung expiratory tissue.Fixed smears- imprints of exfoliative cells of the buccal epithelium were stained with azureeosin according to Romanovsky- Giemsa. By light microscopy of cytological samples,changed cells with nuclear abnormalities were evaluated and the number of cells withmicronuclei and karyological indicators of exfoliative cells of the buccal epithelium per100 cells in the preparation were determined.Results.Pathological changes of buccal epithelium exfoliative cells as protrusion, karyopyknosis,karyorrhexis and apoptosis are directly proportional to the disease severity and reflect exclusivelytoxic effects. Identification of the morphological changes expressiveness in the buccal epitheliumcells can be used as a diagnostic factor in prediction of the severe disease, and accordingly,serves as an impetus in optimization of the conducted treatment strategy.Conclusions. A greater number of pathological cells in smears- imprints of the buccalepithelium, and, accordingly, slightly higher cytogenetic indices are determined in patientswith pneumonia and multisystem inflammatory syndrome associated with SARS-CoV-2, whohad a severe and extremely severe course of the disease. Significantly fewer pathologicallychanged cells are found in patients with acute respiratory tract infection of multiplelocalization and bronchitis, who mostly had a medium- severe course of the disease, whichcan be used to predict the severity of the infectious- inflammatory process in children.

Clinical importance of serum miRNA levels in breast cancer patients

There is limited data on the relationship of miRNAs with parameters that may affect surgical management or reflect tumour prognosis. It was aimed to evaluate serum miRNA levels in breast carcinoma cases and reveal the relationship between these levels and prognosis-related factors such as the histological type of the tumour, estrogen receptor, progesterone receptor, Ki-67 index, HER-2neu, E-cadherin, tumour size, CK5/6, CA15.3 levels, number of tumour foci, number of metastatic lymph nodes, and status of receiving neoadjuvant therapy. Thirty-five patients with a histopathologically confirmed breast carcinoma diagnosis in the case group and 35 healthy individuals in the control group were examined. miR-206, miR-17-5p, miR-125a, miR-125b, miR-200a, Let-7a, miR-34a, miR-31, miR-21, miR-155, miR-10b, miR-373, miR-520c, miR-210, miR-145, miR-139-5p, miR-195, miR-99a, miR-497 and miR-205 expression levels in the serum of participants were determined using the Polymerase Chain Reaction method. While serum miR-125b and Let-7a expression levels were significantly higher in breast cancer patients, miR-17-5p, miR-125a, miR-200a, miR-34a, miR-21, miR-99a and miR-497 levels were significantly lower in them. The Let-7a expression level had a statistically significant relationship with breast cancer histological type and HER-2neu parameters, miR-17-5p, miR-125b, Let-7a, miR-34a, miR-21 and miR-99a levels with E-cadherin, miR-34a, miR-99a and miR-497 with CA15.3, miR-125b, miR-200a and miR-34a with the number of metastatic lymph nodes, miR-125a with the number of tumour foci and miR-200a with the status of having the neoadjuvant therapy. Serum miR-17-5p, miR-125a, miR-125b, miR-200a, Let-7a, miR-34a, miR-21, miR-99a and miR-497 expression levels were determined to have predictive and prognostic importance in breast cancer.

Evaluation of virus removal in membrane bioreactor (MBR) and conventional activated sludge (CAS) processes based on long-term monitoring at two wastewater treatment plants

The membrane bioreactor (MBR) process always offers better wastewater treatment than conventional activated sludge (CAS) treatment. However, the difference in their efficacy of virus reduction remains unknown. To investigate this, we monitored virus concentrations before and after MBR and CAS processes over 2 years. Concentrations of norovirus genotypes I and II (NoV GI and GII), aichivirus (AiV), F-specific RNA phage genotypes I, II, and III (GI-, GII-, and GIII-FRNAPHs), and pepper mild mottle virus (PMMoV) were measured by a quantitative polymerase chain reaction (qPCR) method at two municipal wastewater treatment plants (WWTPs A and B) in Japan. Virus concentration datasets containing left-censored data were estimated by using both maximum likelihood estimation (MLE) and robust regression on order statistics (rROS) approaches. PMMoV was the most prevalent at both WWTPs, with median concentrations of 7.5 to 8.8 log10 copies/L before treatment. Log10 removal values (LRVs) of all viruses based on means and standard deviations of concentrations before and after treatment were consistently higher following MBR than following CAS. We used NoV GII as a model pathogen in a quantitative microbial risk assessment of the treated water, and we estimated the additional reductions required following MBR and CAS processes to meet the guideline of 10−6 DALYs pppy for safe wastewater reuse.

Key Contributions of the Overexpressed Plutella xylostella Sigma Glutathione S-Transferase 1 Gene (PxGSTs1) in the Resistance Evolution to Multiple Insecticides.

The overexpression of insect detoxification enzymes is a typical adaptive evolutionary strategy for insects to cope with insecticide pressure. In this study, we identified a glutathione S-transferase (GST) gene, PxGSTs1, that exhibited pronounced expression in the field-resistant population of Plutella xylostella. By using RNAi (RNA interference), the transgenic fly models, and quantitative real-time polymerase chain reaction (RT-qPCR) methods, we confirmed that the augmented expression of PxGSTs1 mediates the resistance of P. xylostella to various types of insecticides, including chlorantraniliprole, novaluron, λ-cyhalothrin, and abamectin. PxGSTs1 was found to bolster insecticide resistance in two ways: direct detoxification and enhancing antioxidative defenses. In addition, our findings demonstrated that pxy-miR-8528a exerts a pivotal influence on forming insecticide resistance in P. xylostella by downregulating PxGSTs1 expression. In summary, we elucidated the multifaceted molecular and biochemical underpinnings of PxGSTs1-driven insecticide resistance in P. xylostella. Our results provide a new perspective for understanding the insecticide resistance mechanism of P. xylostella.