Polymerase Chain Research Articles

A Cross-Sectional Study of the Use of Antigen Rapid Diagnostic Tests for Community Identification of Severe Acute Respiratory Syndrome Coronavirus-2 in Kenya.

Mass testing with antigen-detecting rapid diagnostic tests (Ag-RDT), including testing of asymptomatic individuals, is expected to improve the identification of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections. Mass testing was offered at large gatherings to determine the SARS-CoV-2 case detection rate and the acceptance and cost of implementing this community testing strategy. In 49 high-attendance venues in Kiambu County, Kenya, from June to September 2022, individuals 2 years and older were offered coronavirus disease 2019 (COVID-19) testing, vaccination, and participation in a survey. Polymerase chain reaction (PCR) testing and genome sequencing were conducted for those testing positive by Ag-RDT and those testing negative but with COVID-19 symptoms. Costs were collected from financial records, budgets, and invoices and estimated from a health systems perspective using a micro-costing method. A total of 4,062 individuals were offered testing. The testing acceptance was 3,174/4,062 (78.1%). The case detection rate was 34/3,174 (1.07%; 95% CI: 0.7-1.4%), and 11/34 (32%) of the positives were asymptomatic. The PCR results were available for 27 Ag-RDT‒positive participants and 14 Ag-RDT‒negative participants with SARS-CoV-2 symptoms and were positive in 24/27 (88.9%) and 4/14 (28.6%), respectively. Circulating variants were identified in 11 participants. Community mobilization was the major cost driver (26%) followed by purchase of SARS-CoV-2 Ag-RDTs (20.5%). The cost per individual tested was USD $15.89, and the cost per individual tested positive for SARS-CoV-2 was USD $1,484. The study demonstrates that SARS-CoV-2 Ag-RDTs could be used for identification of SARS-CoV-2 infections in both symptomatic and asymptomatic individuals at mass gatherings.

Positive impact of a diagnostic stewardship intervention on syndromic panel ordering practices and inappropriate C. difficile treatment.

Multiplex polymerase chain reaction (PCR) panels for stool testing may be used to diagnose Clostridioides difficile, which can circumvent more appropriate targeted C. difficile testing, resulting in treatment of incidentally detected colonization. We sought to reduce C. difficile diagnosis via a gastrointestinal pathogen panel (GIPP). Quasi-experimental, pre/post, retrospective cohort study from January 1, 2022, to January 31, 2024. Mayo Clinic Arizona-a single academic medical center and associated clinics. Adult patients receiving C. difficile testing and/or treatment. Preferred C. difficile testing consisted of glutamate dehydrogenase and toxin antigen immunoassay, followed by toxin gene testing for discrepant results. The GIPP contained 22 targets during the baseline period with C. difficile removed during the postintervention period. Surveys were provided to provider and nursing groups, separately, to identify C. difficile ordering practices and knowledge gaps. At baseline, from January 1, 2022, to January 31, 2023, 2,772 GIPPs were completed for 2,307 unique patients (∼7 per day), primarily for outpatients (1,805 of 2,772, 65%). The most common positive target was C. difficile (517 of 1,018, 51%), which resulted in treatment for C. difficile infection in 94.9% (337 of 355) of cases. Following GIPP C. difficile target removal, GIPP orders decreased from 3.23 to 2.7 per 1,000 patient visits (P < .001). Prescribing of C. difficile treatments decreased in the postintervention period in inpatient and outpatient settings. There were no cases of delayed C. difficile diagnosis during the postintervention period. Removing C. difficile from the GIPP resulted in effective diagnostic and antimicrobial stewardship without resulting in delayed diagnoses.

Single‐cell analyses reveal impaired type B spermatogonia differentiation and meiotic entry in C‐Nap1‐null testes

AbstractSperm development is critical for male reproductive capability; any disruption during the process of spermatogenesis will result in male infertility. In this research, we used the C‐Nap1 encoded by the gene of Cep250 knockout mouse line as the model to evaluate the impact of absent C‐Nap1 on spermatogenesis. To investigate the interaction between C‐Nap1 and spermatogenesis, we utilized single‐cell RNA sequencing to analyze 10,332 C‐Nap1+/+ and 13,308 C‐Nap1−/− testicular cells. We identified five main cell types within seminiferous tubules, including spermatogonia, Sertoli cells, spermatogonia stem cells, Leydig cells, and spermatocytes. We found a critical reduction in testicular spermatogonia and spermatocytes in C‐Nap1‐null testes, compared to its C‐Nap1+/+ controls. By combining uniform manifold approximation and projection clustering and psedotime ordering, we distinguished five spermatogonial stages/subtypes, demonstrating that type B spermatogonia differentiation and meiotic initiation are impaired during C‐Nap1‐null spermatogenesis. Following gene ontology enrichment analysis, meiosis‐specific genes downregulated in the C‐Nap1−/− testicular cells were further verified by reverse transcription polymerase chain reaction (RT‐PCR). Based on the differential gene expression, certain downregulated genes such as Ctnnb1 and Aurka encoding C‐Nap1‐binding potential β‐Catenin and Aurka are encountered, which may account for defective type B spermatogonia differentiation and meiotic entry in C‐Nap1‐null testes.

Identification of potential key circular RNAs associated with Escherichia coli-infected bovine mastitis using RNA-sequencing: preliminary study results.

Escherichia coli (E. coli) is commonly found in dairy farms and can invade mammary gland tissue, often causing acute clinical mastitis. Mammary infections with E. coli have shown a wide range of clinical signs, causing abnormal appearance of the milk, udder inflammation and systemic signs of illness. Circular RNA is a class of endogenous non-coding RNA that plays an important role in the occurrence and development of various inflammatory diseases. However, there is little information on the circRNA associated with bovine mastitis. In this study investigated the involvement of circRNAs in bovine mastitis through the construction of an E. coli-infected bovine mastitis model by injecting of E. coli into the mammary gland of dairy cows, using healthy gland mammary tissue as a control (M_C). High-throughput RNA-seq was performed on the E. coli-infected mammary gland tissue (M_E) and differentially expressed circRNAs between theM_C and M_E groups, followed by an analysis of their potential functions using bioinformatics methods. A total of 164 differentially expressed circRNAs were identified, including 92 downregulated circRNAs and 72 upregulated circRNAs. As shown by Gene Ontology enrichment analysis these DE circRNAs were mostly enriched in ras protein signal transduction, cytoplasmic vesicle parts, and enzyme binding, and Kyoto Encyclopedia of Genes and genome singal pathway enrichment analysis indicated significant associations with phagosome signal pathway. Additionally, the expression of bovine mastitis-related circRNAs, including novel_circRNA_0000128, novel_circRNA_0011103, novel_circRNA_0012656, novel_circRNA_0015099, novel_circRNA_005648, novel_circRNA_000074, and novel_circRNA_0011796 were verified via quantitative reverse-transcription polymerase chain reaction (RT-qPCR). These results provide a new direction for further investigation of the molecular mechanisms underlying bovine mastitis.

The role of Toll-like receptor 4 Asp299Gly genetic polymorphism in rehabilitation of patients with post-COVID syndrome

Introduction. The multivariability of the clinical course of post-covid syndrome (PCS) is associated with genetic heterogeneity of the population, which leads to a high interest in the study of genotypes. Aim. To evaluate the role of Toll-like receptor 4 (TLR4 (Asp229Gly) polymorphism in the formation of PCS and rehabilitation potential. Materials and methods. The study included 92 patients diagnosed with PCS between the ages of 21 and 75. All patients received a comprehensive clinical, laboratory, functional examination and quality of life assessment at admission and discharge. Deoxyribonucleic acid isolation and allele-specific polymerase chain reaction were performed from whole blood using «Lytech» kits (Russia) according to the manufacturer's instructions. Formation of groups for statistical processing was carried out in accordance with the results of genetic analysis. Patients received comprehensive rehabilitation, including: climatotherapy, exercise therapy, massage, aromatherapy, respiratory therapy. Performance was assessed based on the dynamics of the studied indicators. Results and discussion. Of the 92 patients included in the study, 22 (23.9 %) were AA homozygotes, 33 (35.8 %) were GG, and 37 (40.3 %) were AG heterozygotes. By age, the groups were comparable. The gender ratio was predominantly female in all groups. According to the initial characteristics, the condition of patients with more pronounced clinical symptoms was in the group of patients with the AG genotype. All groups showed varying degrees of positive dynamics. The dependence of the severity of PCS and the effectiveness of rehabilitation on genetic predisposition was established. Different severity of initial clinical symptoms and deviations of functional parameters were revealed. Significant differences were found when assessing the effectiveness of rehabilitation. Positive dynamics was observed in all groups, but most successfully in the AG group. This confirms the role of TLR4 in the immunological response to SARS-CoV-2 and its genetic polymorphism in a wide range of manifestations of both acute COVID-19 and PCS. Conclusions. The effectiveness of rehabilitation in patients with PCS is interrelated with the genetic heterogeneity of TLR4. The heterozygous variant of the AG genotype forms more pronounced clinical manifestations of PCS, but also a higher rehabilitation potential, which contributes to the successful completion of rehabilitation. The minor allele TLR4 (Asp299Gly) G is associated with a worsening of the course of PCS. Patients with the homozygous GG genotype need a longer course of rehabilitation.

PCBP2 stabilizes TROAP to promote the malignant progression of gastric cancer.

Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Poly(rC) binding protein 2 (PCBP2), an RNA binding protein, has been reported to be involved in the progression of various cancers. However, the role of PCBP2 in GC and its underlying mechanisms remain elusive. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect mRNA expression, whereas western blotting and immunohistochemistry assays were used to detect protein expression levels. Cell proliferation, migration, and invasion were assessed by EdU assay, wound-healing assay, and transwell assay, respectively. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and iron (Fe2+) were detected using commercial assay kits. RIP assay was conducted to analyze the relationship between PCBP2 and trophinin-associated protein (TROAP). Actinomycin D assay was performed to investigate the effect of PCBP2 depletion on TROAP mRNA stabilization. Finally, a subcutaneous xenograft mouse model was established to validate the effect of PCBP2 depletion on tumor formation. PCBP2 expression was upregulated in GC tissues and cells, and its depletion inhibited GC cell proliferation, migration, and invasion. Additionally, PCBP2 depletion promoted ferroptosis, a novel form of cell death mediated by iron. TROAP expression was also upregulated in GC tissues and cells, and PCBP2 stabilized TROAP mRNA expression in GC cells. Knocking down PCBP2 regulated GC cell proliferation, migration, invasion, and ferroptosis by reducing TROAP expression. Furthermore, PCBP2 knockdown repressed aggressive behavior of GC cells in vivo. Our study demonstrated that PCBP2 stabilized TROAP to promote the malignant progression of gastric cancer. Targeting PCBP2 and its downstream target TROAP may provide a novel therapeutic strategy for the treatment of gastric cancer.

The miR-1269a/PCDHGA9/CXCR4/β-catenin pathway promotes colorectal cancer invasion and metastasis.

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related death. This research focuses on investigating the impact and underlying molecular mechanisms of protocadherin gamma subfamily A, 9 (PCDHGA9) on the invasion and metastasis of CRC, aiming to identify more precise molecular markers for the diagnosis and prognosis of CRC. PCDHGA9 expression was detected using quantitative real-time quantitative polymerase chain reaction (RT-qPCR) in 63 pairs of colorectal cancer tissues. Differential gene expression from high-throughput sequencing was analyzed using ingenuity pathway analysis (IPA) to explore the biological functions of PCDHGA9 and its potential regulated genes. Bioinformatics tools were employed to explore potential upstream regulatory microRNAs of PCDHGA9. Dual-luciferase assays were performed to demonstrate the regulation between PCDHGA9 and miR-1269a. Protein mass spectrometry suggested an interaction between PCDHGA9 and HOXA1. JASPAR predicted that HOXA1 may act as a transcription factor of CXCR4. Coimmunoprecipitation, dual-luciferase assays, and nuclear-cytoplasmic fractionation experiments confirmed the molecular mechanism involving PCDHGA9, CXCR4, HOXA1, and β-catenin. Transwell, wound healing, and western blot assays were conducted to confirm the impact of PCDHGA9, miR-1269a, and CXCR4 on the invasion, metastasis, and epithelial-mesenchymal transition (EMT) functions of CRC cells in in vitro experiments. A whole-body fluorescence imaging system was used to evaluate the combined impact of miR-1269a and PCDHGA9 on the invasion and metastasis of CRC in in vivo experiments. The expression of PCDHGA9 was found to be lower in CRC tissues compared with their corresponding adjacent tissues. Low expression of PCDHGA9 potentially correlated with worse prognosis and increased chances of invasion and metastasis in CRC. miR-1269a was highly expressed in CRC tissues and acted as a negative regulator for PCDHGA9, promoting invasion, migration, and EMT of CRC cells. PCDHGA9's interaction with HOXA1 downregulated CXCR4, a transcription factor, leading to accumulation of β-catenin and further promoting invasion, migration, and EMT of CRC cells. PCDHGA9, acting as a tumor suppressor, is downregulated by miR-1269a. The low level of PCDHGA9 activates the Wnt/β-catenin pathway by releasing its interaction with HOXA1, promoting the expression of CXCR4, and causing invasion, migration, and EMT in CRC.

ELAVL1-dependent SOAT2 exacerbated the pancreatitis-like cellular injury of AR42J cells induced by hyperstimulation with caerulein.

Pancreatitis is a severe inflammatory condition characterized by damage to the pancreas. Sterol o-acyltransferase 2 (SOAT2) has been reported to aggravate acute pancreatitis, however, the underlying mechanism remains to be elucidated. Rat pancreatic exocrine cells (AR42J) were treated with caerulein to induce pancreatitis-like cellular injury. Cell viability was determined using a cell counting kit-8 (CCK-8) assay, while cell proliferation was analyzed through a 5-Ethynyl-2'-deoxyuridine assay. Cell apoptosis was measured using flow cytometry, and enzyme-linked immunosorbent assays were performed to detect levels of pro-inflammatory cytokines IL-6 and TNF-α. Additionally, Fe2+ levels were analyzed using a colorimetric assay kit, reactive oxygen species (ROS) levels were assessed with a Cellular ROS Assay kit, and lipid peroxidation was measured using a malondialdehyde assay kit. Glutathione levels were analyzed with a detection assay. Protein and mRNA expression were evaluated through western blotting and quantitative real-time polymerase chain reaction, respectively. Furthermore, an RNA immunoprecipitation assay was conducted to investigate the association between ELAV-like RNA binding protein 1 (ELAVL1) and SOAT2. Actinomycin D assay was performed to explore the effect of ELAVL1 depletion on the transcript stability of SOAT2 mRNA. SOAT2 and ELAVL1 expression were upregulated in caerulein-exposed AR42J cells. Caerulein treatment induced pancreatitis-like cellular apoptosis, inflammatory response, ferroptosis, and cell proliferation inhibition. Silencing of SOAT2 protected against caerulein-induced AR42J cell injury. Moreover, ELAVL1 stabilized SOAT2 mRNA expression in AR42J cells. SOAT2 overexpression attenuated the effects induced by ELAVL1 silencing in caerulein-exposed AR42J cells. Additionally, ELAVL1 knockdown activated the NRF2/HO-1 pathway by downregulating SOAT2 expression in caerulein-exposed AR42J cells. SOAT2 silencing protected AR42J cells from caerulein-induced injury by inactivating the NRF2 pathway. In conclusion, ELAVL1-dependent SOAT2 exacerbated pancreatic exocrine cell injury by inactivating the NRF2/HO-1 pathway in pancreatitis. These findings provide new insights into the molecular mechanisms underlying pancreatitis and offer potential therapeutic targets for the treatment of this condition.

One-Step Multiplex Polymerase Chain Reaction Assay for the Detection of Major Disease-Transmitting Mosquito Vectors in India.

Mosquitoes are important vectors that transmit viral, protozoan, and helminthic diseases across the world. Climate change and unplanned urbanization are accelerating the spread of these diseases. Controlling vector-borne diseases can be performed most effectively through vector control. Inadequate knowledge of vector bionomics is an impediment and can lead to inappropriate vector control efforts. However, the conventional methods of vector identification are based on morphological differences, demand a significant amount of time and specific skills, and are often misleading. An efficient and affordable solution is needed to quickly and accurately identify pooled samples from vast geographical territories. To ensure the correct identification of distorted or pooled samples in India, a set of definitive steps is required, including the construction of unique primers and the standardization of a one-step assay based on the second internal transcribed spacer gene of the ribosomal DNA. We have successfully developed and confirmed a highly efficient one-step multiplex reverse transcriptase polymerase chain reaction assay for the accurate identification of major mosquito vectors, especially in the cases of both the adult and larval forms of Anopheles sp., Aedes sp., and Culex sp. Hence, the specificity, universality, and uniqueness of these primers could serve as a critical tool for the rapid one-step and one-reaction identification of mosquitoes to control mosquito-borne disease outbreaks and public health emergencies.

Comparative Diagnosis of Schistosomiasis by Standard and Molecular Methods in Turan District of Kwande Local Government Area, Benue State, Nigeria

The study was carried out to compare the diagnosis of schistosomiasis by standard and molecular methods in the Turan District of Kwande LGA. A total of 682 samples were collected, 482 urine and 200 stool samples. The urine samples were filtered on Whatman filter No.3 paper for DNA extraction and amplification. The urine samples were also examined microscopically using centrifugation and the Kato-Katz method. Forty (40) samples of the filtered urine were randomly picked irrespective of being positive or negative by microscopy and amplified by Polymerase Chain Reaction (PCR). Chi-square and Fisher’s exact test were applied to determine the association of infection in relation to location and water contact activity. The Kappa coefficient was also applied to assess the degree of concordance between the diagnostic techniques. From the 482 urine samples analyzed, 111(23.0%) were infected with S. haematobium; and out of the 200 stool samples examined, 3(1.5%) were infected with S. mansoni. In comparison PCR established a prevalence of 12 (30.0%) for S. haematobium and 6 (15%) for S. mansoni. PCR also detected 3 (7.5%) co-infections with S. mansoni and S. haematobium. Concordance between centrifugation and PCR revealed an almost perfect agreement (κ = 0.81) while Kato-Katz and PCR showed a substantial agreement (κ = 0.63). Prevalence of urogenital schistosomiasis by location revealed the highest infection rate of 32 (29.4%) in Yaav, followed by Kumakwagh with prevalence of 31(24.8%) and lowest infection rate of 27 (20.4%) was observed in Mbadura. However, no significant difference was observed in the prevalence of urinary schistosomiasis between the different communities (χ² = 4.68, p = 0.20). The study revealed that detection of Schistosoma parasite-specific DNA in urine by PCR is an effective tool for the diagnosis of schistosomiasis than processing urine and stool by standard methods

Comparative Diagnosis of Schistosomiasis by Standard and Molecular Methods in Turan District of Kwande Local Government Area, Benue State, Nigeria

The study was carried out to compare the diagnosis of schistosomiasis by standard and molecular methods in the Turan District of Kwande LGA. A total of 682 samples were collected, 482 urine and 200 stool samples. The urine samples were filtered on Whatman filter No.3 paper for DNA extraction and amplification. The urine samples were also examined microscopically using centrifugation and the Kato-Katz method. Forty (40) samples of the filtered urine were randomly picked irrespective of being positive or negative by microscopy and amplified by Polymerase Chain Reaction (PCR). Chi-square and Fisher’s exact test were applied to determine the association of infection in relation to location and water contact activity. The Kappa coefficient was also applied to assess the degree of concordance between the diagnostic techniques. From the 482 urine samples analyzed, 111(23.0%) were infected with S. haematobium; and out of the 200 stool samples examined, 3(1.5%) were infected with S. mansoni. In comparison PCR established a prevalence of 12 (30.0%) for S. haematobium and 6 (15%) for S. mansoni. PCR also detected 3 (7.5%) co-infections with S. mansoni and S. haematobium. Concordance between centrifugation and PCR revealed an almost perfect agreement (κ = 0.81) while Kato-Katz and PCR showed a substantial agreement (κ = 0.63). Prevalence of urogenital schistosomiasis by location revealed the highest infection rate of 32 (29.4%) in Yaav, followed by Kumakwagh with prevalence of 31(24.8%) and lowest infection rate of 27 (20.4%) was observed in Mbadura. However, no significant difference was observed in the prevalence of urinary schistosomiasis between the different communities (χ² = 4.68, p = 0.20). The study revealed that detection of Schistosoma parasite-specific DNA in urine by PCR is an effective tool for the diagnosis of schistosomiasis than processing urine and stool by standard methods

Molecular Detection of Integrons I and II in Some Clinical Isolates from Niger Delta University Teaching Hospital, Okolobiri, Nigeria

Aim: This study aimed at detecting the integron-integrase I and II gene in Gram negative bacteria. Study Design: Cross-sectional and descriptive study design were utilized in this study. Place and Duration of Study: Niger Delta University Teaching Hospital Okolobiri and Niger Delta University Molecular Laboratory CHS, Wilberforce Island, Amassoma, between February 2023 and April 2023. Methodology: This study was specifically carried out on clinical isolates with a population figure obtained from 6 different samples collected from Niger Delta University Teaching Hospital, okolobiri in Bayelsa state. Of 500 different clinical samples, 100 clinical isolates were isolated and identified using Standard Bacteriological methods. Genomic DNA was extracted by boiling method. The Integrons I and II genes were detected using Polymerase Chain Reaction. Results: Of the 100 isolates used for molecular analysis, the Int I1 gene was detected in 54 isolates of which 8(44.4%) were Escherichia coli,14(63.63%) were Pseudomonas aeruginosa and 14(70%) were Klebsiella oxytoca. IntI2 gene was detected in 78 isolates of which16(88.88%) were Escherichia coli,20(90.90%) was Pseudomonas aeruginosa and 10(50%) were Klebsiella oxytoca. IntI1 and IntI2 genes were detected in 38 isolates of which9(50%) were Escherichia coli,12(54.54%) was Pseudomonas aeruginosa and 6(30%) were Klebsiella oxytoca. Conclusion: This study showed that there was a high prevalence of class I and II integrons in multi-drug-resistant Gram-negative bacteria, therefore justifying the extent of resistance observed.

Angiotensin-converting enzyme insertion/deletion polymorphism as a potential risk factor of congenital heart disease: insights from a tertiary pediatric cardiac care centre from North India

Background: Our study aims to elucidate the genetic influence of angiotensin-converting enzyme insertion/deletion (ACE I/D) polymorphism on congenital heart disease (CHD) in a north Indian cohort. Methods: 667 CHD cases, including 433 individuals with parental data and 104 controls were enrolled and genotyped by polymerase chain reaction. Case-control association, parental transmission test, and association of patients' and parents' clinical parameters with ACE I/D were explored. Results: Our findings highlight significant associations, notably the increased CHD risk conferred by the DD genotype in females (p=0.036; OR=1.68), its correlation with abnormal hemoglobin levels (p=0.049; OR=1.68), and its impact on primigravida (p=0.05). Conversely, the II genotype was found to significantly elevate the risk of CHD in offspring of tobacco-consuming fathers by 2.5-fold (p=0.029). Notably, cyanotic cases exhibited a heightened prevalence of ACE I/D mutations (p=0.059), with tetralogy of Fallot (TOF) showing the strongest association (p=0.024). Additionally, the DD genotype's involvement in conditions such as stenosis (p=0.026) and pulmonary artery hypertension (PAH) (p=0.05) underscores its clinical relevance. The parent of origin test showed maternal transmission of the D allele in combined (p=0.037) and acyanotic cases (p=0.039) and paternal transmission in ventricular septal defect (p=0.021). Conclusions: This is the first study from India and possibly the only study globally that reports a significant association between ACE I/D and CHD, highlighting the importance of genetic factors in CHD susceptibility.

Epidemiology and Molecular Characterization of Entamoeba spp. in Non-Human Primates in Zoos in China

The genus Entamoeba infects both humans and NHPs. In zoos, visitors feeding significantly increases the frequency of human-to-NHP contact, thereby raising the risk of zoonotic transmission. In this study, six Entamoeba species were investigated and analyzed in the fecal samples of 14 NHP species from zoos in Beijing, Guiyang, Shijiazhuang, Tangshan, and Xingtai in China. A total of 19 out of 84 primate fecal samples tested positive for Entamoeba spp. by polymerase chain reaction (PCR). Among these, 14 samples contained mono-detections of E. coli (7/84), E. dispar (4/84), and E. polecki (3/84). Five samples were found to have mixed detections with two or three species, suggesting the potential for zoonotic transmission; however, no pathogenic E. histolytica, E. moshkovskii, or E. nuttalli were detected. This study provides new insights into parasitic detections in NHPs in Chinese zoos and offers valuable background information for the prevention and control of zoonotic parasitic diseases.

Crispr-Cas9-based long non-coding RNA interference and activation identified that the aberrant expression of Myc-regulated ST8SIA6 antisense RNA 1 promotes tumorigenesis and metastasis in hepatocellular carcinoma

Objective: Long non-coding RNAs (lncRNAs) participate in the formation, progression, and metastasis of cancer. This study aimed to explore the roles of the lncRNA ST8SIA6 antisense RNA 1 (ST8SIA6-AS1) in tumorigenesis and elucidate the underlying molecular mechanism of its upregulation in hepatocellular carcinoma (HCC). Material and Methods: A total of 56 in-house pairs of HCC tissues were examined, and ST8SIA6-AS1 levels were determined through real-time polymerase chain reaction (PCR). The biological behavior of ST8SIA6-AS1 by Crispr-Cas9-based gene repression and activation was determined in vitro and in vivo. The binding sites and biological behavior of Myc proto-oncogene and forkhead box A on chromatin were investigated through luciferase reporter assays, chromatin immunoprecipitation–quantitative PCR, and co-immunoprecipitation (co-IP) assays. The regulatory mechanisms of ST8SIA6-AS1 expression were analyzed with encyclopedia of DNA elements and gene expression profiling interactive analysis. Results: The expression of ST8SIA6-AS1 significantly increased in multiple HCC cell lines and the 56 in-house pairs of HCC tissues (P = 0.0018). Functionally, high-efficiency Crispr-Cas9-based knockdown of ST8SIA6-AS1 revealed that ST8SIA6-AS1 knockdown attenuated the proliferation, migration, and infiltration of HCC cells and considerably reduced the growth rate of subcutaneous and orthotopic HCC tumors. Conversely, ST8SIA6-AS1 overexpression considerably improved the oncogenic characteristics of the HCC cells. Furthermore, ST8SIA6-AS1 upregulation was regulated by the direct binding of transcription factor Myc to the −260 bp to+155 bp and +1003 bp to +1312 bp regions of the ST8SIA6-AS1 transcription start site, which is a segment with high level of H3K27 acetylation. Myc knockdown or treatment with the BET bromodomain inhibitor JQ-1 considerably reduced ST8SIA6-AS1 RNA expression in the HCC cells. Conclusion: Our study has established the oncogenic role of ST8SIA6-AS1 and elucidated the Myc-dependent upregulation mechanism of ST8SIA6-AS1 in HCC, providing a profound theoretical molecular basis for the carcinogenic function of ST8SIA6-AS1 in HCC.

A simple colorimetric detection of telomerase exploiting specific cleavage of exonuclease III coupled with telomeric DNA controlled aggregation of nanogold.

Telomerase activity has piqued scientists' interest for the reason that it has the potential to be employed for early-stage cancer detection, anticancer therapy and studies related to cancer progression and metastasis. Several approaches have been developed to detect telomerase activity. However, these approaches were lengthy, challenging to quantify, of limited sensitivity and prone to polymerase chain reaction (PCR)-related artefacts. We herein developed a novel nanoplasmonic sensing platform for colorimetric detection of telomerase activity relying on the telomere elongation of telomerase at the 3' end, structure-specific cleavage activity of exonuclease III that removes mononucleotides from the 3'-hydroxyl termini of double-stranded DNA, and electrostatic interaction of elongated telomeres with plasmonic nanoparticles. Using HeLa cells as a model for colorimetric detection of telomerase activity, this biosensor could detect telomerase activity with a detection limit of ∼100 cells per reaction by visible inspection and ∼5 cells per reaction by spectroscopic measurement and analysis time within about three hours. The proposed sensing method provided a novel tool for simple, rapid, and low-cost detection of telomerase activity, eliminating the necessity for thermal cycling, primers in PCR-based assays, and amplification of telomerase extension products. It exhibits significant potential as a label-free, simple, ultrasensitive strategy for on-site detection of telomerase activity in proteomics and clinical diagnostics.

Identification of stem melanosis of wheat (Pseudomonas cichorii) by real-time PCR

The phytopathogenic bacterium, Pseudomonas cichorii, affects a wide range of crops in filed and greenhouse production and causes wheat stem melanosis, which has been regulated by importers of Russian grain products. It is crucial to update the identification method based on real-time polymerase chain reaction (PCR) (PCR-RT). This method helps confirming P. cichorii in wheat samples. A 90 base pair long section of the hrcRST pathogenicity gene cluster was used as the target. Positive results were obtained for reference strains, confirmed by amplicon sequencing. Nucleotide sequences were then compared to the typical strain, DSM 50259. As a result of comparative DNA analysis, the sequences of the direct primer and probe were modified. The possibility of using a modification of 6FAM/BHQ1 dye/quencher available on the territory of the Russian Federation was demonstrated. The specificity of the new PCR-RT primer system PscF/PscHrc751R/PscP1 was assessed using 107 bacterial strains of the genus Pseudomonas, including P. cichorii, P. fuscovaginae, P. syringae, P. trivialis, P. viridiflava, P. chlororaphis, P. lutea and P. orientalis. The DNA of 4 strains (P. poae, P. graminis and 2 strains of P. fluorescens) showed a non-specific reaction at the 35–37 cycle threshold, with an accumulation that did not appear exponential. The analytical sensitivity of the test allows for the detection of P. cichorii at a concentration of 10^1 CFU/mL. The PCR-RV PscF/PscHrc751R/PscP1 test can be used as a screening tool for the detection of P. cichorii in plant products and for the characterization of pure bacterial cultures.

Delayed Sowing Reduced Verticillium Wilt by Altering Soil Temperature and Humidity to Enhance Beneficial Rhizosphere Bacteria of Sunflower

Sunflower Verticillium Wilt (SVW) caused by Verticillium dahliae is a significant threat to sunflower production in China. This soilborne disease is difficult to control. It has been observed that delayed sowing reduces the severity of SVW on different varieties and across various locations. Soil was collected from multiple locations with different sowing dates to understand the underlying biological mechanisms driving this phenomenon. The soil bacterial community was characterized through 16S rRNA gene amplicon sequencing performed on the Illumina MiSeq platform, followed by comprehensive bioinformatics analysis. Microsclerotia numbers in soil were detected using both NP-10 selective medium and quantitative polymerase chain reaction (qPCR). By delaying the sowing date, the number of microsclerotia in soil and the biomass of V. dahliae colonized inside sunflower roots were reduced during the early developmental stages (V2–V6) of sunflowers. Amplicon sequencing revealed an increased abundance of bacterial genera, such as Pseudomonas, Azoarcus, and Bacillus in soil samples collected from delayed sowing plots. Five bacterial strains isolated from the delayed sowing plot exhibited strong antagonistic effects against V. dahliae. The result of the pot experiments indicated that supplying two different synthetic communities (SynComs) in the pot did increase the control efficiencies on SVW by 19.08% and 37.82% separately. Additionally, soil temperature and humidity across different sowing dates were also monitored, and a significant correlation between disease severity and environmental factors was observed. In conclusion, delayed sowing appears to decrease microsclerotia levels by recruiting beneficial rhizosphere bacteria, thereby reducing the severity of SVW.

Molecular Design for Cardiac Cell Differentiation Using a Small Data Set and Decorated Shape Features.

The discovery of small organic compounds for inducing stem cell differentiation is a time- and resource-intensive process. While data science could, in principle, streamline the discovery of these compounds, novel approaches are required due to the difficulty of acquiring training data from large numbers of example compounds. In this paper, we present the design of a new compound for inducing cardiomyocyte differentiation using simple regression models trained with a data set containing only 80 examples. We introduce decorated shape descriptors, an information-rich molecular feature representation that integrates both molecular shape and hydrophilicity information. These models demonstrate improved performance compared to ones using standard molecular descriptors based on shape alone. Model overtraining is diagnosed using a new type of sensitivity analysis. Our new compound is designed using a conservative molecular design strategy, and its effectiveness is confirmed through expression profiles of cardiomyocyte-related marker genes using real-time polymerase chain reaction experiments on human iPS cell lines. This work demonstrates a viable data-driven strategy for designing new compounds for stem cell differentiation protocols and will be useful in situations where training data is limited.

Probable Molecular Targeting of Inhibitory Effect of Carvacrol-Loaded Bovine Serum Albumin Nanoparticles on Human Breast Adenocarcinoma Cells.

To entrap carvacrol (CAR) in bovine serum albumin nanoparticles (BSANPs) to form CAR-loaded BSANPs (CAR@BSANPs) and to explore the anti-cancer effects in breast adenocarcinoma cells (MCF-7 cells) treated with CAR and CAR@BSANPs. A desolvation method was used to synthesize BSANPs and CAR@BSANPs. The BSANPs and CAR@BSANPs were characterized by several physicochemical methods, including visual observation, high-resolution field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and high-performance liquid chromatography. MCF-7 cells were used and analyzed after 24 h of exposure to CAR and CAR@BSANPs at half-maximal inhibitory concentration. The anti-proliferative, apoptotic, reactive oxygen species (ROS), and nitric oxide (NO) scavenging activity as well as gene expression analysis were investigated by the cell viability assay, phase-contrast microscopy, 2',7'-dichlorofluorescein-diacetate assay, Griess-Illosvoy colorimetric assay, and quantitative real-time polymerase chain reaction, respectively. CAR and CAR@BSANPs showed anti-proliferative, apoptotic, ROS generation, and NO scavenging effects on MCF-7 cells. Expression profile of B-cell lymphoma 2-like 11 (BCL2L11), vascular endothelial growth factor A (VEGFA), hypoxia inducible factor factor-1α (HIF1A), BCL2L11/apoptosis regulator (BAX), and BCL2L11/Bcl2 homologous antagonist/killer 1 (BAK1) ratios revealed downregulated genes; and BAX, BAK1, and CASP8 were upregulated by CAR and CAR@BSANPs treatment. In vitro anticancer assays of the CAR and CAR@BSANPs showed that CAR@BSANPs demonstrated higher therapeutic efficacy in the MCF-7 cells than CAR. CAR and CAR@BSANPs affect gene expression and may subsequently reduce the growth and proliferation of the MCF-7 cells. Molecular targeting of regulatory genes of the MCF-7 cells with CAR and CAR@BSANPs may be an effective therapeutic strategy against breast cancer.