Real-time Reverse Transcription Research Articles

Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.).

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay

BackgroundAfter the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M).ResultsThe assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen’s correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance.ConclusionsOverall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Overexpression of CBL-Interacting Protein Kinases 23 Improves Tolerance to Low-Nitrogen Stress in Potato Plants

CBL-interacting protein kinases (CIPKs) play important regulatory roles in plant growth development and abiotic stress tolerance. However, the biological roles of these genes in response to low-nitrate (LN) stress in potato plants have not been determined. Here, we reported that StCIPK23 was expressed mainly in roots and leaves. StCIPK23 was located mainly in the cell membrane, nucleus, and cytoplasm. Further research suggested that, compared with wild-type (WT) plants, StCIPK23-overexpressing plants were taller and had significantly greater nitrate and ammonium nitrogen contents under LN stress. StCIPK23 overexpression can increase StAT, StNRT2.1, StNR, StGS1-3, and StGOGAT expression levels in StCIPK23 transgenic seedlings compared to those in WT plants under LN stress. The results of yeast two-hybrid and luciferase complementation imaging experiments suggested that StCIPK23 could interact with StCBL3. Real-time reverse transcription–PCR revealed the StCIPK23 expression level peaked at 6 h and the StCBL3 expression level peaked at 9 h in the roots under LN stress. In conclusion, we found that StCIPK23 and StCBL3 form a complex to regulate the expression of key genes in the nitrogen metabolism pathway to improve LN tolerance in potato plants.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats.

To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats' retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Treg Immunomodulation Contributes to the Anti-atherosclerotic Effects of Huxin Formula in ApoE-/- Mice.

To explore the effects of Huxin formula (HXF) in curtailing atherosclerosis and its underlying mechanism. According to random number table method, 24 specific pathogen free male ApoE-/- mice were randomly divided into model group, HXF low-dose (HXF-L) group (8.4 g/kg daily), HXF high-dose (HXF-H) group (16.8 g/kg daily), and pravastatin (8 mg/kg daily) group in Experiment I (n=6 per group). C57BL/6J mice served as the control group (n=6). ApoE-/- mice in HXF-L, HXF-H, pravastatin groups were fed a Western diet and administered continuously by gavage for 12 weeks, while C57BL/6J mice in the control group were fed conventional lab mouse chow for 12 weeks. Further, Tregs were depleted by weekly intraperitoneal injection of purified anti-mouse CD25 antibody (PC61, 250 µg per mouse) for 4 weeks in Experiment II (n=6 per group). Oil Red O and Masson staining were used to evaluate the plaque area and aortic root fibrosis. The CD4+CD25+Foxp3+Treg counts in the lymph nodes and spleen cells were detected using flow cytometric analysis. The transforming growth factor-β1 (TGF-β1), interleukin (IL)-10, and IL-6 serum levels were examined by MILLIPLEX® MAP technology. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot were utilized to assess the expression of TGF-β mRNA and protein in the aorta. The expression of CD4+T lymphocytes, macrophages and smooth muscle cells in the aortic root were detected by immunofluorescence staining. HXF reduced plaque area in ApoE-/- mice (P<0.01). HXF increased the Treg counts in the lymph nodes and spleen cells (P<0.05 or P<0.01). Moreover, HXF alleviated inflammatory response via elevating IL-10 and TGF-β 1 serum levels (P<0.05), while decreasing the IL-6 serum levels in ApoE-/- mice (P>0.05). Also, HXF upregulated the expression of TGF-β mRNA and protein in the aorta (P<0.05). Additionally, HXF attenuated CD4+T lymphocytes, macrophages and smooth muscle cells in aortic root plaque (P<0.01). Furthermore, the depletion of Tregs with CD25 antibody (PC61) curtailed the reduction in plaque area and aortic root fibrosis by HXF (P<0.01). HXF relieved atherosclerosis, probably by restraining inflammatory response, reducing inflammatory cell infiltration and attenuating aortic root fibrosis by increasing Treg counts.

Genomic identification and expression analysis of acid invertase (AINV) gene family in Dendrobium officinale Kimura et Migo

BackgroundDendrobium officinale Kimura et Migo, a renowned traditional Chinese orchid herb esteemed for its significant horticultural and medicinal value, thrives in adverse habitats and contends with various abiotic or biotic stresses. Acid invertases (AINV) are widely considered enzymes involved in regulating sucrose metabolism and have been revealed to participate in plant responses to environmental stress. Although members of AINV gene family have been identified and characterized in multiple plant genomes, detailed information regarding this gene family and its expression patterns remains unknown in D. officinale, despite their significance in polysaccharide biosynthesis.ResultsThis study systematically analyzed the D. officinale genome and identified four DoAINV genes, which were classified into two subfamilies based on subcellular prediction and phylogenetic analysis. Comparison of gene structures and conserved motifs in DoAINV genes indicated a high-level conservation during their evolution history. The conserved amino acids and domains of DoAINV proteins were identified as pivotal for their functional roles. Additionally, cis-elements associated with responses to abiotic and biotic stress were found to be the most prevalent motif in all DoAINV genes, indicating their responsiveness to stress. Furthermore, bioinformatics analysis of transcriptome data, validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct organ-specific expression patterns of DoAINV genes across various tissues and in response to abiotic stress. Examination of soluble sugar content and interaction networks provided insights into stress release and sucrose metabolism.ConclusionsDoAINV genes are implicated in various activities including growth and development, stress response, and polysaccharide biosynthesis. These findings provide valuable insights into the AINV gene amily of D. officinale and will aid in further elucidating the functions of DoAINV genes.

The Ability to Detect the COVID-19 Genome Using Saliva Swabs in Comparison with Nasopharyngeal Swabs in Baghdad

Abstract Objective Nasopharyngeal swab (NPS) sampling has been recommended by the World Health Organization (WHO) since the start of the COVID-19 pandemic, and real-time reverse transcription polymerase chain reaction (RT–PCR) is used to detect SARS-CoV- 2, the causative agent of COVID-19. This sampling technique is invasive and causes discomfort to the patient. Saliva swabs (SSs) can be used as an alternative noninvasive method; however, there are limited data confirming its suitability for the diagnosis of COVID-19. The aim of this study was to test the ability to detect COVID-19 using SSs in comparison with NPSs in the Baghdad Alkark sector. Materials and Methods Six hundred and fifty patients were included in this study, and written informed consent was obtained from all the study participants. Paired NPSs and SSs were collected at the same time from each participant between days 3 and 5 after disease initiation. SSs were taken from the sublingual area. An RT‒PCR assay was used to detect the viral ribonucleic acid (RNA) of SARS-CoV-2 for the diagnosis of COVID-19. The chi-squared test was used for data analysis, with p &lt; 0.05 considered significant. Results Out of 650 participants with suspected COVID-19 (313 males and 145 females), 313 were confirmed to be positive for COVID-19 by quantitative RT–PCR (RT‒qPCR) using both samples. The ages ranged between 12 and 85 years, with a mean/standard deviation (SD) of 45.45 (16.62) years. All the cases with positive results using NPSs were also positive when SSs were used. Statistically, there was no significant difference between the two groups (p = 0.347). Conclusion RT‒PCR assays conducted on SSs and NPSs performed similarly, indicating that SSs may be a safe, inexpensive diagnostic sampling method and an effective tool for population screening. We recommend more studies to support this finding.

Reliability of the rapid antigen test for diagnosis of SARS-CoV-2 in Tunisia

Background: Early and accurate diagnosis is crucial for preventing the spread of SARS-CoV-2 infection. The rapid antigen test was developed for testing infection, and it was necessary to assess its performance before widespread use in Tunisia. Aim: To evaluate the effectiveness of a rapid antigen test for the detection of SARS-CoV-2 in nasopharyngeal swabs in Tunisia. Methods: Nasopharyngeal samples were taken from COVID-19 suspected cases between October and December 2020 and tested using the Standard Q COVID-19 Ag test (SD-Biosensor, Republic of Korea) and real-time reverse transcription polymerase chain reaction (RT-PCR). Results: Overall, 4539 patients were tested. Of the total study population (N = 4539), 82.5% of positive samples remained positive with the rapid antigen test, while 20.2% (470/2321) of samples that were negative with rapid antigen test were confirmed positive with RT-PCR, giving a negative predictive value of 79.8% for the rapid antigen test. The sensitivity and negative predictive value of the rapid antigen test were 70.2% and 65.8%, respectively. These results improved to 96.4% and 92.8%, respectively, when considering the cycle threshold value by RT-PCR below 25. Conclusion: Although the rapid antigen test was less sensitive than RT-PCR, its ability to rapidly detect individuals with high viral loads makes it suitable for use during an epidemic.

MicroRNA-671-5p regulates the inflammatory response of periodontal ligament stem cells via the DUSP8/p38 MAPK pathway.

MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced. microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/μL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation &amp;lt;1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

Rotavirus strains in children less than 5 years of age: A case control study

BackgroundGlobally, rotavirus A (RV) is a leading cause of acute gastroenteritis (AGE) in young children under 5 years. The main aim of this study was to describe the prevalence of RV in children with and without diarrhea whose age ranged from 3 months to 5 years from two different hospitals of Nepal. We also described the diversity of rotavirus circulating during the pre-vaccination period in Nepal between 2006–2009. Materials and methodsA total of 2400 stool samples collected from children's years with diarrhea (cases, N = 1200) and without diarrhea (controls, N = 1200) were tested for the presence of RV by both enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction (RT-PCR). All samples positive for RT PCR and/or ELISA were further characterized for G and P genotypes by conventional RT-PCR. ResultsIn total, 500 of the 2398 (20.8 %) samples (424 cases and 76 controls), were positive for RV. The most frequently detected G-types were G12, G1, and G9 and P-types were P[8] and P[6]. The predominant genotypes G12P[6], G12P[8], G1P[8] and G9P[8] were identified in 27.8 %, 24.2 %, 11.8 % and 10.8 % of the samples, respectively. ConclusionThis study observed high prevalence of G12P[6] rotavirus circulating in Nepal before vaccine introduction and they may pose an emerging challenge to current and future vaccine. Therefore, continuous molecular surveillance of circulating genotypes and emerging rotavirus strain should be carried out to better understand the effectiveness of vaccination program.

The role of semaphorin 3A on chondrogenic differentiation.

Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100ng/mL) was examined using real-time reverse transcription polymerase chain reaction. Further, to assess the deposition of total glycosaminoglycans during chondrogenic differentiation, ATDC5 cells were stained with Alcian Blue. Moreover, the amount of hyaluronan in the culture supernatant was measured by enzyme-linked immunosorbent assay. The addition of Sema3A to cultured ATDC5 cells increased the expression of Sox9, Runx2, COL2A1, ACAN, HAS2, and COL10A1 during chondrogenic differentiation. Moreover, it enhanced total proteoglycan and hyaluronan synthesis. Further, Sema3A was upregulated in the early stages of chondrogenic differentiation, and its secretion decreased later. Sema3A increases extracellular matrix production and promotes chondrogenic differentiation. To the best of our knowledge, this is the first study to demonstrate the role of Sema3A on chondrogenic differentiation.

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

Risk factors and circulation pattern of respiratory syncytial virus in children under 2years in Maputo, Mozambique.

Evidence on risk factors for respiratory syncytial virus (RSV) in low-resource settings is limited. In Mozambican children <2y of age with severe acute respiratory infection (SARI), we explored risk factors for RSV, described its seasonal variation and assessed associations between RSV and a life-threatening condition. We retrospectively included participants presenting in 2017-2018 in two hospitals in Maputo. RSV was detected and subtyped using real-time quantitative reverse transcription polymerase chain reaction on nasopharyngeal swabs. We used logistic regression and χ2 tests to assess associations and Spearman's correlation coefficient to assess the correlation between weather measurement and RSV positivity. RSV was detected in 23.1% (n=109) of 472 included children and in 50.0% (20/40) of those <3months old. Being <3months (vs >1y) was associated with RSV (adjusted odds ratio 4.3 [95% confidence interval 2.1-8.5]). RSV status was not associated with experiencing a life-threatening condition. RSV A and B co-circulated during the study period, but one type predominated in each year. In 2017, the RSV positivity rate was correlated with monthly average temperature (r=0.793, p=0.002) and precipitation (r=0.596, p=0.041). In Mozambican children with SARI, RSV was prevalent, especially in neonates. However, RSV was not associated with a life-threatening condition.

Development of a real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primers for rapid detection of rubella virus

Rubella virus infection during early pregnancy sometimes causes severe birth defects termed congenital rubella syndrome. Although there are safe and effective live-attenuated vaccines, rubella has only been certified as eliminated in the Americas within the six World Health Organization regions. Rubella remains an endemic disease in many regions, and outbreaks occur wherever population immunity is insufficient. There are two main methods for diagnosis of rubella: detection of anti-rubella IgM antibodies by enzyme immunoassay and detection of the viral genome by real-time RT-PCR. Both of these methods require substantial time and effort. In the present study, a rapid rubella detection assay using real-time fluorescent reverse transcription loop-mediated isothermal amplification with quenching primers was developed. The time required for the new assay was one-half that required for a real-time RT-PCR assay. The assay had 93.6% positive percent agreement and 100% negative percent agreement for clinical specimens compared with the real-time RT-PCR assay. The new assay is considered useful for diagnosis of rubella in areas where rubella is endemic.

Evaluation of differences in expression pattern of three isoforms of the transforming growth factor beta in patients with lumbosacral stenosis

ABSTRACT The study investigates molecular changes in the lumbosacral (L/S) spine’s yellow ligamentum flavum during degenerative stenosis, focusing on the role of transforming growth factor beta 1–3 (TGF-β-1–3). Sixty patients with degenerative stenosis and sixty control participants underwent molecular analysis using real-time quantitative reverse transcription reaction technique (RTqPCR), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analysis (IHC). At the mRNA level, study samples showed reduced expression of TGF-β-1 and TGF-β-3, while TGF-β-2 increased by only 4%. Conversely, at the protein level, the study group exhibited significantly higher concentrations of TGF-β-1, TGF-β-2, and TGF-β-3 compared to controls. On the other hand, at the protein level, a statistically significant higher concentration of TGF-β-1 was observed (2139.33 pg/mL ± 2593.72 pg/mL vs. 252.45 pg/mL ± 83.89 pg/mL; p < 0.0001), TGF-β-2 (3104.34 pg/mL ± 1192.74 pg/mL vs. 258.86 pg/mL ± 82.98 pg/mL; p < 0.0001), TGF-β-3 (512.75 pg/mL ± 107.36 pg/mL vs. 55.06 pg/mL ± 9.83 pg/mL, p < 0.0001) in yellow ligaments obtained from patients of the study group compared to control samples. The study did not establish a significant correlation between TGF-β-1–3 concentrations and pain severity. The findings suggest that molecular therapy aimed at restoring the normal expression pattern of TGF-β-1–3 could be a promising strategy for treating degenerative stenosis of the L/S spine. The study underscores the potential therapeutic significance of addressing molecular changes at the TGF-β isoforms level for better understanding and managing degenerative spinal conditions.

Molecular Epidemiology of SARS-CoV-2 Variants in Vaccinated and Non-Vaccinated Patients of Erbil Province, Kurdistan Region-Iraq

Multiple new SARS-CoV-2 variants of concern (VOC) have emerged globally since the onset of the COVID-19 pandemic. With the virus continuing to evolve, more are expected. This emphasizes the need for rapid diagnostic methods for the detection of circulating lineages. Variants-specific real-time reverse transcription (rRT)-PCR method can be used as an alternative to genome sequencing, which is expensive and labored for identifying these variants, especially in settings with limited resources. We assessed the prevalence of various SARS-CoV-2 variants spreading in the Erbil province using a diagnostic screening RT-PCR-based method. A total of 144 SARS-CoV-2 positive samples were prospectively tested for known SARS-CoV2 variants using ViroQ® SC2 Variant rRT-PCR. Furthermore, the technique was validated using 25 SARS-CoV-2 negative nasal samples. Out of 144 SARS-CoV-2 positive samples, 118 (81.9%) were B.1.617.2 (Delta), 5 (3.5%) were Epsilon B.1.427/B.1.429, 1(0.7%) was Eta B.1.525, 2(1.4%) were SARS-CoV-2 Wild type, while 18 (12.5%) were undefined variant, and the delta strain was the most prevalent SARS-CoV-2 strain. Our study showed that variant-specific RT-PCR could be a useful tool for the rapid screening of SARS-CoV-2 variants.

Spatio-temporal dynamics and drivers of highly pathogenic avian influenza H5N1 in Chile.

Highly pathogenic avian influenza A H5N1 clade 2.3.4.4b (hereafter H5N1) is causing vast impacts on biodiversity and poultry around the globe. In Chile, lethal H5N1 cases have been reported in a wide range of wild bird species, marine mammals, backyard and industrial poultry, and humans. This study describes the spatio-temporal patterns of the current epizootic of H5N1 in Chile and test drivers that could be associated with outbreak occurrence. We used H5N1 cases reported by the Chilean National Animal Health Authority from 5 December 2022 to 5 April 2023. These included wild bird cases confirmed through an avian influenza-specific real-time reverse transcription PCR assay (RT-qPCR), obtained from passive and active surveillance. Data were analyzed to detect the presence of H5N1 clusters under space-time permutation probability modeling, the association of H5N1 with distance and days since the first outbreak through linear regression, and the correlation of H5N1 presence with a number of ecological and anthropogenic variables using general linear modeling. From 445 H5N1 identified outbreaks involving 613 individual cases in wild birds, a consistent wave-like spread of H5N1 from north to south was identified, which may help predict hotspots of outbreak risk. For instance, seven statistically significant clusters were identified in central and northern Chile, where poultry production and wildlife mortality are concentrated. The presence of outbreaks was correlated with landscape-scale variables, notably temperature range, bird richness, and human footprint. In less than a year, H5N1 has been associated with the unusual mortality of >100,000 individuals of wild animals in Chile, mainly coastal birds and marine mammals. It is urgent that scientists, the poultry sector, local communities, and national health authorities co-design and implement science-based measures from a One Health perspective to avoid further H5N1 spillover from wildlife to domestic animals and humans, including rapid removal and proper disposal of wild dead animals and the closure of public areas (e.g., beaches) reporting high wildlife mortalities.

Nanoscale dihydroartemisinin@zeolitic imidazolate frameworks for enhanced antigiardial activity and mechanism analysis.

An artificial semisynthetic material can be derived from artemisinin (ART) called dihydroartemisinin (DHA). Although DHA has enhanced antigiardial potential, its clinical application is limited because of its poor selectivity and low solubility. The drug's absorption has a direct impact on the cell, and mechanism research is limited to its destruction of the cytoskeleton. In this study, we used the zeolitic imidazolate framework-8 and loaded it with DHA (DHA@Zif-8) to improve its antigiardial potential. DHA@Zif-8 can enhance cellular uptake, increase antigiardial proliferation and encystation, and expand the endoplasmic reticulum compared with the DHA-treated group. We used RNA sequencing (RNA-seq) to investigate the antigiardial mechanism. We found that 126 genes were downregulated and 123 genes were upregulated. According to the KEGG and GO pathway analysis, the metabolic functions in G. lamblia are affected by DHA@Zif-8 NPs. We used real-time quantitative reverse transcription polymerase chain reaction to verify our results using the RNA-seq data. DHA@Zif-8 NPs significantly enhanced the eradication of the parasite from the stool in vivo. In addition, the intestinal mucosal injury caused by G. lamblia trophozoites markedly improved in the intestine. This research provided the potential of utilizing DHA@Zif-8 to develop an antiprotozoan drug for clinical applications.

Active Anaerobic Methane Oxidation in the Groundwater Table Fluctuation Zone of Rice Paddies

Rice paddies are globally important sources of methane emissions and also active regions for methane consumption. However, the impact of fluctuating groundwater levels on methane cycling has received limited attention. In this study, we delved into the activity and microbial mechanisms underlying anaerobic oxidation of methane (AOM) in paddy fields. A comprehensive approach was employed, including 13C stable isotope assays, inhibition experiments, real-time quantitative reverse transcription PCR, metagenomic sequencing, and binning technology. Geochemical profiles revealed the abundant coexistence of both methane and electron acceptors in the groundwater table fluctuation (GTF) zone, at a depth of 40–60 cm. Notably, the GTF zone exhibited the highest rate of AOM, potentially linked to the reduction of iron oxides and nitrate. Within this zone, Candidatus Methanoperedens (belonging to the ANME-2d group) dominated the Archaea population, accounting for a remarkable 85.4%. Furthermore, our results from inhibition experiments, RT-qPCR, and metagenome-assembled genome (MAG) analysis highlighted the active role of Ca. Methanoperedens GTF50 in the GTF zone. This microorganism could independently mediate AOM process through the intriguing “reverse methanogenesis” pathway. Considering the similarity in geochemical conditions across different paddy fields, it is likely that Ca. Methanoperedens-mediated AOM is prevalent in the GTF zones.