RT-PCR Assay Research Articles

Cycle threshold (Ct)values of SARS-CoV2 vary with the type of symptoms and comorbidities of the patients.

Literature depicts no association of cycle threshold values for SARS-CoV-2 with the patient's demographics or clinical history. However, the severity of the COVID-19 symptoms showed some associations with Ct values in previous studies. We aimed to study the relationship of Ct values with type of the symptoms and comorbidities of the patients. We examined the individuals (n = 8660) that consulted a private diagnostic center for COVID-19 testing. Nasopharyngeal samples were collected from the patients and SARS-CoV-2 was detected by RT-PCR assays using Sansure Biotech kit. Clinical and demographic information was collected by the attending healthcare professional. The differences between groups were analyzed by t-test (unpaired). P-values < 0.05 were considered statistically significant. We found that the mean age of asymptomatic patients (41.47 ± 17.01 years) and symptomatic patients (47.75 ± 17.51) varies significantly (p value < 0.0001). Among the most prevalent symptoms were fever (77.74%) and cough (66.46%). The Ct values of COVID-19 patients with symptoms (25.70 for Orf1ab, 24.25 for N gene) were significantly lower than the Ct values of the patients without symptoms (28.99 for Orf1ab, 28.17 for N gene) (p value < 0.0001). Patients having co-occurrence of cough with fever (24.67 for Orf1ab, 22.86 for N gene) and solely fever (25.12 for Orf1ab, 24.01 for N gene) showed significantly lower Ct values than the patients without symptoms (28.99 for Orf1ab, 28.17 for N gene, p value < 0.0001). However, this difference was independent of patients sex and dependent upon patient's age and SARS-CoV-2 detected gene. The most prevalent comorbidities among COVID-19 patients were blood pressure (64.7%) and diabetes (50.5%). The patients having blood pressure and diabetes together showed significantly lower Ct values (23.37 for Orf1ab, 23.33 for N gene) than patients without comorbidities (27.65 for Orf1ab, 26.75 for N gene, p value < 0.001). We conclude that the Ct values of the SARS-CoV-2 detected gene (either Orf1ab or N) is associated with the types of symptoms and comorbidities of the COVID-19 patients. Furthermore, the relationship between Ct values and symptomology or comorbidity of the patients is independent of patient's sex but dependent upon patient's age and SARS-CoV-2 detected gene.

A high-throughput, polymerase-targeted RT-PCR for broad detection of mammalian filoviruses.

Filoviruses are some of the most lethal viruses in the modern world, and increasing numbers of filovirus species and genera have been discovered in recent years. Despite the potential severity of filovirus outbreaks in the human population, comparably few sensitive pan-filovirus RT-PCR assays have been described that might facilitate early detection and prevention. Here, we present a new pan-filovirus RT-PCR assay targeting the L polymerase gene for detection of all known mammalian filoviruses. We demonstrate the detection of 10 synthetic filovirus RNA templates with analytical sensitivity ranging from 178 to 3,354 copies/mL, without cross-reactivity on 10 non-filoviral human viral species. We verified assay performance on 10 inactivated filovirus isolates, yielding initial sensitivities of 0.012-44.17 TCID50/mL. We coupled this broadly reactive RT-PCR with a deep sequencing workflow that is amenable to high-throughput pooling to maximize detection and discovery potential. In summary, this pan-filovirus RT-PCR assay targets the most conserved filovirus gene, offers the widest breadth of coverage to date, and may help in the detection and discovery of novel filoviruses.IMPORTANCEFiloviruses remain some of the most mysterious viruses known to the world, with extremely high lethality rates and significant pandemic potential. Yet comparably few filovirus species and genera have been discovered to date and questions surround the definitive host species for zoonotic infections. Here, we describe a novel broadly reactive RT-PCR assay targeting the conserved L polymerase gene for high-throughput screening for filoviruses in a variety of clinical and environmental specimens. We demonstrate the assay can detect all known mammalian filoviruses and determine the sensitivity and specificity of the assay on synthetic RNA sequences, inactivated filovirus isolates, and non-filoviral species.

Sensitive and Accurate Quantification of Enterovirus-D68 (EV-D68) Viral Loads Using Droplet Digital PCR (ddPCR).

Enterovirus-D68 (EV-D68) is a reemerging virus that has been associated with numerous outbreaks in children in the past 10 years. Most assays examining viral infection kinetics have relied on the use of quantitative RT-PCR (qRT-PCR) assays as an assay of choice. Though valuable, there are inherent limitations that introduce variability, thereby reducing its value when comparing results across the field. Unlike the qRT-PCR assay that uses a standard curve to determine the copy number of viral RNA, the droplet digital PCR assay (ddPCR) directly quantifies the absolute number of copies within a given sample, which in turn makes the assay highly sensitive and accurate. Here, we have developed an EV-D68-specific ddPCR assay that effectively quantifies EV-D68 RNA copies in both cells and supernatants within a dynamic range of 6.7 × 10-3 copies/μL to 1.2 × 104 copies/μL of the sample. The assay was highly specific for a broad range of EV-D68 isolates (Fermon, US/MO/14-18947, US/MO/14-18949, US/KY/14-18953, USA/2018-23088, USA/2020-23336 and EV-D68-infected human nasal turbinate samples from the 2022 outbreak) without cross-reactivity to other viruses such as Enterovirus-A71 (EV-A71), Human Parechovirus (HPeV)-1 and -2, Coxsackievirus (CV)-B1, Human Coronavirus (HCoV)-NL63, SARS-CoV-2, Influenza-A and B, Rhinovirus, and Respiratory Syncytial Virus (RSV)-A2, which are known to cause infection in children. The assay was able to readily quantify EV-D68 in infected cells and supernatants along with nasal turbinate samples collected from children during the 2022 outbreak. Our results suggest that the assay can be readily translated to accurately quantify viral loads in tissues and body fluids such as plasma and lung or nasal aspirates.

RBM5 suppresses proliferation, metastasis and glycolysis of colorectal cancer cells via stabilizing phosphatase and tensin homolog mRNA.

RNA binding motif 5 (RBM5) has emerged as crucial regulators in many cancers. To explore more functional and mechanistic exploration of RBM5 since the lack of research on RBM5 in colorectal cancer (CRC) dictates that is essential. Through Gene Expression Profiling Interactive Analysis, we analyzed RBM5 expression in colon adenocarcinoma and rectum adenocarcinoma tissues. For detecting the mRNA expression of RBM5, quantitative real time-polymerase chain reaction was performed. Protein expression levels of RBM5, hexokinase 2, lactate dehydrogenase A, phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), phosphorylated-protein kinase B (p-AKT), and AKT were determined via Western blot. Functionally, cell counting kit-8 and 5-ethynyl-2'-deoxyuridine (EDU) assay were performed to evaluate proliferation of CRC cells. Invasiveness and migration of CRC cells were evaluated through conducting transwell assays. Glucose consumption, lactate production and adenosine-triphosphate (ATP) production were measured through a glucose assay kit, a lactate assay kit and an ATP production assay kit, respectively. Besides, RNA immunoprecipitation assay, half-life RT-PCR and dual-luciferase reporter assay were applied to detect interaction between RBM5 and PTEN. To establish a xenotypic tumor mice, CRC cells were subcutaneously injected into the right flank of each mouse. Protein expression of RBM5, Ki67, and PTEN in tumor tissues was examined using immunohistochemistry staining. Haematoxylin and eosin staining was used to evaluate tumor liver metastasis in mice. We discovered down-regulation of RBM5 expression in CRC tissues and cells. RBM5 overexpression repressed proliferation, migration and invasion of CRC cells. Meantime, RBM5 impaired glycolysis in CRC cells, presenting as decreased glucose consumption, decreased lactate production and decreased ATP production. Besides, RBM5 bound to PTEN mRNA to stabilize its expression. PTEN expression was positively regulated by RBM5 in CRC cells. The protein levels of PI3K and p-AKT were significantly decreased after RBM5 overexpression. The suppressive influences of RBM5 on glycolysis, proliferation and metastasis of CRC cells were partially counteracted by PTEN knockdown. RBM5 suppressed tumor growth and liver metastasis in vivo. This investigation provided new evidence that RBM5 was involved in CRC by binding to PTEN, expanding the importance of RBM5 in the treatment of CRC.

The Vps21 signalling pathway regulates white-opaque switching and mating in Candida albicans

ABSTRACT Candida albicans is able to switch between two epigenetic cell types, namely white and opaque. Multiple conserved signalling pathways control the switch between white and opaque cell types in response to environmental changes. Here, we report the regulatory roles of the endosomal Rab family GTPase Vps21 and associated key components of the Vps21 signalling pathway in white-opaque switching and mating in C. albicans. Deletion of VPS21 promoted a switch from the white to the opaque phenotype in the presence of N-acetyl-glucosamine (GlcNAc). Consistently, inactivation of the guanine nucleotide exchange factor of Vps21 (Vps9) and downstream components in the Vps21 pathway (Vps3, Vac1, and Pep12) had similar promoting effects on phenotypic switching. The mating efficiency of opaque cells is much higher than that of white cells under standard laboratory culture conditions. However, compared to the wildtype strain, the vps21/vps21, vps9/vps9, vps3/vps3, vac1/vac1, and pep12/pep12 mutant strains exhibited dramatically reduced mating efficiencies. Quantitative RT-PCR assays demonstrated that inactivation of the Vps21 signalling pathway led to downregulation of pheromone expression and mating response pathway associated genes. Taken together, our findings indicate that the conserved Vps21 signalling pathway plays critical roles in the regulation of cell-type switching and mating in C. albicans.

Anadenanthera colubrina regulated LPS-induced inflammation by suppressing NF-κB and p38-MAPK signaling pathways

We aimed to determine the chemical profile and unveil Anadenanthera colubrina (Vell.) Brenan standardized extract effects on inflammatory cytokines expression and key proteins from immunoregulating signaling pathways on LPS-induced THP-1 monocyte. Using the RT-PCR and Luminex Assays, we planned to show the gene expression and the levels of IL-8, IL-1β, and IL-10 inflammatory cytokines. Key proteins of NF-κB and MAPK transduction signaling pathways (NF-κB, p-38, p-NF-κB, and p-p38) were detected by Simple Western. Using HPLC-ESI-MSn (High-Performance Liquid-Chromatography) and HPLC-HRESIMS, we showed the profile of the extract that includes an opus of flavonoids, including the catechins, quercetin, kaempferol, and the proanthocyanidins. Cell viability was unaffected up to 250 µg/mL of the extract (LD50 = 978.7 µg/mL). Thereafter, the extract's impact on the cytokine became clear. Upon LPS stimuli, in the presence of the extract, gene expression of IL-1β and IL-10 were downregulated and the cytokines expression of IL-1β and IL-10 were down an upregulated respectively. The extract is involved in TLR-4-related NF-κB/MAPK pathways; it ignited phosphorylation of p38 and NF-κB, orchestrating a reduced signal intensity. Therefore, Anadenanthera colubrina's showed low cytotoxicity and profound influence as a protector against the inflammation, modulating IL-1β and IL-10 inflammatory cytokines gene expression and secretion by regulating intracellular NF-κB and p38-MAPK signaling pathways.

Evaluation of the sensitivity of a measles diagnostic real-time RT-PCR assay incorporating recently observed priming mismatch variants, 2024.

We investigated a variant of measles virus that encodes three mismatches to the reverse priming site for a widely used diagnostic real-time RT-PCR assay; reduction of sensitivity was hypothesised. We examined performance of the assay in context of the variant using in silico data, synthetic RNA templates and clinical specimens. Sensitivity was reduced observed at low copy numbers for templates encoding the variant sequence. We designed and tested an alternate priming strategy, rescuing the sensitivity of the assay.

Attenuated Salmonella typhimurium L forms suppress tumor growth and promote apoptosis in murine ovarian tumors

To study the effects of attenuated Salmonella typhimurium L forms on the in vivo tumorigenicity and apoptosis of murine epithelial ovarian cancer cells, as well as the related mechanisms. Attenuated Salmonella typhimurium VNP20009 was induced into bacterial L forms by using antibiotic ceftriaxone. CCK-8 cell proliferation assay showed that attenuated S. typhimurium L forms can inhibit the proliferation of murine ovarian epithelial cancer ID8 cells. Attenuated ST L forms can induce apoptosis and inhibit invasion ability of epithelial ovarian cancer cells in vitro. TUNEL assay showed that attenuated ST L forms can induce apoptosis of ID8 cells in murine ovarian tumors. Meanwhile, attenuated ST L forms inhibit tumor growth in murine ovarian tumors. The tumorigenicity-related proteins of xenograft tumors detected by immunohistochemistry and fluorescence quantitative RT-PCR assays showed that attenuated ST L forms can reduce the expression of proteins that promote tumor growth and metastasis, such as Lgals9 and MMP9. This study confirmed that attenuated ST L forms can suppress tumor growth and promote apoptosis in murine ovarian tumors. Attenuated ST L forms may serve as a novel biological agent for bacterial-mediated tumor therapy in epithelial ovarian cancer.

Comparison of Bordetella species identification among differing rt-PCR assays in the United States.

When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS481-based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii. The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States.

Salvianolic Acid B Accelerates Osteoporotic Fracture Healing via LncRNA-MALAT1/miR-155-5p/HIF1A Axis

Background: Osteoporotic fracture is a pathological fracture secondary to osteoporosis, causing disabilities and a heavy burden to the patients. In the previous study, we found that salvianolic acid B (SalB) promoted the osteogenesis of Mesenchymal Stem Cells (MSCs). Objective: This study aimed to explore the role of SalB in osteoporotic fracture healing, as well as the potential molecular mechanism. Methods: Human bone marrow mesenchymal stem cells (hMSCs) were treated with SalB or PBS in vitro, and an osteoporotic fracture model in Sprague-Dawley (SD) rats was established successfully. SalB or PBS was locally injected at the fracture. Eight weeks later, microCT was used to compare the healing of osteoporotic fractures with or without SalB. The relative expressions of mRNAs were measured by qRT-PCR. Bioinformatics analysis, RT-PCR, and dual luciferase reporter assay were utilized to detect the interrelation of genes. Immunohistochemistry staining was used to test expressions of proteins. Results: In the present study, we found that SalB significantly increased the level of lncRNA-- MALAT1 in a dose-dependent manner. Additionally, silencing lncRNA-MALAT1 inhibited the expressions of osteogenesis-related marker genes and abolished the effect of SalB on osteogenesis. Also, we found that lncRNA-MALAT1 sponged miR-155-5p, and miR-155-5p directly targeted HIF-1α. Using the osteoporotic fracture healing model, our result demonstrated that local administration of SalB could promote both bone and type H vessel formation in the calluses. The mechanical test further showed that SalB could improve the mechanical properties of fractured femurs. Conclusion: Taken together, our study reported that SalB could promote osteogenesis and type H vessel formation to accelerate osteoporotic fracture healing through the lncRNA-- MALAT1/miR-155-5p/HIF-1α axis.

Descriptive study of perineuronal net in enteric nervous system of humans and mice.

Perineuronal nets (PNN) are highly specialized structures of the extracellular matrix around specific groups of neurons in the central nervous system (CNS). They play functions related to optimizing physiological processes and protection neurons against harmful stimuli. Traditionally, their existence was only described in the CNS. However, there was no description of the presence and composition of PNN in the enteric nervous system (ENS) until now. Thus, our aim was to demonstrate the presence and characterize the components of the PNN in the enteric nervous system. Samples of intestinal tissue from mice and humans were analyzed by RT-PCR and immunofluorescence assays. We used a marker (Wisteria floribunda agglutinin) considered as standard for detecting the presence of PNN in the CNS and antibodies for labeling members of the four main PNN-related protein families in the CNS. Our results demonstrated the presence of components of PNN in the ENS of both species; however its molecular composition is species-specific.

Reduction in ACE2 expression in peripheral blood mononuclear cells during COVID-19 – implications for post COVID-19 conditions

BackgroundSevere COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC).MethodsThe IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10.ResultsForty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals.ConclusionsThis work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.

Curcumin inhibits growth of Porphyromonas gingivalis by arrest of bacterial dipeptidyl peptidase activity

ABSTRACT Background Curcumin is a multi-functional polyphenol with anti-bacterial and anti-inflammatory effects and may have potential for treatment of periodontal diseases. The present study was conducted to examine the molecular basis of the anti-bacterial effect of curcumin against Porphyromonas gingivalis using metabolome analysis. Materials and Methods P. gingivalis were incubated with 10 µg/mL curcumin, and then metabolites were analyzed with CE-TOF/MS. Expression levels of sigma factors were also evaluated using RT-PCR assays. The activities of dipeptidyl peptidases (DPPs) were assessed by examining the degradation reactions of MCA-labeled peptides. Results The relative amounts of various glycogenic amino acids were significantly decreased when P. gingivalis was incubated with curcumin. Furthermore, the metabolites on the amino acid degradation pathway, including high-energy compounds such as ATP, various intermediate metabolites of RNA/DNA synthesis, nucleoside sugars and amino sugars were also decreased. Additionally, the expression levels of sigma-54 and sigma-70 were significantly decreased, and the same results as noted following nutrient starvation. Curcumin also significantly suppressed the activities of some DPPs, while the human DPP-4 inhibitors markedly inhibited the growth of P. gingivalis and activities of the DPPs. Conclusions Curcumin suppresses the growth of P. gingivalis by inhibiting DPPs and also interferes with nucleic acid synthesis and central metabolic pathways, beginning with amino acid metabolism.

IGFBP7 promotes endothelial cell repair in the recovery phase of acute lung injury.

IGFBP7 has been found to play an important role in inflammatory diseases, such as acute lung injury (ALI). However, the role of IGFBP7 in different stages of inflammation remains unclear. Transcriptome sequencing was used to identify the regulatory genes of IGFBP7, and endothelial IGFBP7 expression was knocked down using Aplnr-Dre mice to evaluate the endothelial proliferation capacity. The expression of proliferation-related genes was detected by Western blotting and RT-PCR assays. In the present study, we found that knockdown of IGFBP7 in endothelial cells significantly decreases the expression of endothelial cell proliferation-related genes and cell number in the recovery phase but not in the acute phase of ALI. Mechanistically, using bulk-RNA sequencing and CO-IP, we found that IGFBP7 promotes phosphorylation of FOS and subsequently up-regulates YAP1 molecules, thereby promoting endothelial cell proliferation. This study indicated that IGFBP7 has diverse roles in different stages of ALI, which extends the understanding of IGFBP7 in different stages of ALI and suggests that IGFBP7 as a potential therapeutic target in ALI needs to take into account the period specificity of ALI.

Exploring immuno-genetic resistance in pigs to the food-borne zoonotic pathogen, Toxoplasma gondii: A serological study in Central Italy

Toxoplasmosis is a re-emerging food-borne zoonosis of warm-blooded animals caused by Toxoplasma gondii, an obligate intracellular parasite with a complex biological cycle. Until now, the genotypic approach for discovering putative resistance immuno-genetic markers has never been adopted for T. gondii infection. Thus, this study aimed, for the first time, to deepen these novel insights. In particular, pigs, reared in different farms in Central Italy, were phenotypically characterized by serological assays performed on diaphragm meat juice. Out of 179 tested animals, 98 resulted seropositive (54.74%), 57 seronegative (31.84%), and 24 doubtful (13.40%), underlining a possible re-emerging diffusion of this protozoan in the investigated areas. Contextually, an RT-PCR assay, followed by Sanger sequencing for IL-18, a pro-inflammatory cytokine with a key role upstream of the infection, was developed. At this stage, for this interleukin, significant polymorphic variations were not detected compared to the reference sequence, except in a seropositive animal. However, the starting outcomes of this novel and preliminary study will be investigated in depth with different approaches also on other target genes, with a crucial activity in the immunity pathway towards T. gondii, to unveil the possible presence of resistance genetic mechanisms and, finally, to make pork derived food safer.

Fish Oil Supplementation Mitigates High-Fat Diet-Induced Obesity: Exploring Epigenetic Modulation and Genes Associated with Adipose Tissue Dysfunction in Mice.

This study investigated the effects of fish oil (FO) treatment, particularly enriched with eicosapentaenoic acid (EPA), on obesity induced by a high-fat diet (HFD) in mice. The investigation focused on elucidating the impact of FO on epigenetic modifications in white adipose tissue (WAT) and the involvement of adipose-derived stem cells (ASCs). C57BL/6j mice were divided into two groups: control diet and HFD for 16 weeks. In the last 8 weeks, the HFD group was subdivided into HFD and HFD + FO (treated with FO). WAT was removed for RNA and protein extraction, while ASCs were isolated, cultured, and treated with leptin. All samples were analyzed using functional genomics tools, including PCR-array, RT-PCR, and Western Blot assays. Mice receiving an HFD displayed increased body mass, fat accumulation, and altered gene expression associated with WAT inflammation and dysfunction. FO supplementation attenuated these effects, a potential protective role against HFD-induced obesity. Analysis of H3K27 revealed HFD-induced changes in histone, which were partially reversed by FO treatment. This study further explored leptin signaling in ASCs, suggesting a potential mechanism for ASC dysfunction in the obesity-rich leptin environment of WAT. Overall, FO supplementation demonstrated efficacy in mitigating HFD-induced obesity, influencing epigenetic and molecular pathways, and shedding light on the role of ASCs and leptin signaling in WAT dysfunction associated with obesity.

Genetic Characterization of Palyam Serogroup Viruses Isolated in Japan from 1984 to 2018 and Development of a Real-Time RT-PCR Assay for Broad Detection of Palyam Serogroup Viruses and Specific Detection of Chuzan (Kasba) and D'Aguilar Viruses.

We performed whole genome sequencing (WGS) of 15 Palyam serogroup virus (PALV) strains isolated from cattle or Culicoides biting midges in Japan from 1984 to 2018. We found that the PALV strains consisted of Chuzan (Kasba) virus (CHUV), D'Aguilar virus (DAGV), Bunyip Creek virus, and another PALV, Marrakai virus (MARV). The Japanese MARV strains isolated in 1997 were closely related to Australian PALV strains isolated in 1968-1976 in genome segments 2 and 10, but they were most closely related to other Japanese PALV strains in the other genome segments. Our data suggest that the Japanese MARV strains were reassortant viruses between Asian and Australian PALVs. In addition to the WGS, we developed a real-time reverse-transcription polymerase chain reaction assay that can broadly detect PALV and specifically detect CHUV and DAGV, utilizing the data obtained by the WGS in this study. We detected the DAGV gene in bovine stillborn fetuses and congenitally abnormal calves in 2019 using the newly developed assay. To our knowledge, this is the first report of isolation of MARV outside of Australia and the first report of detection of PALV in bovine fetuses or calves with congenital abnormality outside of Africa.

Detection of Cucumber Mosaic Virus (CMV) in Cucurbits by TaqMan Real-Time RT-PCR in Hakkari Province, Türkiye

Cucumber mosaic virus (CMV) is among the important viruses that can infect many crops. In 2022, this study was conducted in the Kırıkdağ, Otluca, and Merzan areas of the Hakkari province of Türkiye to investigate the presence of CMV in cucurbits exhibiting viral disease symptoms (yellowing, necrosis, mosaic, and deformity). A total of 50 samples; 7 watermelons (Citrullus lanatus), 3 melons (Cucumis melo L.), 17 cucumbers (Cucumis sativus L.), 8 zucchinis (Cucurbita pepo L.), 9 pumpkins (Cucurbita moschata), 2 gourds (Lagenaria siceraria) and 4 gherkins (Cucumis melo var. flexuous) were collected. The presence of CMV in the samples was investigated by TaqMan real-time RT-PCR using probes and primers specific to the 105 bp region of partial the coat protein gene of the virus. CMV-specific viral RNA was detected in 6 (12%) of the 50 samples analyzed. CMV was detected most commonly in the Otluca area and melon (33.3%) among cucurbit varieties. CMV agent was not detected in the samples collected from the Merzan area. The TaqMan real-time RT-PCR assay used in this study demonstrated that the method can be widely used for the general detection of CMV in a wide range of hosts. This is the first report on the detection of CMV in cucurbits in the Hakkari province of Türkiye.

The Impact of Azadirachta Indica Extract on the Expression Profile of Esp Gene in Treated Enterococcus faecalis

Background and Objectives Enterococcus faecalis is a bacterium often associated with persistent endodontic infections, posing challenges for effective root canal disinfection and treatment outcomes due to its resilience in adverse root canal conditions and resistance to antimicrobial therapies. This study aimed to investigate the effects of Azadirachtaindica's active plant compounds at subinhibitory concentrations on the expression of the virulence gene (esp) in Enterococcus faecalis strains (ATCC 29212). Materials and Methods Azadirachta indica (neem) specimens were obtained in Erbil, Northern Iraq, and the active compounds from the dried plant samples were extracted. Dry extracts were dissolved in Dimethylsulphoxide (DMSO) until reaching 100 mg/ml. Bacterial samples were treated with the extracted plant material and incubated. The assessment of relative esp gene expression in treated Enterococcus faecalis strains (ATCC 29212) began with total RNA extraction using the RNXplus kit, followed by a Quantitative RT-PCR assay. Results The minimum concentration of Azadirachta indica's ethyl acetate fraction 3 (30 mg/ml) inhibited the growth of the targeted bacteria. In contrast, the highest concentration inhibiting the target bacteria was 50 mg/ml of fraction 6 (Azadirachta indica-water). All fractions of Azadirachta indica decreased the expression of the target gene, with fraction 5 (ethyl alcohol) showing the highest fold change. Conclusion The ethyl alcohol extraction of Azadirachta indica inhibited the expression of the virulence gene (esp) by 2.84-fold, suggesting a potential decrease in (esp) gene expression, which could be considered as an alternative to classical irrigants.

AMPK activation reduces cancer cell aggressiveness via inhibition of monoamine oxidase A (MAO-A) expression/activity

AimAMPK can be considered as an important target molecule for cancer for its unique ability to directly recognize cellular energy status. The main aim of this study is to explore the role of different AMPK activators in managing cancer cell aggressiveness and to understand the mechanistic details behind the process. Main methodsFirst, we explored the AMPK expression pattern and its significance in different subtypes of lung cancer by accessing the TCGA data sets for LUNG, LUAD and LUSC patients and then established the correlation between AMPK expression pattern and overall survival of lung cancer patients using Kaplan-Meire plot. We further carried out several cell-based assays by employing different wet lab techniques including RT-PCR, Western Blot, proliferation, migration and invasion assays to fulfil the aim of the study. Key findings•Expression of AMPK is correlated with survival and prognosis of lung cancer patients.•AMPK activators like Metformin and Phenformin downregulate A549 and HCT-116 cell proliferation and migration via repression of p38MAPK activity, subsequent augmentation of R1 repressor and corresponding downregulation of MAO-A expression/activity resulting reduction in the intracellular ROS.•SRT-1720 directly activates AMPK in LKB1 mutant A549 cells either alone or in combination with Metformin resulting regulation of cancer cell aggressiveness. SignificanceThis study identifies the importance of AMPK activators as a repurposing agent for combating lung and colon cancer cell aggressiveness. It also suggests SRT-1720 as a potent repurposing agent for cancer treatment especially in NSCLC patients where a point mutation is present in LKB1.