Reverse Assay Research Articles

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.

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.

Brachyury promotes extracellular matrix synthesis through transcriptional regulation of Smad3 in nucleus pulposus.

Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that the transcription factor Brachyury (Bry) has the potential to promote the synthesis of collagen II and aggrecan, while the specific mechanism is still unknown. In this study, we used a lipopolysaccharide (LPS)-induced model of nucleus pulposus cell (NPC) degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which Bry affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of Bry exacerbated the decrease in collagen II and aggrecan expression in the lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that Smad3 may participate in the regulatory pathway of ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that Bry enhances the transcription of Smad3 by interacting with a specific motif on the promoter region. In addition, Western blot and reverse transcription-qPCR assays demonstrated that Smad3 positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that Bry-mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from the in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that Bry positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3.NEW & NOTEWORTHY Mechanically, in the nucleus, Bry enhances the transcription of Smad3, leading to increased expression of Smad3 protein levels; in the cytoplasm, elevated substrate levels further lead to an increase in the phosphorylation of Smad3, thereby regulating collagen II and aggrecan expression. Further in vivo experiments provide additional evidence that Bry can alleviate IVDD through this mechanism.

Interleukin-10 supplementation protects against oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages

Approximately 10% of the United States population will experience a kidney stone (KS). Dietary oxalate promotes calcium oxalate (CaOx) crystals, which are precursors to CaOx KS. Macrophages rely on Interleukin-10 (IL-10) anti-inflammatory cytokine signaling to phagocytose and remove CaOx crystals. We recently reported that oxalate causes macrophages to become M1-like (pro-inflammatory) as well as have increased Interleukin-6 (IL-6; pro-inflammatory) and reduced IL-10 cytokine secretion. The purpose of this study was to assess the effects of IL-10 supplementation on mouse kidneys and bone-marrow derived macrophages exposed to oxalate. We hypothesized that exogenous IL-10 treatment prevents oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages (BMDMs). Mice with a C57BL/6 background were fed Hydroxy-L-proline (HLP; an oxalate precursor) for 7 or 14 days ± IL-10 intraperitoneal injections (25 μg/kg for 2 days) before collecting kidneys and BMDMs. Real-Time Quantitative Reverse Transcription PCR, fluorescence assays, and histological analyses were performed. Mice fed HLP had increased IL-6 and M1 macrophage expression in their kidneys over time. These mice also had reduced IL-10 and M2 macrophage gene expression. In contrast, mice fed HLP with IL-10 supplementation had increased M2 macrophage levels as well as reduced inflammation in their kidneys. Consistent with these findings, BMDM isolated from mice fed HLP displayed similar results. These cells also had reduced mitochondrial membrane polarization and lysosomal staining and increased cellular ROS; whereas, exogenous IL-10 treatment protected BMDMs from oxalate mediated injury. These findings suggest that IL-10 signaling may be important for preventing oxalate-induced injury and inflammation in mouse kidneys and BMDMs. NIH R01 DK129885 (TM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Development of one-step reverse transcription PCR assay for detection of porcine epidemic diarrhoea virus in pigs

Development of one-step reverse transcription PCR assay for detection of porcine epidemic diarrhoea virus in pigs

Mitochondrial antioxidant elamipretide improves learning and memory impairment induced by chronic sleep deprivation in mice.

The inflammation and synaptic dysfunction induced by mitochondrial dysfunction play essential roles in the learning and memory impairment associated with sleep dysfunction. Elamipretide (SS-31), a novel mitochondrion-targeted antioxidant, was proven to improve mitochondrial dysfunction, the inflammatory response, synaptic dysfunction, and cognitive impairment in models of cerebral ischemia, sepsis, and type 2 diabetes. However, the potential for SS-31 to improve the cognitive impairment induced by chronic sleep deprivation (CSD) and its underlying mechanisms is unknown. Adult c57BL/6J mice were subjected to CSD for 21 days using an activity wheel accompanied by daily intraperitoneal injection of SS-31 (5mg/kg). The novel object recognition and Morris water maze test were used to evaluate hippocampus-dependent cognitive function. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were used to determine the effects of CSD and SS-31 on markers of mitochondria, inflammation response, and synaptic function. Enzyme-linked immunosorbent assays were used to examine the levels of proinflammatory cytokines. SS-31 could improve the cognitive impairment induced by CSD. In particular, SS-31 treatment restored the CSD-induced decrease in sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator alpha levels and the increase in levels nuclear factor kappa-B and inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha. Furthermore, SS-31 significantly increased the levels of brain-derived neurotrophic factor, postsynaptic density protein-95, and synaptophysin in CSD mice. Taken together, these results suggest that SS-31 could improve CSD-induced mitochondrial biogenesis dysfunction, inflammatory response, synaptic dysfunction, and cognitive impairment by increasing SIRT1 expression levels.

Development of a reverse transcription loop-mediated isothermal amplification assay with novel quantitative pH biosensor readout method for SARS-CoV-2detection.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a molecular amplification method that can detect SARS-CoV-2 in a shorter time than the current gold-standard molecular diagnostic reverse transcription-polymerase chain reaction (RT-PCR). However, previously developed RT-LAMP assays have mostly relied on highly subjective visual colorimetric interpretation. In this study, an RT-LAMP assay was developed with quantitative measurement of reaction pH using a novel portable pH biosensor compared to qualitative colorimetric interpretation and gel electrophoresis, with 57 clinical COVID-19 samples used for validation of the test. The LoD of the assay is 103 copies/μL. The highest sensitivity was found in the qualitative methods (93.75%), while the highest specificity and likelihood ratio was found in the pH sensor (87.5% and 6.72). On the sensor measurement, a significant difference (p < 0.0001) was observed between the average pH of the RT-PCR (+) COVID-19 (6.15 ± 0.27), while the average pH of the RT-PCR (-) samples (6.72 ± 0.22). Correlation analysis revealed a strong correlation (r = 0.78, p < 0.0001) between the Ct values obtained from RT-PCR with the biosensor pH readout. RT-LAMP with the quantitative pH sensor readout method has the potential to be further developed as an objective molecular assay for rapid and simple detection of SARS-CoV-2.

In vitro toxicological evaluation of glo menthol and non-menthol heated tobacco products

Heated tobacco products (HTPs) are non-combustible, inhaled tobacco products that generate an aerosol with fewer and lower levels of toxicants, with a potential to reduce risk relative to cigarette smoking. Here, we assessed in vitro toxicological effects of three menthol (glo neo neoCLICK, neo Smooth Menthol and Fresh Menthol) and one non-menthol (neo Smooth Tobacco) variants of glo HTP, along with market comparators for cigarettes and HTPs. Limited chemical characterization of the study products revealed significantly lower levels of acetaldehyde, acrolein, crotanaldehyde and formaldehyde in test samples from HTPs than those from cigarettes. The glo HTPs were non-mutagenic in the bacterial reverse mutagenesis assay. Although, the whole aerosol exposures of glo HTPs were classified as genotoxic in the in vitro micronucleus assay, and cytotoxic in the NRU (monolayer) and MTT (3 dimensional EpiAirway™ tissues) assays, the cigarette comparators were the most toxic study products in each of these assessments. Further, glo HTPs elicited oxidative stress responses only at the highest dose tested, whereas the cigarette comparators were potent inducers of oxidative stress at substantially lower doses in the EpiAirway tissues. The comparator (non-glo) HTP results were similar to the glo HTPs in these assays. Thus, the glo HTPs exhibit substantially lower toxicity compared to cigarettes.

Regulation mechanism of the quorum sensing regulator AphA on the type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis

Objective: To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods: Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), ΔaphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter-lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp (tssD2) and vgrG (tssI2), and the quorum sensing regulator HapR in WT and ΔaphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results: The mRNA expression levels of tssB2, hcp(tssD2), vgrG (tssI2), and hapR as well as the protein expression and secretion levels of Hcp in ΔaphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in ΔaphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions: AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).

Profile of non-tuberculous mycobacteria amongst tuberculosis presumptive people in Cameroon

BackgroundCameroon is a tuberculosis (TB) burden country with a 12% positivity among TB presumptive cases. Of the presumptive cases with a negative TB test, some are infected with Non-tuberculous Mycobacteria (NTM). However, the diagnosis of NTM infections remains difficult due to the lack of tools in many laboratories, particularly in resource limited laboratories and remote setting. The present study was undertaken to determine NTM profile and associated comorbidities among TB presumptive people.MethodsA retrospective study was conducted from December 2018 to December 2019 in the Tuberculosis-National Reference Laboratory (TB-NRL) for Bacteriological analysis of samples and Jamot Hospital of Yaounde (JHY) for clinical evaluation of confirmed NTM patients.We included in this study data of 5267 TB presumptive people previously diagnosed using three consecutive samples and having culture and SD Bioline results with or without Microscopy and reverse hybridization-based Line Probe Assay(LPA) results. The data on co-morbidities or history of people infected with NTM were then collected from the three participants with available clinical data.ResultsWe collected data of 5267 presumptive TB people. Among them, 3436 (65.24%), have a positive culture with 3200 (60.75%) isolates belong to Mycobacterium tuberculosis Complex (MBTC) and 236 (4.48%) to NTM. Our results showed that, 123 (52.11%) NTM were isolated from people with negative microscopy and 113 (47.88%) from people with positive microscopy. Among the 236 NTM, 108 (45.8%) isolates were identified using LPA. M. fortuitum was the most represented species (32.41%) followed by M. intracellulare (19.44%). Sputum had the highest proportion of NTM (56%), followed by bronchial aspirations (31%). The extra-pulmonary samples presented lower proportions of isolates compared to pulmonary samples. Some patients affected with NTM presented comorbidities as HIV infection, Pulmonary tuberculosis, Type 2 diabetes, Chronic bronchitis and Alveolar pneumonia.ConclusionsOur study showed the presence of NTM strains among presumptive TB people with a predominance of M. fortuitum and M. intracellulare. It is important to implement a surveillance system of NTM in TB burden country and also to develop a point-of-care test for NTM identification in limited-resource settings.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021–Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.

A bacterial biosensor based on gold nanoparticles functionalized by a hydrophobin-chimera and combined with machine learning for user-friendly detection

A chimeric protein, in which a cationic antimicrobial peptide was fused to the adhesive self-assembling moiety of a fungal class I hydrophobin, was produced and used to easily functionalize gold nanoparticles (f-AuNPs). f-AuNPs are able to bind Escherichia coli and Staphylococcus epidermidis bacteria, tested as model organisms. A reverse colorimetric assay was then set up based on the induced aggregation of the free f-AuNPs. This leads to changes in optical properties of the solution, visible even by naked eyes. This approach allows detection of 10 CFU mL−1 of E. coli within 15 min and can be applicable to detect bacterial contamination in real matrices. Notably, the colorimetric variation was combined to machine-learning methodologies inferring data from photos of the colored solutions, acquired by smartphones. This fast, highly practical and flexible system, would enable on-site measurements and, therefore, help to prevent significant contaminations.

Development and field validation of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the rapid detection of chikungunya virus in patient and mosquito samples

ObjectivesWe aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil. MethodsWe optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases. ResultsOur RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in −1.18 PFU (confidence interval [CI] ranging from –2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20–30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97–100.00%), clinical specificity of 96.72% (95% CI, 88.65–99.60%), and overall accuracy of 98.00% (95% CI, 92.96–99.76%). DiscussionTaken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries.

Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus.

Among broilers, the main pathogen that leads to swollen head syndrome (SHS) is the subgroup C avian metapneumovirus (aMPV-C). The aMPV-C infection can lead to an upsurge in the rate of soft-shell eggs, resulting in reduced egg production and seriously affecting the economy of the livestock industry. Therefore, a rapid method for aMPV-C detection needs to be invented. According to the N gene of aMPV-C, we designed the specific probe and primer and created a reverse transcription recombinase-aided amplification assay (RT-RAA) for the detection of aMPV-C. aMPV-C could be detected quickly and specifically by this method at 41 °C for 30 min. The sensitivity assay inferred that the minimum detection threshold of RT-RAA was 3.38 × 101 copies/μL. A specificity assay showed that the RT-RAA method did not cross-react with other subgroups (aMPV-A, aMPV-B, aMPV-D) or other viruses (H9N2, NDV, IBV, IBDV). Forty samples of known clinical background were tested by RT-RAA and RT-qPCR. The two approaches had a 100% correlation rate. In conclusion, this research successfully created an RT-RAA assay for aMPV-C.

Melatonin Inhibits AGS Cell Proliferation by Binding to the ATP Binding Site of CDK2 Under Hyperglycemic Conditions.

Cancer cells utilize glucose as their primary energy source. The aggressive nature of cancer cells is therefore enhanced in hyperglycemic conditions. This study has been adopted to investigate the therapeutic potential of melatonin against such aggressive proliferation of AGS cells-a human gastric cancer cell line, under hyperglycemic conditions. AGS cells were incubated with high glucose-containing media, and the effects of melatonin have been evaluated, therein. Cell proliferation, ROS generation, flow-cytometric analysis for cell cycle and apoptosis, wound healing, immunoblotting, zymography, reverse zymography assays, in-silico analysis, and kinase activity assays were performed to evaluate the effects of melatonin. We observed that melatonin inhibited the hyperglycemia-induced cell proliferation in a dose-dependent manner. It further altered the expression and activity of MMP-9 and TIMP-1. Moreover, melatonin inhibited AGS cell proliferation by arresting AGS cells in the G0/G1 phase after binding in the ATP binding site of CDK-2, thereby inhibiting its kinase activity. In association, a significant decrease in the expression of cyclin D1, cyclin E, CDK-4, and CDK-2 were observed. In conclusion, these findings suggest that melatonin has anti-gastric cancer potential. Melatonin could therefore be included in future drug designs for gastric cancer-hyperglycemia co-morbidity treatment.

NCAPD3 promotes diffuse large B-cell lymphoma progression through modulating SIRT1 expression in an H3K9 monomethylation-dependent manner

IntroductionCondensin, a family of structural maintenance of chromosome complexes, has been shown to regulate chromosome compaction and segregation during mitosis. NCAPD3, a HEAT-repeat subunit of condensin II, plays a dominant role in condensin-mediated chromosome dynamics but remains unexplored in lymphoma. ObjectivesThe study aims to unravel the molecular function and mechanism of NCAPD3 in diffuse large B-cell lymphoma (DLBCL). MethodsThe expression and clinical significance of NCAPD3 were assessed in public database and clinical specimens. Chromosome spreads, co-immunoprecipitation (co-IP), mass spectrometry (MS), and chromatin immunoprecipitation (ChIP) assays were conducted to untangle the role and mechanism of NCAPD3 in DLBCL. ResultsNCAPD3 was highly expressed in DLBCL, correlated with poor prognosis. NCAPD3 deficiency impeded cell proliferation, induced apoptosis and increased the chemosensitivity. Instead, NCAPD3 overexpression facilitated cell proliferation. In vivo experiments further indicated targeting NCAPD3 suppressed tumor growth. Noteworthily, NCAPD3 deficiency disturbed the mitosis, triggering the formation of aneuploids. To reveal the function of NCAPD3 in DLBCL, chromosome spreads were conducted, presenting that chromosomes became compact upon NCAPD3 overexpression, instead, loose, twisted and lacking axial rigidity upon NCAPD3 absence. Meanwhile, the classical transcription-activated marker, H3K4 trimethylation, was found globally upregulated after NCAPD3 knockout, suggesting that NCAPD3 might participate in chromatin remodeling and transcription regulation. MS revealed NCAPD3 could interact with transcription factor, TFII I. Further co-IP and ChIP assays verified NCAPD3 could be anchored at the promoter of SIRT1 by TFII I and then supported the transcription of SIRT1 via recognizing H3K9 monomethylation (H3K9me1) on SIRT1 promoter. Function reversion assay verified the oncogenic role of NCAPD3 in DLBCL was partially mediated by SIRT1. ConclusionThis study demonstrated that dysregulation of NCAPD3 could disturb chromosome compaction and segregation and regulate the transcription activity of SIRT1 in an H3K9me1-dependent manner, which provided novel insights into targeted strategy for DLBCL.

Morphological changes and hub genes screening in rats with anorectal malformations during hindgut development

BackgroundAnorectal malformations (ARMs) are among the most common congenital digestive tract malformations worldwide. Although there have been extensive efforts for elucidating their pathogenesis, the specific molecular mechanisms underlying ARMs remain unclear. MethodsWe generated ARM Wistar rat models using ethylenethiourea. Nine pregnant rats were allocated to the ARM and control groups, respectively. Hindgut tissue was isolated from embryos collected on gestational days 14, 15, and 16, representing the key timepoints of anorectal development. High-throughput sequencing was used to identify differentially expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the obtained genes. Module analysis was performed via Search Tool for the Retrieval of Interacting Genes (STRING), and a protein-protein interaction (PPI) network was constructed using Cytoscape software. Gene Set Enrichment Analysis (GSEA) was conducted to verify the enrichment of development/apoptosis/epithelium/morphogenesis/Wnt pathway-related core gene sets and identify ARM hub genes. Reverse transcription-quantitative polymerase chain reaction assay was employed to verify mRNA levels of hub genes. Results313 GSEA core genes form ten core gene sets were selected. Based on the 281 differentially expressed circRNAs (|Fc| > 2, FDR < 0.05), 42 PPI core genes were obtained. According to the integration of GSEA analysis and PPI network, three hub genes (Cul1, Gli3, and Osr2) highly associated with ETU-induced ARMs were identified. The qRT-PCR validation data were consistent with the sequencing results. ConclusionsThese results provide a theoretical basis for the further identification of potential diagnostic and therapeutic targets in ARMs.

The impact of wound-healing assay, phorbol myristate acetate (PMA) stimulation and siRNA-mediated FURIN gene silencing on endogenous retroviral ERVW-1 expression level in U87-MG astrocytoma cells

PurposeHuman endogenous retroviruses (HERVs) are ubiquitous genomic sequences. Normally dormant HERVs, undergo reactivation by environmental factors. This deregulation of HERVs’ transcriptional equilibrium correlates with medical conditions such as multiple sclerosis (MS). Here we sought to explore whether exposing the U-87 MG astrocytoma cells to traumatic injury deregulates the expression of HERV-W family member ERVW-1 encoding syncytin-1. We also examined the expression of FURIN gene that is crucial in syncytin-1 synthesis. Material and methodsScratch assay was used as a model of cells injury in U-87 MG cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and migration assay using Boyden chamber were used. Phorbol 12-myristate 13-acetate (PMA) and small interfering RNA (siRNA) were used for cell stimulation and gene expression inhibition, respectively. ResultsResults revealed reduced ERVW-1 expression in cells exposed to injury (p ​< ​0.05) while GFAP gene - a marker of active astrocytes, was upregulated (p ​< ​0.01). These findings were confirmed by both WB and RT-qPCR. Expression of FURIN gene was not altered after injury, but cell stimulation by PMA strongly increased FURIN expression, simultaneously downregulating ERVW-1 (p ​< ​0.01). SiRNA-mediated expression inhibition of ERVW-1 and FURIN influenced the mRNA level for SLC1A5 (ASCT2) - primary syncytin-1 receptor, that was significantly lower. FURIN inhibition by siRNA caused strong upregulation of ERVW-1 expression (p ​< ​0.01). ConclusionResults showed that mechanical impact affects the expression of endogenous retroviruses in U-87 MG astrocytoma cells by scratch assay. Regulation of FURIN, a crucial enzyme in ERVW-1 turnover may support the therapy of some neurological conditions.

Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay

BackgroundTraditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control.MethodsWe aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/μl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China.ResultsThe established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/μl for S. aureus, 103 copies/μl for S. flexneri, 105 copies/μl for A. caviae, 105 copies/μl for V. vulnificus, 100 copies/μl for S. enterica, 105 copies/μl for P. vulgaris, and 100 copies/μl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species.ConclusionsOverall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.Graphical

Fabrication of biocompatible and biodegradable cloth-based sweat sensors using polylactic acid (PLA) via stencil transparent film-printing

Cloth-based sensors are gaining attention for creating microfluidic wearable sensors for continuous monitoring of sweat biomarkers because of their flexibility, low cost, and natural hydrophilicity making them suitable for integration into everyday products. Here, we present a novel, low-cost and simple fabrication approach to create a cloth-based sensor ready for use as a sweat sensor while being environmentally friendly using biocompatible and biodegradable material, polylactic acid (PLA), and a stencil transparent film-printing method. The method provided high-resolution structures with narrowest hydrophilic channels and hydrophobic barriers measuring 1.90 ± 0.34 mm and 1.23 ± 0.14 mm, respectively. Various designs for detecting sweat biomarkers, including pH, lactate, and chloride, were generated by simply adjusting the stencil transparent film designs. For pH detection, a square-shaped cloth-based sensor was constructed and different natural indicators was used as biocompatible and non-toxic reagents to measure pH providing the detection in the pH range of 1–14. In lactate determination, an enzymatic assay with nanoceria as colorimetric probes was used in a spot format, offering a lactate detection range of 2.5–25 mM, effectively distinguishing between normal and abnormal muscle fatigue patients at a cut-off level of 12.5 mM sweat lactate. For chloride, a distance-based format was employed using the reverse Mohr titration assay, resulting in a detection range of 10–100 mM. Finally, the analysis of lactate and chloride in artificial sweat was successfully demonstrated yielding the recovery in the acceptable range demonstrating accurate measurements of the fabricated cloth-based sensors.