Real-time Reverse Transcription-polymerase Chain Reaction Research Articles

TGFBR3 inhibits progression of papillary thyroid cancer by inhibiting the PI3K/AKT pathway and EMT.

Transforming growth factor beta receptor III (TGFBR3) has been shown to play a tumor-suppressive role in a variety of cancers. However, its role in papillary thyroid cancer (PTC) remains unknown. TGFBR3 expression levels in PTC were analyzed utilizing The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Edu, wound healing, and Transwell assays were used to evaluate cell proliferation, migration, and invasion. Transcriptome sequencing, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), and Western blotting were used to detect the underlying mechanism of TGFBR3 in PTC progression. This study demonstrated that TGFBR3 expression was significantly down-regulated in PTC compared to normal thyroid tissues. Low expression of TGFBR3 was associated with poor prognosis of patients with PTC. Furthermore, TGFBR3 expression positively correlated with thyroid differentiation score. In investigating the biological impact of TGFBR3 overexpression in PTC cell lines, we found that the proliferation, migration, and invasion of PTC cells were significantly inhibited in response to TGFBR3 overexpression. Moreover, we also demonstrated that overexpression of TGFBR3 inhibited the PI3K/AKT pathway and epithelial-mesenchymal transformation processes. Lastly, TGFBR3 expression was found to be involved in tumor immune infiltration, highlighting its potential influence on immune dynamics within the tumor microenvironment in PTC. TGFBR3 plays a tumor-suppressive role in PTC progression by inhibiting the PI3K/AKT pathway and epithelial mesenchymal transformation.

CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.

Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.

Genome-wide identification and expression analysis of the ZRT, IRT-like protein (ZIP) family in Nicotiana tabacum.

Iron (Fe) and Zinc (Zn) are essential micronutrients for plant growth and development. ZIP (ZRT, IRT-like protein) transporters, known for their role in the regulation of Zinc and Iron uptake, are pivotal in facilitating the absorption, transport, and maintenance of Fe/Zn homeostasis in plants. Nicotiana tabacum has been widely used as a model plant for gene function analysis; however, the tobacco ZIP genes have not been identified systematically. In this study, we have identified a comprehensive set of 32 NtZIP genes, which were phylogenetically categorized into three distinct clades. The gene structures, characterized by their exon/intron organization, and the protein motifs are relatively conserved, particularly among genes within the same clade. These NtZIP genes exhibit an uneven distribution across 12 chromosomes. The gene localization analysis revealed the presence of 11 pairs of homeologous locus genes and 7 pairs of tandem duplication genes within the genome. To further explore the functionality of these genes, real-time quantitative reverse transcription PCR was employed to assess their expression levels in roots subjected to metal deficiency. The results indicated that certain NtZIP genes are specifically upregulated in response to either Fe or Zn deficiency. Additionally, the presence of specific cis-elements within their promoter regions, such as the E-box associated with Fe deficiency response and the ZDRE box linked to Zn deficiency response, was identified. This study lays a foundational groundwork for future research into the biological functions of NtZIP genes in tobacco in micronutrient regulation and homeostasis.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

MicroRNA-1307-3p contributes to breast cancer progression through PRM2.

Despite advances in screening and therapy, breast cancer (BC) remains the predominant cancer in women globally. Dysregulation of microRNAs (miRNAs) is pivotal in carcinogenesis across various cancers, including BC. Evidence indicates that miR-1307-3p is upregulated in BC tumors, yet its target genes are not fully elucidated. This study aimed to explore how miR-1307-3p regulates BC proliferation, migration, invasion, and angiogenesis and to identify potential target genes. Basal miR-1307-3p levels were quantified in BC cell lines MDA-MB-231 and MCF-7, as well as MCF-10A using quantitative real-time reverse transcription-PCR (RT-qPCR). The impact of miR-1307-3p inhibition on BC cell proliferation, migration, invasion, and angiogenesis was assessed. Nine miRNA-target prediction databases identified potential miR-1307-3p targets. Target expression was validated using RT-qPCR, Western blot, and dual-luciferase reporter assays. MiR-1307-3p was overexpressed in MDA-MB-231 and MCF-7 compared to MCF-10A. Inhibiting miR-1307-3p significantly reduced BC cell proliferation, migration, invasion, and angiogenesis. Bioinformatics analysis identified 17 potential miR-1307-3p targets, with protamine 2 (PRM2) overexpression confirmed via Western blot and dual-luciferase assays. MiR-1307-3p overexpression in BC promotes proliferation, migration, invasion, and angiogenesis. PRM2 emerges as a novel miR-1307-3p target in BC.

Performance of CO-RADS in the diagnosis of COVID-19 in hemodialysis patients.

Different data systems have been used to reach a common decision in the interpretation of chest computed tomography (CT) scans for the detection of COVID-19 infection. The aim of our study was to determine the usefulness of the COVID-19 Reporting and Data System (CO-RADS) for COVID-19 in patients undergoing hemodialysis. We included 90 hemodialysis patients who underwent chest CT and had samples available for real-time reverse transcription-polymerase chain reaction (RT-PCR). The files of the patients were retrospectively reviewed and the data were recorded. Image interpretation and CO-RADS staging were performed retrospectively by two radiologists experienced in COVID-19 patients, blinded to the RT-PCR results. The RT-PCR results were then compared with the CO-RADS stages obtained. The success of CO-RADS in diagnosing COVID-19 was evaluated according to its prediction of a positive RT-PCR result. At the same time, the relationship between CO-RADS stages and prognosis was also evaluated. Patients were divided into two groups according to the RT-PCR results, of which 38 (42.2%) had positive results. Validity tests of CO-RADS were performed according to the RT-PCR test. While 71.1% of patients with CO-RADS ≥4 were RT-PCR (+), 13.2% of patients with CO-RADS ≤2 were RT-PCR (+) (p < 0.001). CO-RADS ≥4 detected COVID-19 with 71.1% sensitivity and 51.9% specificity. ROC analysis confirmed the diagnostic performance of CO-RADS for predicting RT-PCR positivity with AUC = 0.73 (95% CI: 0.63-0.84). The relationship between CO-RADS stages and mortality was not significant (p = 0.21). CO-RADS is moderately sensitive and poorly specific for the diagnosis of COVID-19 in hemodialysis patients. In these patients, CO-RADS would be more useful to exclude other infections than to diagnose COVID-19.

Lysine (K)-specific methyltransferase 2A (KMT2A) rearrangements among Iraqi de novo acute myeloid leukemia

Abstract: BACKGROUND: The classification of acute myeloid leukemia (AML) has evolved extensively over the last 20 years significantly, impacting the diagnosis and prognosis of the patients. The lysine (K)-specific methyltransferase 2A (KMT2A) gene, which has more than 80 gene fusions, is present in approximately 10% of all leukemias. Most KMT2A rearrangements are associated with adverse prognosis and need heavy chemotherapy protocol upfront. OBJECTIVE: This study aims to study the prevalence of KMT2A gene fusion among Iraqi patients with AML and its association with clinical and hematological parameters and patients’ outcomes. PATIENTS, MATERIALS, AND METHODS: A prospective cohort study conducted between December 2020 and May 2022 enrolled 115 Iraqi adults newly diagnosed with AML at the Hematology Unit of Baghdad Teaching Hospital. The patients were also monitored at this facility during the study period. Genetic rearrangements were detected using the Leukemia Q-Fusion Screening Kit through Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) analysis. RESULTS: KMT2A rearrangements were identified in 23 (20%) patients. The most common was t(10;11) which presented in 15 (13%) patients, followed by t(9;11) in 5 (4.3%) patients and t(11;17) in 3 (2.6%) patients. Patients with KMT2A rearrangements were significantly older and more likely to have splenomegaly. At 1-month posttreatment, they had significantly lower red blood cell counts and hemoglobin levels and higher blast percentages. Only 4.3% achieved complete remission (CR) compared to 76.1% without KMT2A rearrangements, with a significantly higher mortality rate (30.4% vs. 5.4%, P = 0.0001). Regarding the treatment response, no significant differences were observed among the different fusion types of KMT2A rearrangements. CONCLUSION: KMT2A rearrangements are more prevalent among Iraqi AML patients compared to the global trend and are associated with older age, higher rates of splenomegaly, poorer hematological recovery, and worse outcomes, regardless of the KMT2A rearrangement fusion type.

Screening and functional characterization of isocitrate lyase AceA in the biofilm formation of Vibrio alginolyticus.

Biofilm is a well-known sessile lifestyle for bacterial pathogens, but a little is known about the mechanism on biofilm formation in Vibrio alginolyticus. In this study, we screened V. alginolyticus strains with strong biofilm formation ability from coastal seawater. The antibiotic resistance of the biofilm cells (BFs) was higher than that of the planktonic cells (PTs). To study the genes and pathways involved in biofilm formation, we performed transcriptome analysis of the BFs and PTs of V. alginolyticus R9. A total of 685 differentially expressed genes (DEGs) were upregulated, and 517 DEGs were downregulated in the BFs. The upregulated DEGs were significantly enriched in several pathways including glyoxylate and dicarboxylate metabolism, while the downregulated genes were significantly enriched in the flagellar assembly pathways. The key gene involved in glyoxylate shunt, aceA, was cloned, and ΔaceA mutant was constructed to determine the function of AceA in carbon source utilization, biofilm formation, and virulence. Real-time reverse transcription PCR showed that the expression of aceA was higher at the mature stage but lower at the disperse stage of biofilm formation, and the expression of the flagellar related genes was upregulated in ΔaceA. This is the first study to illustrate the global gene expression profile during the biofilm formation of V. alginolyticus, and isocitrate lyase AceA, the key enzyme involved in glyoxylate shunt, was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the late stage.IMPORTANCEBiofilms pose serious public problems, not only protecting the cells in it from environmental hazard but also affecting the composition and abundance of bacteria, algae, fungi, and protozoa. The important opportunistic pathogen Vibrio alginolyticus is extremely ubiquitously present in seawater, and it also exhibited a strong ability to form biofilm; thus, investigation on the biofilm formation of V. alginolyticus at molecular level is fundamental for the deeper exploration of the environmental concerns arose by biofilm. In this study, transcriptome analysis of biofilm cells (BFs) and planktonic cells (PTs) from V. alginolyticus was performed and AceA was screened to play an important role in biofilm formation. AceA was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the disperse stage. This method was helpful to further understand the ability and mechanism of V. alginolyticus biofilm formation and provide clues for prevention of V. alginolyticus infection.

An integrative analysis of SARS-CoV 2 during the first and second waves of COVID-19 in Salta, Argentina

Wastewater surveillance has been extensively applied to provide information about SARS-CoV-2 circulation in the community. However, its applicability is limited in regions lacking adequate sewerage infrastructure, without wastewater treatment plants (WWTP) or with insufficient coverage. During the COVID-19 pandemic, from July 2020 to September 2021, comprehensive epidemiological data encompassing positive, recovered, and deceased cases were collected alongside precipitation records. Additionally, wastewater samples from 13 main sewersheds and river water from two points (up- and downstream the main WWTP), in the city of Salta, were gathered. A total of 452 water samples were analyzed for quantitative detection of SARS-CoV-2 using reverse transcription real-time PCR. Across the 62-week study period, two distinct waves of COVID-19 were identified. The dynamics of deceased cases showed peaks 10 and 28 days after the peaks of positive cases in the first and second waves, respectively. Downstream river water exhibited higher fecal contamination than the upstream samples, evincing the impact of the WWTP discharges. Viral concentration in river waters mirrored those from wastewater, reflecting the progression of cases. Despite the lower reported number of cases during the first wave in comparison to the second (5420 vs. 8516 cases at the respective peaks), higher viral concentrations were detected in water samples (1.97 × 107 vs. 2.36 × 106 gc/L, respectively), suggesting underreporting during the first wave, and highlighting the positive effect of vaccination during the second. To the best of our knowledge, this is the first study that simultaneously and systematically analyzed surface water and wastewater over a prolonged period, the effect of precipitations were considered for the variations in the concentrations, and the findings compared with epidemiological information. Environmental surveillance was demonstrated to be a great tool to obtain valuable information about the circulation patterns of SARS-CoV-2, especially under resource constraints to massively test the population, thus, underreporting cases. Furthermore, the methodology employed herein can be easily expanded to the community-level surveillance of other pathogens excreted in urine and feces, encompassing viruses, bacteria, and protozoa.

Effect of recombinant irisin on recombinant human bone morphogenetic protein-2 induced osteogenesis and osteoblast differentiation

Osteoporotic fragility fractures substantially impact aging societies, necessitating long-term care and increasing healthcare costs. Myokine irisin, secreted by skeletal muscle, influences bone metabolism; however, a comprehensive understanding of the mechanisms by which irisin affects bone metabolism is still lacking. Therefore, this study aimed to explore the effects of irisin on osteogenesis and osteoblast differentiation triggered by bone morphogenetic protein-2 (BMP-2). We used 4-week-old male ICR mice and implanted polyethylene glycol pellets containing recombinant human BMP-2 (rh-BMP-2) into the left dorsal muscle pouch. Mice received weekly intraperitoneal injections of either phosphate-buffered saline or recombinant irisin (re-irisin). Ectopic bone formation was evaluated 3 weeks post-surgery using micro-computed tomography (μ-CT) and histological analysis. In vitro experiments, C2C12 cells were treated with or without rh-BMP-2 and re-irisin, and we assessed osteoblast differentiation markers, e.g., runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osteopontin, using real-time reverse transcription-polymerase chain reaction. The μ-CT analyses showed that re-irisin significantly increased bone mineral content and bone volume of ectopic bones newly formed by rh-BMP-2. The gene expressions of the osteoblast markers were significantly increased by rh-BMP-2 and further upregulated by re-irisin. The treatment of cyclic AMP response element-binding protein (CREB) small interfering RNA attenuated these effects, suggesting that CREB signaling pathway was involved in rh-BMP-2/re-irisin-induced osteoblastic differentiation. This study demonstrates the potential of irisin to enhance osteogenesis through BMP signaling, offering insights for osteoporosis treatment and highlighting irisin as a promising therapeutic target for improving bone health and extending a healthy lifespan.

Role of miR-455-3p in the alleviation of LPS-induced acute lung injury by allicin

Acute lung injury (ALI) is a type of diffuse lung injury that seriously affects the survival of critically ill patients. MicroRNAs (miRNAs) can serve as promising therapeutic targets or offer insights for the development of potential therapeutic strategies against ALI. In our previous study, we demonstrated the protective effect of allicin in ALI, but the role of miRNAs in the alleviation of ALI by allicin remains unclear. This study aimed to investigate whether miRNAs mediate the effects of allicin on ALI. Cell viability and proliferation were determined using CCK-8 and EdU assays, respectively, while cellular apoptosis was analyzed by flow cytometry. The claudin-4 protein was detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting. The binding of miR-455 with claudin-4 was determined by bioinformatics analysis and validated by dual luciferase reporter assays. The lung wet/dry ratio of lipopolysaccharide (LPS)-treated rats was determined by hematoxylin and eosin (HE) and TUNEL staining of the pulmonary tissues. The levels of myeloperoxidase (MPO), interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were determined by enzyme-linked immunosorbent assay (ELISA). We observed that allicin alleviated LPS-induced injury in A549 cells, and claudin-4 knockdown reversed the protective effect of allicin in ALI. Claudin-4 is a direct target of miR-455-3p, and miR-455-3p overexpression partially reversed the protective effect of allicin in LPS-treated A549 cells. Subsequent in vivo experiments confirmed that allicin protects against LPS-induced ALI by regulating the miR-455-3p/claudin-4 axis. The study revealed that the protective effect of allicin in ALI is mediated via miR-455-3p, which suppresses the expression of claudin-4.

Radiosensitivity-related Variation in MicroRNA-34a-5p Levels and Subsequent Neuronal Loss in the Hilus of the Dentate Gyrus after Irradiation at Postnatal Days 10 and 21 in Mice.

The radiosensitivity of mice differs between postnatal days 10 (P10) and 21(P21); these days mark different stages of brain development. In the present study, Ki67 and doublecotin (DCX) immunostaining and hematoxylin staining was performed, which showed that acute radiation exposure at postnatal day 10 induced higher cell apoptosis and loss in the hilus of the dentate gyrus at day 1 postirradiation than postnatal day 21. MicroRNA (miRNA) sequencing and real-time quantitative reverse transcription PCR (qRT-PCR) analysis indicated the upregulation of miRNA-34a-5p at days 1 and 7 after irradiation at postnatal day 10, but not at postnatal day 21. Down-regulation of T-cell intracytoplasmic antigen-1 pathway (Tia1) was indicated by qRT-PCR at day 1 day but not day 7 after irradiation at postnatal day 10. Neurobehavioral testing in mature mice irradiated at postnatal day 10 demonstrated the impairment of short-term memory in novel object recognition and spatial memory, compared to those irradiated at postnatal day 21. Combined with our previous luciferase assay showing the direct interaction of miRNA34a-5p and Tia1, these findings suggest that radiation-induced abnormal miR-34a-5p/Tial interaction at day 1 after irradiation at postnatal day 10 may be involved in apoptosis of the dentate gyrus hilar, impairment of neurogenesis and subsequent short-term memory loss as observed in the novel object recognition and Barnes maze tests.

RRFERV stabilizes TEAD1 expression to mediate nasopharyngeal cancer radiation resistance rendering tumor cells vulnerable to ferroptosis.

Long noncoding RNAs (lncRNAs) regulate various essential biological processes, including cell proliferation, differentiation, apoptosis, migration, and invasion. However, in nasopharyngeal carcinoma (NPC), the clinical significance and mechanisms of lncRNAs in malignant progression are unknown. RNA sequencing and bioinformatic analysis were used to determine the potential function of RRFERV (radiation-resistant but ferroptosis vulnerable), and its biological effects were investigated using in vitro and in vivo experiments. Western blotting, quantitative real-time reverse transcription PCR, RNA immunoprecipitation (RIP) assays, and flow cytometry detected RRFERV expression. Ferroptosis and lipid peroxidation were added to evaluate the relationship between it and radiotherapy resistance. LncRNA-RRFERV was both highly expressed in NPC tissues and radiation-resistant cells. RRFERV is associated with poor clinical outcomes of NPC patients and stabilizes TEAD1 by competitive binding with microRNA-615-5p and microRNA-1293. RRFERV-TEAD1 signaling axis leads to malignant progression and radiotherapy resistance of NPC. Furthermore, we observed that NPC radiotherapy resistance cells exist in a fragile oxidative stress equilibrium, which makes them more sensitive to ferroptosis inducers. Surprisingly, we found that RRFERV-TEAD1 signaling axis also plays a key role in mediating the lipid peroxidation levels of NPC radiotherapy resistance cells through transcriptional activation of ACSL4/TFRC. RRFERV serves as an independent prognostic factor in NPC. During the malignant progression of NPC caused by high expression of RRFERV, ferroptosis can be induced to effectively kill cancer cells and reverse the radiotherapy resistance of NPC cells, suggesting a potential treatment approach for recurrent and refractory NPC.

Discriminating North American Swine Influenza Viruses with a Portable, One-Step, Triplex Real-Time RT-PCR Assay, and Portable Sequencing.

Swine harbors a genetically diverse population of swine influenza A viruses (IAV-S), with demonstrated potential to transmit to the human population, causing outbreaks and pandemics. Here, we describe the development of a one-step, triplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay that detects and distinguishes the majority of the antigenically distinct influenza A virus hemagglutinin (HA) clades currently circulating in North American swine, including the IAV-S H1 1A.1 (α), 1A.2 (β), 1A.3 (γ), 1B.2.2 (δ1) and 1B.2.1 (δ2) clades, and the IAV-S H3 2010.1 clade. We performed an in-field test at an exhibition swine show using in-field viral concentration and RNA extraction methodologies and a portable real-time PCR instrument, and rapidly identified three distinct IAV-S clades circulating within the N.A. swine population. Portable sequencing is used to further confirm the results of the in-field test of the swine triplex assay. The IAV-S triplex rRT-PCR assay can be easily transported and used in-field to characterize circulating IAV-S clades in North America, allowing for surveillance and early detection of North American IAV-S with human outbreak and pandemic potential.

Intracellular delivery of antiviral shRNA using penetratin-based complexes effectively inhibits respiratory syncytial virus replication and host cell apoptosis

BackgroundCell-penetrating peptides (CPPs) are effective for delivering therapeutic molecules with minimal toxicity. This study focuses on the use of penetratin, a well-characterized CPP, to deliver a DNA vector encoding short hairpin RNA (shRNA) targeting the respiratory syncytial virus (RSV) F gene into infected cells. RSV is known to cause severe lower respiratory infections in infants and poses significant risks to immunocompromised individuals and the elderly. We evaluated the antiviral efficacy of the penetratin-shRNA complex by comparing its ability to inhibit RSV replication and induce apoptosis with ribavirin treatment.MethodsPenetratin-shRNA complexes were prepared at different ratios and analyzed using gel retardation assays, dynamic light scattering, and zeta potential measurements. The complexes were tested in HEp-2 and A549 cells for transfection efficiency, cytotoxicity, viral load, and apoptosis using plaque assays, real-time reverse transcription-polymerase chain reaction (RT-PCR), DNA fragmentation, propidium iodide staining, and caspase 3/7 activation assays.ResultsThe gel shift assay determined that a 20:1 CPP-to-shRNA ratio was optimal for effective complexation, resulting in particles with a size of 164 nm and a zeta potential of 8.7 mV. Transfection efficiency in HEp-2 cells was highest at this ratio, reaching up to 93%. The penetratin-shRNA complex effectively silenced the RSV F gene, reduced viral titers, and decreased DNA fragmentation and apoptosis in infected cells.ConclusionPenetratin effectively delivers shRNA targeting the RSV F gene, significantly reducing viral load and preventing apoptosis without toxicity. This approach surpasses Lipofectamine and shows potential for future therapeutic interventions, especially when combined with ribavirin, against RSV infection.Graphical

Frequency of COVID-19 Among Pregnant Patients at a Tertiary Care Hospital, Lahore: A Cross-Sectional Study

AbstractCoronavirus Disease 2019 (COVID-19) among pregnant women is a major health concern. Determining the incidence of COVID-19 among pregnant women is crucial for providing appropriate care. This study was conducted to determine the frequency of COVID-19 in pregnant females at a Tertiary care Hospital, Lahore. It was a cross-sectional study conducted among 150 pregnant females through convenient sampling from Gynecology &amp; Obstetrics outpatient department (OPD) of tertiary care hospital, Lahore. Data was obtained through questionnaire. COVID-19 status was confirmed by nasopharyngeal swabs for real-time reverse transcription polymerase chain reaction (RT-PCR). Data was entered and analyzed using SPSS version 24.0. Chi-square test was used, with p-value &lt;0.05 was considered significant. The frequency of COVID-19 was 4.7% among 150 pregnant females. About 130(86.7%) females were vaccinated for COVID-19, among them only 2 % were found positive (p=0.000). About 30(20.0%) females were symptomatic however only 1.3% were found positive (p=0.561). There were 23(15.3%) pregnant females who had risk factors but only 1.3% were found positive (p=0.319) for COVID-19.Education and vaccination status were significant factors for COVID-19 infection. The study concluded that the frequency of COVID-19 was 4.7% among pregnant females, with education and vaccination status being significant factors.

The effects of apelin-13 in a mouse model of post-traumatic stress disorder.

The objective is to investigate the effects of apelin-13 in models of post-traumatic stress disorder (PTSD). Mature male CD1 mice were subjected to the single prolonged stress method to induce PTSD-related behaviors. These behaviors were then evaluated using the elevated plus maze test, Morris water maze test, and open field test. Hippocampal neural cell death was assessed using propidium iodide labeling. The expression of hippocampal autophagy pathway-associated proteins was determined through immunoblotting analysis, and LC3 levels were also measured via quantitative real-time reverse transcription-PCR. The results demonstrate that administration of apelin-13 suppressed PTSD-induced hippocampal neural cell death and alleviated PTSD-related behaviors in mice. Additionally, PTSD led to an up-regulation of LC3 and FoxO3a, and down-regulation of P62, p-PI3K, p-Akt, and p-FoxO3a in the hippocampus. However, these changes were reversed by apelin-13 treatment. These findings support the hypothesis that apelin-13 prevents the development of PTSD-like behavior and inhibits autophagy of neuronal cells in a mouse model of PTSD. Apelin-13 may hold potential as a therapeutic agent for PTSD in clinical applications.

Functional characterization of Arabidopsis hydroxynitrile lyase in response to abiotic stress and the regulation of flowering time.

Hydroxynitrile lyases (HNLs) are a class of hydrolytic enzymes from a wide range of sources, which play crucial roles in the catalysis of the reversible conversion of carbonyl compounds derived from cyanide and free cyanide in cyanogenic plant species. HNLs were also discovered in non-cyanogenic plants, such as Arabidopsis thaliana, and their roles remain unclear even during plant growth and reproduction. The pattern of expression of the HNL in A. thaliana (AtHNL) in different tissues, as well as under abiotic stresses and hormone treatments, was examined by real-time quantitative reverse transcription PCR (qRT-PCR) and an AtHNL promoter-driven histochemical β-glucuronidase (GUS) assay. AtHNL is highly expressed in flowers and siliques, and the expression of AtHNL was dramatically affected by abiotic stresses and hormone treatments. The overexpression of AtHNL resulted in transgenic A. thaliana seedlings that were more tolerance to mannitol and salinity. Moreover, transgenic lines of A. thaliana that overexpressed this gene were less sensitive to abscisic acid (ABA). Altered expression of ABA/stress responsive genes was also observed in hnl mutant and AtHNL-overexpressing plants, suggesting AtHNL may play functional roles on regulating Arabidopsis resistance to ABA and abiotic stresses by affecting ABA/stress responsive gene expression. In addition, the overexpression of AtHNL resulted in earlier flowering, whereas the AtHNL mutant flowered later than the wild type (WT) plants. The expression of the floral stimulators CONSTANS (CO), SUPPRESSOR OF OVER EXPRESSION OF CO 1 (SOC1) and FLOWERING LOCUS T (FT) was upregulated in plants that overexpressed AtHNL when compared with the WT plants. In contrast, expression of the floral repressor FLOWERING LOCUS C (FLC) was upregulated in AtHNL mutants and downregulated in plants that overexpressed AtHNL compared to the WT plants. This study revealed that AtHNL can be induced under abiotic stresses and ABA treatment, and genetic analysis showed that AtHNL could also act as a positive regulator of abiotic stress and ABA tolerance, as well as flowering time.

The oncogenic ADAMTS1–VCAN–EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma

BackgroundMetastasis, the leading cause of renal cell carcinoma (RCC) mortality, involves cancer cells resisting anoikis and invading. Until now, the role of the matrix metalloproteinase (MMP)-related enzyme, A disintegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1), in RCC anoikis regulation remains unclear.MethodsThe clinical significance of ADAMTS1 and its associated molecules in patients with RCC was investigated using data from the Gene Expression Omnibus (GEO) and TCGA datasets. Human phosphoreceptor tyrosine kinase (RTK) array, luciferase reporter assays, immunoprecipitation (IP) assays, western blotting, and real-time reverse-transcription quantitative polymerase chain reaction (RT–qPCR) were used to elucidate the underlying mechanisms of ADAMTS1. Functional assays, including anoikis resistance assays, invasion assays, and a Zebrafish xenotransplantation model, were conducted to assess the roles of ADAMTS1 in conferring resistance to anoikis in RCC.ResultsThis study found elevated ADAMTS1 transcripts in RCC tissues that were correlated with a poor prognosis. ADAMTS1 manipulation significantly affected cell anoikis through the mitochondrial pathway in RCC cells. Human receptor tyrosine kinase (RTK) array screening identified that epidermal growth factor receptor (EGFR) activation was responsible for ADAMTS1-induced anoikis resistance and invasion. Further investigations revealed that enzymatically active ADAMTS1-induced versican V1 (VCAN V1) proteolysis led to EGFR transactivation, which in turn, through positive feedback, regulated ADAMTS1. Additionally, ADAMTS1 can form a complex with p53 to influence EGFR signaling. In vivo, VCAN or EGFR knockdown reversed ADAMTS1-induced prometastatic characteristics of RCC. A clinical analysis revealed a positive correlation between ADAMTS1 and VCAN or the EGFR and patients with RCC with high ADAMTS1 and VCAN expression had the worst prognoses.ConclusionsOur results collectively uncover a novel cyclic axis involving ADAMTS1–VCAN–EGFR, which significantly contributes to RCC invasion and resistance to anoikis, thus presenting a promising therapeutic target for RCC metastasis.

A systematic review on the selection of reference genes for gene expression studies in rodents: are the classics the best choice?

Rodents are commonly used as animal models in studies investigating various experimental conditions, often requiring gene expression analysis. Quantitative real-time reverse transcription PCR (RT-qPCR) is the most widely used tool to quantify target gene expression levels under different experimental conditions in various biological samples. Relative normalization with reference genes is a crucial step in RT-qPCR to obtain reliable quantification results. In this work, the main reference genes used in gene expression studies among the three rodents commonly employed in scientific research-hamster, rat, and mouse-are analyzed and described. An individual literature search for each rodent was conducted using specific search terms in three databases: PubMed, Scopus, and Web of Science. A total of 157 articles were selected (rats = 73, mice = 79, and hamsters = 5), identifying various reference genes. The most commonly used reference genes were analyzed according to each rodent, sample type, and experimental condition evaluated, revealing a great variability in the stability of each gene across different samples and conditions. Classic genes, which are expected to be stably expressed in both samples and conditions analyzed, demonstrated greater variability, corroborating existing concerns about the use of these genes. Therefore, this review provides important insights for researchers seeking to identify suitable reference genes for their validation studies in rodents.