Reverse Transcription-polymerase Chain Reaction Assay Research Articles

Research on the improvement effect of Saposhnikovia divaricata (Trucz.) Schischk on rheumatoid arthritis based on the “component-target-pathway” association

ObjectiveTo investigate the therapeutic effect and mechanism of the traditional Chinese medicine Saposhnikovia divaricata (Trucz.) Schischk in rats with complete Freund's adjuvant-induced rheumatoid arthritis (RA). MethodsThe chemical targets and RA targets of Saposhnikovia divaricata (Trucz.) Schischk were acquired by the network pharmacological method. The complete Freund's adjuvant-induced rat RA model was used to further explore the mechanism of Saposhnikovia divaricata (Trucz.) Schischk in improving RA. Pathological changes in the volume of toes, body weight and synovial tissues of joints as well as serum inflammatory factor levels before and after the intervention of Saposhnikovia divaricata (Trucz.) Schischk were investigated. The key metabolic pathways were screened by correlations between metabolites and key targets. Finally, a quantitative analysis of key targets and metabolites was experimentally validated. ResultsSaposhnikovia divaricata (Trucz.) Schischk administration increased body weight, mitigated foot swelling and downregulated inflammatory cytokine levels in model rats. The histopathology showed that treatment with Saposhnikovia divaricata (Trucz.) Schischk can induce inflammatory cell infiltration and synovial hyperplasia and obviously reduce cartilage injuries, thus improving arthritis symptoms in rats. According to the network pharmacology-metabonomics association analysis results, the purine metabolic signaling pathway might be the key pathway for RA intervention with Saposhnikovia divaricata (Trucz.) Schischk. Targeted metabonomics, Western blotting (WB) and reverse transcription-polymerase chain reaction (RT‒PCR) assays showed that the recombinant adenosine deaminase (ADA) mRNA expression level and metabolic level of inosine in Saposhnikovia divaricata (Trucz.) Schischk administration group were lower than those of the model group. This reflected that Saposhnikovia divaricata (Trucz.) Schischk could improve RA by downregulating ADA mRNA expression levels and the metabolic level of inosine in the purine signaling pathway. ConclusionBased on the “component-disease-target” association analysis, this study concludes that Saposhnikovia divaricata (Trucz.) Schischk improves complete Freund's adjuvant-induced RA symptoms in rats mainly by downregulating ADA mRNA expression levels in the purine metabolic signaling pathway, mitigating foot swelling, improving the levels of serum inflammatory factors (IL-1β, IL-6 and TNF-α), and decreasing the ADA protein expression level to intervene in purine metabolism.

Investigation of SARS-CoV-2 Detection Method Applicability and Virus Occurrence in Food and Food Packaging.

While it is clear that SARS CoV-2 coronavirus is the primary respiratory virus, there are no entirely clarified ways of transmission. Foodborne transmission has remained an unexplained path. Therefore, the goals of this paper are to examine and present an assessment of the most appropriate of the four selected kits for RNA extraction for the testing and detection of SARS-CoV-2 on food packaging surfaces, food surfaces, and in food. This will enable to indicate the possibility of infection through contact or direct food consumption. Finding the best technique is vital as RNA extraction is one of the essential elements in detecting SARS-CoV-2. This was achieved through an experiment with four commercial kits following the original manufacturers' protocols, and with a modification of the original protocols that included the use of ethanol and isopropanol. The selected kit was used for RNA extraction from the swabs of packaging surfaces, food surface, and ready-to-eat food samples. The coronavirus was then identified using real-time reverse transcription-polymerase chain reaction (RT-PCR) assays to determine whether the SARS-CoV-2 virus or viral particles are present in the food chain with the overall purpose of demonstrating the possibility that food can contribute as a vehicle for the transmission of the virus. The findings of this investigation made the most effective extraction kit and protocol stand out. The results of the applicability of the kit indicated a significant share of positive samples of viral SARS-CoV-2 virus particles on surfaces from the environment where infected persons with 'silent' COVID-19 infection, with mild symptoms or no symptoms, were present. However, according to the findings of the second part of the study, the virus was not detected on the examined samples of food packaging surfaces, food surfaces, and food. The presented results distinguished one of the most suitable protocols for isolating RNA from environmental surface samples. The main contribution of the study is in the presentation of the results, that is, the examination of samples that are primarily related to the food chain, food packaging, food surfaces, and ready-to-eat food. The results of this study could also be helpful for further determination of the potential of food as a vector for the transmission of coronaviruses.

Overexpression of RAB31 in gastric cancer is associated with released exosomes and increased tumor cell invasion and metastasis.

Gastric cancer (GC) is one of most common cancers worldwide. Several studies have suggested that Rab31 functions as a membrane vesicle transport regulator; however, the mechanism by which RAB31 regulates exosome secretion and promotes metastasis remains to be clarified. We examined the expression of RAB31 protein and mRNA in GC tissue samples via immunohistochemistry and reverse transcription-polymerase chain reaction assays, respectively. We elucidated the function of RAB31 in GC cells by constructing a cell model and a pulmonary metastatic model of GC with overexpression of RAB31. Protein mass spectrometry was used to identify the exosomal protein. RAB31 expression increased at both the protein and mRNA levels with the development of GC. Cells overexpressing RAB31 showed an enhanced ability to migrate in both the in vitro cell model and the pulmonary metastatic model of GC. Exosome nanoparticle tracking analysis and electron microscopy revealed that the both the number and size of the exosomes secreted by GC cells were reduced when RAB31 expression was depleted. Injection of exosomes derived from RAB31 overexpressing cells promoted pulmonary metastasis in vivo. Analysis of the exosomal proteins revealed that PSMA1 was overexpressed in GC tissue in accordance with RAB31 expression. PSMA1 overexpression was highly associated with poor prognosis of GC patients. Our findings revealed a key role for RAB31 in GC metastasis through regulation of exosome secretion.

Hu'po Anshen decoction promotes fracture healing in mice with traumatic brain injury through BMP2-COX2-ATF4 signaling pathway.

Hu'po Anshen decoction (HPASD), a traditional Chinese medicine used to treat concussion and fracture, could regulate the expression of bone morphogenetic protein 2 (BMP2). However, whether HPASD affects the fracture healing of traumatic brain injury (TBI) combined with a fracture through BMP2 and its downstream signals remains obscure. The chondrocyte-specific BMP2 conditional knockout mice and chondrocyte-specific cyclooxygenase-2 (COX2) overexpression mice were generated. BMP2 conditional knockout mice were treated with fracture surgery, fracture combined with TBI, or fracture combined with TBI followed by different doses of HPASD (2.4, 4.8, and 9.6 g/kg), respectively. TBI was induced by Feeney's weight-drop technique. The fracture callus formation and fracture sites were determined by X-ray, micro-CT, and histological analyses. The expressions of chondrocyte-, osteoblast-, and BMP2/COX2 signal-related targets were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot assays. The specific absence of BMP2 in chondrocytes led to the prolonged formation of cartilage callus, a delay in the osteogenesis initiation and the downregulation of RUNX2, Smad1/5/9, EP4, ERK1/2, RSK2, ATF4. Overexpression of COX2 partially reverses the effects of chondrocyte-specific BMP2 knockout mice. HPASD promoted cartilage callus formation and osteogenesis initiation, as accompanied by upregulated expression levels of RUNX2, Smad1/5/9, EP4, ERK1/2, RSK2, and ATF4 in a time-dependent and concentration-dependent manner in chondrocyte-specific BMP2 knockout mice. Overall, our findings demonstrated that HPASD induced COX2 transcription through the BMP2-Smad1/5/9-RUNX2 axis, and then affected fracture healing through the COX2-mediated EP4-ERK1/2-RSK2-ATF4 axis.

Evaluation of Inconclusive Results on the Cepheid X-Pert Xpress Platform (GXP) for the Diagnosis of Severe Acute Respiratory Syndrome-2: A Narrative Literature Review

Introduction:Molecular assay for diagnosing and detecting SARS-COV-2 is an essential tool in pandemic management, allowing for early informed decision-making. Worldwide, the gold standard for testing SARS-COV-2 includes real-time reverse transcription-polymerase chain reaction assay (RT-PCR). The Cepheid Xpert-Xpress was authorized for emergency diagnosis of SARS-COV-2. This platform demonstrated various advantages, including faster results, due to a decreased turnaround time, and decreased contamination risk. However, inconclusive results often leave clinicians uncertain regarding individual patient management decisions. Often leading to more confusion than answers.The aim of this literature review includes the following: identify the frequency and clinical implications of inconclusive results for SARS-COV-2 diagnosis utilizing GeneXpert assaywhether inconclusive results should be interpreted as negativeassessing the reliability of the GeneXpert platform to diagnose SARS-COV-2Method:A narrative literature review was conducted with eight critically appraised articles which met the inclusion criteria.After the initial data collection, the SANRA Framework was implemented to aid in the sorting and filtering of data. The analysis of data was conducted with a critical appraisal tool.Results:The GeneXpert SARS-COV-2 assay demonstrated high sensitivity and specificity. Studies indicated that inconclusive results associated with a high cycle-threshold value (CT-value) of more than thirty-five on the Cepheid Xpert Xpress were associated with a decreased viral load and, thus, decreased infectivity. However, numerous factors influence the CT-value, such as specimen integrity. Thus, results must not be interpreted in isolation.Conclusion:This narrative literature review demonstrated the need for institutions to assist clinicians with decision-making regarding inconclusive results. A flow diagram grading a patient’s risk of having SARS-COV-2 with an inconclusive result could be of immense value. The flow diagram should incorporate the current epidemiology in the area, patient symptomology and risk and duration of exposure.

Single-step multiplex reverse transcription-polymerase chain reaction for the detection and differentiation of QX-like infectious bronchitis virus from the Thai variant and vaccine strains H120, Ma5, and 4/91.

QX-like infectious bronchitis virus (IBV) is a highly infectious avian coronavirus that causes respiratory and kidney disease. It is linked to increased mortality and loss of performance in infected chickens worldwide, including Thailand. Thus, a simple and rapid diagnostic method for the diagnosis of QX-like IBV is needed. This study aimed to develop a single-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay to detect and differentiate QX-like IBV from Thai IBV and vaccine strains used in the poultry industry (H120, Ma5, and 4/91). Primer sets specific for QX-like and Thai IBV were designed to target the S1 gene. The specificity of the technique was verified using nine isolates of QX-like IBV, four isolates of Thai IBV, and other avian viral respiratory pathogens. The detection limit was evaluated using a serial ten-fold dilution of QX-like and Thai IBV. The results showed that single-step mRT-PCR could detect QX-like IBV and differentiate it from Thai IBV and the vaccine strains H120, Ma5, and 4/91. The limit of detection of the developed assay was 102.2 embryo infectious dose (EID)50/mL for QX-like IBV and 101.8 EID50/mL for Thai IBV. Interestingly, the developed assay could identify mixed infection by both IBVs in a single sample. The single-step mRT-PCR assay developed in this study can potentially discriminate QX-like IBV from Thai IBV and the vaccine strains H120, Ma5, and 4/91 in a single reaction. It is also suitable for use in all laboratories with access to conventional PCR equipment.

Hypoxia-induced Wnt/β-catenin signaling activation in subchondral bone osteoblasts leads to an osteoarthritis-like phenotype of chondrocytes in articular cartilage.

Background: Osteoarthritis (OA) is a whole-joint disease and characterized by alterations in the articular cartilage, subchondral bone, ligaments, and synovial membrane. The crosstalk between cartilage and subchondral bone plays a crucial role in the pathogenesis and progression of OA. Hypoxia has been reported to play an important role in cartilage degradation and subchondral bone remodeling in OA. In this study, we aimed to identify the involvement of hypoxia in modifying the osteoblast phenotypes and determine whether these alterations could influence the metabolism of chondrocytes. Methods: First, the levels of Hif-1α in subchondral bone of different compartments in patients with OA were assessed using immunohistochemistry (IHC). In in vitro, human primary osteoblasts were cultured under hypoxic and normoxic conditions, and the hypoxic or normoxic conditioned media (HCM and NCM) were used to culture human primary chondrocytes. Then, phenotypic changes in osteoblasts were assessed using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Furthermore, the expression of type II collagen (COL2A1), aggrecan (ACAN), SRY-related high-mobility group-box gene 9 (SOX9), matrix metalloproteinase 13 (MMP13), and matrix metalloproteinase 3 (MMP3) in chondrocytes was measured using RT-PCR. Finally, the serum levels of Wnt-related proteins were determined using ELISA. Results: Hif-1α was significantly increased in severely sclerotic subchondral bone compared to less damaged subchondral bone. β-Catenin and SOST were identified as upregulated and downregulated in hypoxic osteoblasts, respectively. The hypoxia-induced results were confirmed by ELISA. Stimulating human primary chondrocytes with HCM significantly induced MMP13 and MMP3 and inhibited COL2A1, ACAN, and SOX9 mRNA expression. The serum levels of DKK-1 were significantly increased in human OA. Conclusion: Together, these findings revealed that hypoxia in subchondral bone is a key factor in the crosstalk between chondrocytes and osteoblasts and facilitates the shift of chondrocytes toward an OA-like phenotype probably by activating the Wnt/β-catenin signaling pathway in osteoblasts.

Expression of lncRNA DLX6-AS1 in patients with hepatic carcinoma and its prognostic value

ABSTRACT Hepatic carcinoma (HCC) is one of the most common malignant tumors worldwide, and the prognosis of HCC patients is often poor. Long-chain non-coding RNA (lncRNA) distal-less homeobox 6 antisense 1 (DLX6-AS1) has been shown to be involved in the pathogenesis of various cancers. This study aims to investigate the expression of DLX6-AS1 in HCC patients and its prognostic value. The serum DLX6-AS1 was quantified using a reverse transcription-polymerase chain reaction (RT-PCR) assay in both HCC patients and healthy individuals, and the correlation of DLX6-AS1 with clinicopathological features of HCC patients, as well as the diagnostic and prognostic value of DLX6-AS1 for HCC patients, were analyzed. The results showed that the expression of serum DLX6-AS1 in HCC patients was significantly higher than that of healthy individuals (P < 0.05), and DLX6-AS1 was related to tumor differentiation, pathological staging, and lymph node metastasis (all P < 0.05). Patients with high DLX6-AS1 expression showed significantly higher mortality than those with low DLX6-AS1 expression, and the DLX6-AS1 expression in dead patients was significantly higher than that in living patients. Furthermore, the AUC of DLX6-AS1 for poor prognosis of HCC patients was larger than 0.8. The univariate analysis revealed that the poor prognosis of HCC patients was related to pathological staging, lymph node metastasis, differentiation, and DLX6-AS1 expression (all P < 0.05), and the Cox multivariate analysis revealed that pathological staging, lymph node metastasis, differentiation, and DLX6-AS1 expression were independent risk factors for poor prognosis of HCC patients (all P < 0.05). These findings suggest that DLX6-AS1 may be a promising target for diagnosis, treatment, and prognosis prediction of HCC patients.

Pretreatment of kaempferol-3-O-rutinoside protects H9c2 cells against LPS-induced inflammation through the AMPK/SIRT1 pathway

Kaempferol-3-O-rutinoside (KR) is a flavonoid glycoside derived from traditional Chinese medicine, plants, and tea, and has good myocardial protection. This study focuses on the mechanism of myocardial protection with KR and whether myocardial protection can be achieved by activating the adenosine monophosphate-activated protein kinase–silent mating-type information regulation 2 homolog 1 (sirtuin) (AMPK/SIRT1) signal pathway. Molecular docking technique was used to predict the binding affinity of KR to AMPK. The inflammatory injury model of H9c2 cells was established by lipopolysaccharide (LPS) induction. H9c2 cell proliferation was detected by cell counting kit -8 assay. The apoptosis rate was measured by flow cytometry. Levels of interlukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked-immunosorbent serologic assay. AMPK and SIRT1 expression levels were quantified through reverse transcription-polymerase chain reaction and Western blotting assay. Results indicated that KR has a certain binding affinity with AMPK. KR could increase the growth of H9c2 inhibited by LPS, reduce apoptosis rate, and reverse the elevated levels of IL-1β, IL-6, and TNF-α. Furthermore, KR could suppress the expression levels of AMPK and SIRT1, and AMPK-specific inhibitor (Compound C) could significantly counteract the activation of KR, indicating that its anti-inflammatory effect is achieved by regulating the AMPK/SIRT1 signaling pathway.

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

West Nile Virus and Epizootic Hemorrhagic Disease Virus Co-Infection in a Novel Host at the Nashville Zoo.

On August 30, 2017, one of five bontebok in a mixed-species exhibit at the Nashville Zoo at Grassmere exhibited acute hind-limb ataxia and altered demeanor. Pathological examination demonstrated meningoencephalitis and spinal myelitis. Coinfection of West Nile virus (WNV) and epizootic hemorrhagic disease virus (EHDV) was revealed by quantitative real-time and traditional reverse transcription-polymerase chain reaction assays and virus isolation/whole genome sequencing from brain tissue, respectively. Whole genome sequencing was conducted for EHDV. Mosquito testing from September 19 to October 13, 2017, demonstrated a higher WNV infection rate in mosquitoes at the zoo compared with the rest of Nashville-Davidson County. EHDV is endemic in wild white-tailed deer (family Cervidae) in Tennessee, and the prevalence in wildlife depends on environmental influences. This case illustrates the potential susceptibility of exotic zoo animals to endemic domestic arthropod-borne viruses (arboviruses) and reinforces the importance of cooperative antemortem and postmortem surveillance strategies among human, wildlife, and domestic animal health agencies.

Baicalin can enhance odonto/osteogenic differentiation of inflammatory dental pulp stem cells by inhibiting the NF-κB and β-catenin/Wnt signaling pathways.

Scutellaria baicalensis Georgi is a famous traditional Chinese medicine, which is widely used in treating fever, upper respiratory tract infection and other diseases. Pharmacology study showed it can exhibit anti-bacterial, anti-inflammation and analgesic effects. In this study, we investigated the effect of baicalin on the odonto/osteogenic differentiation of inflammatory dental pulp stem cells (iDPSCs). iDPSCs were isolated from the inflamed pulps collected from pulpitis. The proliferation of iDPSCs was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazolium bromide (MTT) assay and flow cytometry. Alkaline phosphatase (ALP) activity assay, alizarin red staining, Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were conducted to examine the differentiation potency along with the involvement of nuclear factor kappa B(NF-κB) and β-catenin/Wnt signaling pathway. MTT assay and cell-cycle analysis demonstrated that baicalin had no influence on the proliferation of iDPSCs. ALP activity assay and alizarin red staining demonstrated that baicalin could obviously enhance ALP activity and calcified nodules formed in iDPSCs. RT-PCR and Western blot showed that the odonto/osteogenic markers were upregulated in baicalin-treated iDPSCs. Moreover, expression of cytoplastic phosphor-P65, nuclear P65, and β-catenin in iDPSCs was significantly increased compared with DPSCs, but the expression in baicalin-treated iDPSCs was inhibited. In addition, 20µM Baicalin could accelerate odonto/osteogenic differentiation of iDPSCs via inhibition of NF-κB and β-catenin/Wnt signaling pathways. Baicalin can promote odonto/osteogenic differentiation of iDPSCs through inhibition of NF-κB and β-catenin/Wnt pathways, thus providing direct evidence that baicalin may be effective in repairing pulp with early irreversible pulpitis.

Potential anti-cancer effects of hibernating common carp (Cyprinus carpio) plasma on B16-F10 murine melanoma: In vitro and in vivo studies

Melanoma is the major type of skin cancer, which its treatment is still a challenge in the world. In recent years, interest in hibernation-based therapeutic approaches for various biomedical applications has been increased. Many studies indicated that some factors in the blood plasma of hibernating animals such as alpha-2-macroglobulin (A2M) cause anti-proliferative effects. Considering that, the present study was conducted to investigate the anti-cancer effects of hibernating common carp plasma (HCCP) on murine melanoma (B16-F10) in vitro and in vivo. The effect of HCCP on cell viability, migration, apoptosis rate, and cell cycle distribution of B16-F10 cells, tumor growth, and rate of survival were evaluated. To investigate the role of A2M in the anti-cancer effects of HCCP, the gene of interest and proteins in HCCP and non-hibernating common carp plasma (NHCCP) were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analysis. Based on our findings, HCCP significantly decreased B16-F10 cell viability. Moreover, HCCP caused morphological alternations, inhibition of migration, induction of apoptosis, and significantly induced the cell cycle arrest at the G2/M phase. In addition, A2M level was significantly increased in HCCP compared with NHCCP. Taken together, our findings suggested that HCCP had the potential to be a promising novel therapeutic target for cancer treatment because of its anti-cancer properties.

Protocatechuic aldehyde promotes the functional recovery of spinal cord injury by activating the Wnt/β-catenin signaling pathway

Context/objective This study aimed to explore the anti-inflammatory and neuroprotective effects of protocatechuic aldehyde (PCA) in rats with spinal cord injury (SCI) and to clarify the molecular mechanisms underlying its pharmacological effects. Design Male Sprague Dawley rat model of moderate spinal cord contusion were established. Setting Third-class first-class hospital. Outcome measures The Basso, Beattie, and Bresnahan scores and performance on the inclined plane test were evaluated. Histological analyses were performed via hematoxylin and eosin staining. Apoptosis in the spinal cord and neurons was detected by 5 terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining. Apoptotic factors (Bax, Bcl-2, and cleaved caspase-3) were also evaluated. INOS, IL-1β, IL-10, TNF-α, Wnt-3α, β-catenin, iBA-1, and NeuN were assessed by real-time reverse transcription-polymerase chain reaction (RT–PCR), western blotting (WB), and enzyme-linked immunosorbent assay. Cell viability and the immunofluorescence of IL-1β were measured in PC-12 cells. Results Using WB and quantitative reverse transcription-PCR, we confirmed that PCA treatment activated the Wnt/β-catenin signaling axis in vivo and in vitro. Hematoxylin and eosin staining and hindlimb motor functional evaluation revealed that treatment with PCA improved tissue protection and functional recovery via the Wnt/β-catenin axis. The upregulation of TUNEL-positive cells, downregulation of neurons, elevated apoptosis-associated factors in rats, and increased apoptotic rates were observed in microglia and PC-12 after PCA application. Finally, PCA mitigated SCI-induced inflammation by targeting the Wnt/β-catenin axis. Conclusion This study provided preliminary evidence that PCA inhibits neuroinflammation and apoptosis through the Wnt/β-catenin pathway, thereby attenuating the secondary injury after SCI and promoting the regeneration of injured spinal tissues.

Reinfections from SARS-CoV-2: A Retrospective Study from the Gyncentrum Genetic Laboratory in Sosnowiec, Poland, April 2020 to July 2022.

BACKGROUND The increasing number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfections has opened a new research direction related to analyzing long-term immune response and accurately characterizing individual cases of reinfection to understand its mechanism and estimate the risk of widespread reinfection both locally and globally. This retrospective study from the Gyncentrum Genetic Laboratory in Sosnowiec, Poland aimed to evaluate reinfections from SARS-CoV-2 between April 2020 and July 2022. MATERIAL AND METHODS The study extended the previously published report on SARS-CoV-2 infection cases in Poland by analyzing 8041 reinfections diagnosed with real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Data were collected on the amount of time elapsed from the first infection to the next and, based on these data, all results were divided into several groups for statistical analysis: 0-44, 45-90, 91-200, 201-310, 311-420, and >420 (days). RESULTS The study showed that of the 8041 patients who experienced reinfection, the vast majority (5505) became reinfected more than 310 days after the original infection, even though the average time between infections was 354.3 days. Statistical analysis revealed that the risk of SARS-CoV-2 reinfection increases with time and that this relationship becomes statistically significant after the 200th day following the initial infection (p<0.01). CONCLUSIONS We have shown that acquired immunity to SARS-CoV-2 infection is relatively short-lived - it starts diminishing about 6 months after the initial positive test. Moreover, the risk of reinfection is very high more than 1 year after the initial infection.

Indirect regulation of HIPPO pathway by miRNA mediates high-intensity intermittent exercise to ameliorate aging skeletal muscle function.

Exercise-induced microRNA (miRNA) and HIPPO pathways participate in the regulation of skeletal muscle plasticity but their underlying mechanisms remain unclear. We aimed to investigate the effect of high-intensity interval training (HIIT) on miRNA expression and the HIPPO pathway in the skeletal muscle of aging rats to determine its role in the amelioration of muscle aging. Thirty-six 18-month-old female rats were randomly divided into sedentary control (SED, n=12), moderate-intensity continuous training (MICT, n=12), and HIIT (n=12) groups, with continuous exercise for 8 months. Quantitative reverse transcription-polymerase chain reaction, immunoblotting, KEGG enrichment, and dual-luciferase assays were performed on the target skeletal muscle. Compared with the SED group, the MICT and HIIT groups showed a significant trend of improvement in Lee's index and grip strength and a marked increase in skeletal muscle mitochondrial function, apoptosis, antioxidant, and lipolysis-related protein expression. They also exhibited PI3K/AKT pathway activation and a decrease in expression of HIPPO pathway-related proteins; 20 miRNAs were differentially expressed and enriched in the exercise group compared with the SED group, including the HIPPO pathway and metabolic pathways. Further analysis of L6 cells confirmed that miR-182 may target PTEN, which indirectly regulates HIPPO signaling, but not Mob1. the combined application of HIIT and MICT increased the antioxidant and lipolytic capacities of skeletal muscle and improved atrophy of aging skeletal muscle; HIIT was more effective than MICT. This may be related to HIIT-mediated AKT pathway activation and HIPPO pathway inhibition by miRNAs (miR-486 and miR-182).

The Outcome of COVID-19 Infection in Patients With Gastrointestinal Diseases: An Experience at a Tertiary Center.

Observing the impact of the coronavirus disease 2019 (COVID-19) pandemic on digestive diseases in hospitalized patients at the Department of Gastroenterology-Hepatology in "Mother Teresa" University Hospital Center (UHC),Tirana. This retrospective study was carried out from June 2020 to December 2021 involving 41 cases of patients >18 years who were positive for COVID-19 infection detected by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) assays of nasopharyngeal swab specimens. The severity of COVID-19 infection was evaluated by hematological/biochemical parameters, blood oxygenation/need for oxygen, radiological data on pulmonary CT imaging. Out of 2527 hospitalized cases, 1.6% (41) were positive for the infection. The average age was 60.05 +/- 15.008 years. The group of age with more patients (48.8%) was 41-60 years. Infected males were higher than females (p<0.001). Out of the total, 21% were vaccinated at the diagnosis. Most patients came from urban areas, more than a half from the capital. Frequency of the digestive diseases was: cirrhosis 31.7%, pancreatitis 21.9%, alcoholic liver disease 21.9%, gastrointestinal hemorrhage 19.5%, digestive cancer 14.6%, biliary diseases 7.3%, inflammatory bowel disease (IBD) 2.4%, other digestive diseases 4.8%. Fever (90%) and fatigue (78.04%) were the dominant clinical signs. Biochemical and hematological parameters showed elevation of average value of aspartate amino transferase (AST), alanine transaminase (ALT) (AST>ALT, p<0.001), and bilirubin in all the patients. Higher levels of creatinine andsignificantly predictive value of systemic inflammation indices NLR (neutrophil to lymphocyte ratio) and MLR (monocyte to lymphocyte ratio) were found in the fatality cases. Patients with cirrhosis had more severe form of COVID-19, lower blood oxygenationand needed treatment by O2-therapy (p<0.046). Death rate was 12%. A strong correlation was found between the need for O2-therapy and deaths (p<0.001) and between characteristic findings for COVID-19 in pulmonary CT imaging and low blood oxygenation (p<0.003). Comorbiditywith chronic diseases, such as liver cirrhosis, has an important impact on the severity and mortality of the patients with COVID-19 infection. Inflammatory indices, such as NLR (neutrophil to lymphocyte ratio) and MLR (monocyte to lymphocyte ratio), are useful tools in predicting the evolution toward severe forms of the disease.

Identification of Usutu Virus Africa 3 Lineage in a Survey of Mosquitoes and Birds from Urban Areas of Western Spain

Usutu virus (USUV) is an emerging zoonotic arbovirus that has caused an increasing number of animal and human cases in Europe in recent years. Understanding the vector species and avian hosts involved in the USUV enzootic cycle in an area of active circulation is vital to anticipate potential outbreaks. Mosquitoes were captured in 2020, while wild birds were sampled in both 2020 and 2021 in Extremadura, southwestern Spain. The presence of USUV in the mosquito vectors was assessed by a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay and confirmed by sequencing amplicons from two generic RT-PCR sets for flaviviruses. Sequences were analysed phylogenetically. Bird sera were screened for flavivirus antibodies with a blocking ELISA kit and subsequently tested for virus-specific antibodies with a micro-virus-neutralization test. Overall, 6,004 mosquitoes belonging to 13 species were captured, including some well-known flavivirus vectors (Culex pipiens, Cx. perexiguus, and Cx. univittatus). Of the 438 pools tested, USUV was detected in two pools of Cx. pipiens. Phylogenetic analysis using a fragment of the NS5 gene assigned the USUV detected the Africa 3 lineage. Out of 1,413 wild birds tested, USUV-specific antibodies were detected in 17 birds (1.2%, 10 males and 7 females) from eight species. The first detection of USUV Africa 3 lineage in mosquitoes from Spain, together with serologically positive resident wild birds in urban and rural areas, indicates active circulation and a possible risk of exposure for the human population, with necessity to establish specific surveillance plans.

Association of the Interleukin 1B-31*C Proinflammatory Allele with the Severity of COVID-19 Patients: A Preliminary Report.

Individuals with no known comorbidities or risk factors may develop severe coronavirus disease 2019 (COVID-19). The present study assessed the effect of certain host polymorphisms and viral lineage on the severity of COVID-19 among hospitalized patients with no known comorbidities in Mexico. The analysis included 117 unrelated hospitalized patients with COVID-19. Patients were stratified by whether they required intensive care unit (ICU) admission: the ICU group (n = 40) and non-ICU group (n = 77). COVID-19 was diagnosed on the basis of a positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription-polymerase chain reaction (RT-PCR) assay and clinical and radiographic criteria. The presence of the IL1B-31 (T/C) polymorphism was determined for all patients using PCR and nucleotide sequencing. Genotyping of the IL-4 (-590, T/C) and IL-8 (-251, T/A) polymorphisms was performed by the amplification refractory mutation system-PCR method. Genotyping of IL1-RN was performed using PCR. Viral genome sequencing was performed using the ARTIC Network amplicon sequencing protocol using a MinION. Logistic regression analysis identified the carriage of IL-1 B*-31 *C as an independent potential risk factor (odds ratio [OR] = 3.1736, 95% confidence interval [CI] = 1.0748-9.3705, p = 0.0366) for ICU admission and the presence of IL-RN*2 as a protective factor (OR = 0.4371, 95% CI = 0.1935-0.9871, p = 0.0465) against ICU admission. Under the codominant model, the CC genotype of IL1B-31 significantly increased the risk of ICU admission (OR: 6.38, 95% CI: 11.57-25.86, p < 0.024). The IL1B-31 *C-IL-4-590 *T haplotype increased the risk of ICU admission (OR = 2.53, 95% CI = 1.02-6.25, p = 0.047). The 42 SARS-CoV-2 genomes sequenced belonged to four clades, 20A-20D. No association was detected between SARS-CoV-2 clades and ICU admission or death. Thus, in patients with no known comorbidities or risk factors, the IL1B-31*C proinflammatory allele was observed to be associated with the risk of ICU admission owing to COVID-19.

Performance of a SARS-CoV-2 RT-PCR Assay with Non-Traditional Specimen Types

During the first two years of the coronavirus disease 2019 (COVID-19) pandemic, nasopharyngeal (NP) specimens were the gold standard for clinical diagnostic testing. As information about the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the pandemic continued to be shared, it was clear that the virus could be detected in other specimen types during an active infection. The University of Louisville Infectious Diseases Laboratory accepted non-traditional specimen types, most without a paired, positive NP result, for research purposes only to support local epidemiology efforts. A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay originally validated for NP specimens was used for non-traditional specimen types using a variety of specimen preparation methods. Limit of detection (LOD) studies allowed for direct comparison between NP, sputum, and breast milk specimen types. The primary aim of the study was to determine whether SARS-CoV-2 RNA could be detected in different human specimen types. The results showed that the non-traditional specimens were not inherently inhibitory since SARS-CoV-2 RNA was detected in 36 (14.5%) out of 249 non-traditional specimens, and the limit of detection for SARS-CoV-2 in breast milk and sputum was the same as for NP specimens. SARS-CoV-2 was not detected in 15 breast milk specimens from mothers with positive SARS-CoV-2 NP results. In addition, a direct comparison study showed that NP specimens performed better than paired nasal specimens. In conclusion, by analyzing real-time RT-PCR test results for these non-traditional specimen types, two benefits were realized. Health care providers gained additional epidemiologic information (since information was not to be used for managing or treating patients), and the laboratory gathered important information about specimen types for which complete method validation studies could be pursued in the future.