Establishment and application of triple PCR method for CPV, CCoV and CRV

Canine parvovirus (CPV), canine coronavirus (CCoV) and canine rotavirus (CRV) are the three main causative viruses of diarrhea in dogs with similar clinical symptoms;thereby it is necessary to establish a high effective molecular detection method for differentiating the above pathogens. By optimizing the primer concentration and annealing temperature, a triple PCR method was established for simultaneous detection of CPV, CCoV and CRV, and then the specificity, sensitivity and repeatability of the method were tested. The results showed that the target fragments of CPV VP2 gene (253 bp), CCoV ORF-1b gene (379 bp) and CRV VP6 gene (852 bp) could be accurately amplified by the triple PCR method with high specificity, the detection limits of CPV, CCOV and CRV were 6.44x10-1 pg/L, 8.72x10-1 pg/L and 8.35x10-1 pg/L respectively with high sensitivity, and the method had good stability. Using this triple PCR method, 135 canine diarrhea fecal samples collected in Chengdu region from 2019 to 2020 were detected, and compared with those of single PCR method. The detection rates of CPV, CCoV and CRV were 16.30%, 20.74% and 4.44%, respectively, and the total infection rate was 51.11% (65/135) with 20.00% (13/65) co-infection rate. The detection results were consistent with three single PCR methods. In conclusion, CPV/CCoV/CRV triple PCR method successfully established in this paper can be applied as an effective molecular method to detection of related pathogens and to the epidemiological investigation.

Establishment of a duplex fluorescent quantitative PCR assay for detection of Porcine circovirus type 2 and type 3

To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3), specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested. Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies.L-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181), the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181). The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181). The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.

Silent superbug killers in a river near you how factory farms contaminate public water courses on three continents

Water downstream from factory farms harbours an invisible threat to people's health which could eclipse the COVID-19 crisis. The threat? Antibiotic Resistance Genes (ARGs) which are driving antimicrobial resistance the world's superbug crisis - projected to kill up to 10 million people annually by 2050. This publication reports the presence of ARGs in animal waste discharged from industrial farms into public waterways or onto soil (or crops) in four countries. Gauge community impact and sentiment regarding the issue was also highlighted. The water and sediment from public water courses connected to effluent discharges from 6-10 pig farms were tested in each of four countries (Canada, Spain, Thailand and the USA).

Effects of Porcine deltacoronavirus on the transduction pathway of the innate immune signal

Porcine deltacoronavirus (PDCOV) is a new type of pig intestinal coronavirus, which targets pig small intestinal epithelial cells to cause severe enteritis. After infecting the host, PDCoV finishes its proliferation in the host cell by antagonism or escape the innate immune signaling transduction pathway. In order to understand the action mechanism of PDCOV 0n the congenital immune signal transduction pathways, this paper reviews the effects of PDCOV on RLR, Jak-STAT, MAPK and mitochondrial signaling pathway to clarify the relationship between PDCOV and host innate immune signaling transduction pathways in order to provide help for the prevention and treatment of PDCOV infection.

Effect of Drebrin protein on the proliferation of PEDV in Vero cells

Previous studies have revealed that developmental regulated brain protein (Drebrin) is involved in cell- to-cell communication, nerve transmission, tumor metastasis, spermatogenesis and other life activities, but there are few studies on viruses. The aim of the current research was therefore, to study the function of Drebrin and its effect on the proliferation of porcine epidemic diarrhea virus (PEDV). The Drebrin gene was cloned according to the Drebrin gene sequence (XM_008015438.2) of Chlorocebus sabaeus registered by GenBank, and the phylogenetic tree was constructed to analyze its homology. The results showed that the CDS region of Vero cells Drebrin gene was 2088 bp long, encoding 695 amino acids, and was relatively conserved and had high homology with all species. To investigate the effect of Drebrin on the proliferation of PEDV in Vero cells, the eukaryotic expression vector pcDNA3.1-Drebrin-Flag was constructed. After transfection of Vero cells with different concentrations of pcDNA3.1-Drebrin-Flag, cells were infected with PEDV. Our results showed that overexpression of Drebrin in Vero cells could significantly inhibit the intracellular PEDV mRNA level and N protein expression, reduce the extracellular virus titer and inhibit the proliferation of PEDV. Further study on the interaction between Drebrin and PEDV S proteins by laser confocal technique was also performed. The results showed that Drebrin and S protein were co-located in the cytoplasm, suggesting that the two proteins may interact with each other. This study demonstrated for the first time that Drebrin can inhibit PEDV proliferation in Vero cells, laying a foundation for further research in to Drebrin function and provides a valuable information for anti-PEDV research.

Development and application of a multiplex fluorescent quantitative RT-PCR for detection of Porcine epidemic diarrhea virus, porcine coronavirus and porcine acute diarrhea syndrome coronavirus

To develop a multiplex fluorescent quantitative RT-PCR for the detection of porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV), in this study, specific primers/probes were designed based on the conserved regions of M, M and N gene sequences of PEDV, PDCoV and SADS-CoV, respectively. After optimization of the reaction conditions, a multiplex fluorescent quantitative RT-PCR for PEDV, PDCoV and SADS-CoV was established. The results of specificity assay showed that the method was positive for detection of PEDV, PDCoV and SADS-CoV, and negative for detection of porcine transmissible gastroenteritis virus, porcine rotavirus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine parvovirus, classical swine fever virus and foot-and-mouth disease virus. The results of sensitivity assay showed that the detection limit of this method for PEDV, PDCoV, and SADS-CoV plasmids standard was 1.0x101 copies/L, and had a good linear relationship with their Ct values in the range of 101 copies/L to 106 copies/L. The results of repeatability assay showed that the coefficients of variation (CVs) of intra- and inter-assay reproducibility ranged from 0.33% to 2.53%, indicating good repeatability and stability. To evaluate the effects of the developed method, 100 clinical samples collected from different parts of Henan province were used for detection of these three viruses and compared with those of single RT-PCR and standard methods. The results of multiplex fluorescent quantitative RT-PCR showed that the positive rates of PEDV, PDCoV and SADS-CoV were 38% (38/100), 14% (14/100) and 5% (5/100), respectively. There was no mixed infection. The coincidence rate with the standard detection methods of PEDV and PDCoV was 100%, and the sensitivity was higher than that of single RT-PCR. In this study, a specific, sensitive and rapid multiplex fluorescent quantitative RTPCR method was established for the first time, which could be used for the differential detection of PEDV, PDCoV and SADS-CoV, and laid a foundation for the differential diagnosis and control of porcine diarrheal diseases.

Blood Transfusion in Covid-19 Patients: One Year Survey

Introduction: Due to the COVID-19 outbreak, many chronic patients and elective surgical procedures have been postponed to create spaces for the hospitalization of COVID-19 patients, raising issues related to this change. The objective of this study is to assess the effect of the COVID-19 pandemic on the demand for blood products transfusion. Materials ant methods: The study presents the results of a retrospective study of blood transfusions in COVID-19 patients admitted to the Constanta County Emergency Clinical Hospital. The period of study was January-December 2021. We compared the transfusion requirement for each type of blood component in COVID 19 patients versus patients with non-COVID pathology. Results and discussions: During 2021, we transfused 282 COVID-19 patients;150 patients had only Covid pneumonia (of which 19 patients with severe forms needed intensive care in ICU-Covid), and 132 patients had various co-morbidities. The maximum blood requests was registered in the period February - April 2021, with a peak of 63 patients in April 2021. The main co-morbidities in patients with Covid 19 were: severe anemia in patients with malignant hemopathies. Anemia at admission in patients with Covid pneumonia is reported in more than 40% of patients. Moderate anemia (Hb <11 g/dL) is considered as an independent risk factor for the severe course of COVID-19 infection and mortality in these patients. The transfusion requirement in these patients was greater than 1.43 RBC (units/patient), 0.81 Plasma units/patient, 0.40 Platelets concentrate units + single donor platelet concentrate units/patient, in accordance with the associated pathology. Conclusion(s): The most requested product was packed red blood cells, the correction of anaemia being an important factor in preventing the severe course of the disease. The platelet requirement was 0.15 units/patient, thrombocytopenia being present in patients with severe evolution of the infection (hospitalized in ICU-COVID). The most requested blood groups were O+ and A+. COVID-19 transfusion data will help plan and prepare for the use of blood resources during the pandemic.Copyright © 2022 Sevigean Ali et al., published by Sciendo.

Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate. Materials and Methods: B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Results: The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high- frequency distribution was improved to 85%. Expression of recombinant protein in E. coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000. Conclusion: According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.

Defining the pediatric response to SARS-CoV-2 variants.

The global population has been severely affected by the coronavirus disease 2019 (COVID-19) pandemic, however, with older age identified as a risk factor, children have been underprioritized. This article discusses the factors contributing to the less severe response observed in children following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including, differing viral entry receptor expression and immune responses. It also discusses how emerging and future variants could present a higher risk to children, including those with underlying comorbidities, in developing severe disease. Furthermore, this perspective discusses the differential inflammatory markers between critical and non-critical cases, as well as discussing the types of variants that may be more pathogenic to children. Importantly, this article highlights where more research is urgently required, in order to protect the most vulnerable of our children.

Protocol for studying co-infection between SARS-CoV-2 and Staphylococcus aureus in vitro.

Bacterial co-infection is one of the most common complications of SARS CoV-2 infection. Here, we present a protocol for the in vitro study of co-infection between SARS CoV-2 and Staphylococcus aureus. We describe steps for quantifying viral and bacterial replication kinetics in the same sample, with the optional extraction of host RNA and proteins. This protocol is applicable to many viral and bacterial strains and can be performed in different cell types. For complete details on the use and execution of this protocol, please refer to Goncheva et al.1.

Cloning, expression and identification of truncated spike gene of IBV (Sf200) and chicken granulocyte- macrophage colony stimulating factor (GM-CSF)

The aim of this study was to clone, express and identify the truncated S1 gene of nephrotropic infectious bronchitis virus (IBV) and granulocyte-monocyte colony stimulating factor (GM-CSF) of chicken. Firstly, two genes were amplified by polymerase chain reaction (PCR) and cloned into pMD18-T vector. The truncated S1 gene designated as Sf200 containing five antigenic sites of S1 glycoprotein on amino acid residues (aa) 24-61, (aa) 291-398 and (aa) 497-543 and GM-CSF were then amplified from the respective recombinant pMD18-T plasmids and cloned into pET-32a (+) vector resulting pET-Sf200, pET-GM which were identified by restriction enzyme digestion and sequencing analysis. The in vitro expression of truncated Sf200 and GM-CSF constructs were later expressed in E. coli BL21 with a molecular mass of approximately 38 kDa and 29 kDa respectively as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polyclonal antibodies were developed by injecting E. coli expressed Sf200 and GM-CSF into the SPF mice and were used to identify the recombinant proteins by Western blot analysis. These findings indicated that the polyclonal antibodies produced in mice could be used to detect the recombinant truncated Sf200 and GM-CSF and vice versa.

Glycyrrhizin inhibits swine acute diarrhea syndrome coronavirus infection via the HMGB1/TLR4/JNK signal pathway

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly discovered enteric coronavirus, is the etiological agent that causes severe clinical diarrhea and intestinal pathological damage in piglets. In this study, Vero E6 and IPI-2I cells were pretreated with different concentrations of glycyrrhizin (GLY) for 2 hours, and then infected with different concentrations of SADSCoV, aiming to investigate the inhibitory effect of GLY on SADS-CoV. Western blot and TCID50 results revealed a significantly decreased N protein expression and viral titer, indicating that GLY can inhibit the infection of SADS-CoV. Vero E6 and IPI-2I cells were pretreated with different concentrations of GLY for 2 hours and infected with SADS-CoV. Western blot results showed that when the concentration of GLY was 0.8 mmol/L, the expression of N protein decreased significantly, indicating that GLY inhibited the invasion of the virus. At first, cells were treated with 0.4 mmol/L GLY, and cell samples were collected at 2 hours, 6 hours and 12 hours after being infected with SADS-CoV for analysis, and the expression of N protein were found to be significantly reduced at all points, indicating that GLY had a significant inhibitory effect on the replication of the virus. GLY is a competitive inhibitor of high mobility group box 1 (HMGB1), and the receptors of HMGB1 mainly include TLR4 and RAGE. Based on this fact, the mutant plasmid at the key sites of HMGB1 (C45S, C106S, C45/106S) and the siRNA of the RAGE receptor were transfected to Vero E6 cells and infected with SADS-CoV, and the cell supernatant and samples were harvested. The western blot and TCID50 results showed that the expression of N protein and the virus titer were decreased, suggesting that GLY exerts its function by affecting the binding of HMGB1/TLR4/RAGE during SADS-CoV infection. To further explore the signaling pathway through which GLY functions, Vero E6 and IPI-2I cells were inoculated with SADS-CoV, and cell samples were harvested, western blot was used to detect the changes of MAPK proteins. The results showed that the protein expression levels of p-p38, p-JNK and p-ERK were up-regulated in the early and late stages, indicating that the MAPK pathway was activated by SADS-CoV infection. Vero E6 and IPI-2I were pretreated with different concentrations of GLY and TLR4 inhibitor TAK for 2 hours and infected with SADS-CoV. Protein samples were harvested and analysed by western blot which showed a decreased p-JNK and N proteins, while other proteins showed no significant changes. These results indicated that GLY and TAK regulated the phosphorylation of JNK but did not regulate the phosphorylation of p38 and ERK. Also, Vero E6 cells were treated with HMGB1 antibody, the siRNA of HMGB1 and HMGB1 mutants plasmid, and infected with SADS-CoV. Protein samples were harvested, western blot results showed that phosphorylation of JNK decreased, indicating that HMGB1 affected JNK phosphorylation. Finally, Vero E6 and IPI-2I cells were pretreated with different concentrations of JNK inhibitor SP600125 to infect SADS-CoV, western blot, TCID50 and IFA results showed that the expression of N protein and virus titer, as well as virus replication were reduced, indicating that SP600125 inhibited virus replication. In conclusion, our results revealed that GLY can inhibit in vitro replication of SADS- CoV, mainly through the HMGB1/TLR4/JNK signaling pathway. The discovery of this pathway provides theoretical support for the research of novel anti-SADS-CoV drugs.

Establishment and applications of a duplex quantitative real-time RT-PCR assay for simultaneous detection of bovine rhinitis A virus and bovine rhinitis B virus

Bovine rhinitis virus (BRV) is an important pathogen responsible for the bovine respiratory disease complex (BRDC) and can be divided into two genotypes (BRAV and BRBV). To establish a duplex quantitative real-time RT-PCR assay for simultaneous detection of BRAV and BRBV, specific primers and TaqMan probes targeting the 5'NTR of BRAV and 3'NTR of BRBV were designed. A duplex quantitative real- time RT- PCR assay for simultaneous detecting BRAV and BRBV was preliminarily established by optimizing reaction conditions for each step. The assay specifically detects BRAV and BRBV, and no crossreaction with other common bovine respiratory pathogens, including IDV, BCoV, BVDV-1, BRSV, BPIV-3, BAdV-3, mycoplasma bovis, Pasteurella multocida, Mannheimia haemolytica, Escherichia coli, and Salmonella, was observed. In addition, the sensitivity test showed that the detection limits of this assay were 3.2x101 copies/L for both BRAV and BRBV plasmid standards. Besides, the repeatability test showed that the variation coefficients of this assay were less than 0.05 from both lot-to-lot and intra-lot. These results showed that the assay has high specificity, extreme sensitivity, and good repeatability. Moreover, a total of 43 nasal swabs of BRDC cattle were tested by our assay and four other quantitative real-time RT-PCR assays, including 3 BRAV assays and 4 BRBV assays. The results showed that the detection rates of our assay were 32.56%(14/43) for BRAV and 30.23%(13/43) for BRBV, and the detection rates of other quantitative real-time RT-PCR assays were 0(0/43), 2.33%(1/43), 23.26%(10/43) for BRAV and 27.91% (12/43), 27.91%(12/43), 27.91%(12/43), 27.91%(12/43) for BRBV, indicating that our assay has a more substantial detection capability than other assays. This study firstly established a duplex quantitative real-time RT-PCR assay for simultaneous detection of BRAV and BRBV, and the assay exhibited high specificity, sensitivity, and stability. Moreover, the study firstly confirmed the existence of BRAV in China, contributing to the prevention and control of BRDC.

Circular medicine. a paradigm of sustainability and resilience in education and professional development

Circular economy is a model of economic, social and environmental production and consumption, aimed to build a sustainable society in tune with the available resources. In the Brundtland Report, (1987, United Nations) sustainability it is defined as an approach focused on "meeting the needs of the present without compromising the ability of future generations to meet their own needs." The European Medical Association (EMA), that has currently more than 50 000 members, is involved in the advancement of health, pursuing educational and professional goals addressed to the quality of environment, lifestyle and civil society through a circular medicine approach. This is a comprehensive strategy, integrating best medical practice, bioinformatics and molecular biology with economy, artificial intelligence and machine learning. Circular Medicine is the resilient framework linking all these topics with circular economy methods. Innovative possibilities of circular medicine are many: it is a comprehensive paradigm shift, countering a non-medicine based on simplistic algorithms with guidelines of insufficient effectiveness, reliability and generalizability, often passed off as innovative Artificial-Intelligence approaches. We launched in 2020 a still ongoing survey aimed at the dissemination of information and at a greater sensitivity of all our members and followers on these topics, striving to build circular medicine in the real-world. This position document is based on its preliminary results: 1. Studying and investigating the links among health, climate change, biodiversity, circular economy, robust Artificial Intelligence and Machine learning support are goals and needed practice suitable to be pursued by EMA and other medical and scientific associations. Circular economy will be fruitfully used in health facilities, including hospitals, with health professionals and life science researchers acting also in the role of influencers and opinion leaders. 2. The interventions based on epidemiology, environmental sciences, best practice in medicine, sustainable technologies and molecular biology, the pillars of a potential framework of circular medicine, need a robust bioinformatics and Artificial Intelligence support. 3. Academic curricula and health professionals CME courses should provide stronger digital knowledge, reliable procedures trainings and expertise along these lines. Daily routine of clinical observation and participatory dialogue are key elements for the progress toward a culture, practice and accomplishments of circular economy and medicine. 4. Molecular biology, still the most innovative field of medical and life science, has a role for strengthening the pathways of the circular economy, as a reliable and resilient basis of "circular medicine." 5. The lessons of COVID19, the ongoing battles for healthier lifestyles dealing with nutrition, exercise and against alcohol and cigarette smoking, and the concept of smart cities are some of the cornerstones of the proposed strategy for a real-world circular medicine. Debunking interventions against the misuse of scientific and medical are means for the development of circular economy and medicine. Effective roadmaps, guidelines and grids for recognizing and counteracting the overlap of bullying, imposter's fear, insufficient expertise and knowledge, fake assertions and evaluations are actively developed by EMA's ad-hoc workshops. AI knowledge and skills should be implemented within innovative molecular biology and medical best-practice academic-CME curricula.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Teaching in the time of post-COVID-19

Although it can be argued as to whether the COVID-19 pandemic is completely behind us, it does seem that the worst may be over. Regardless, the effects still loom large, and colleges and universities are working to return to normalcy, educators are confronted with unprecedented challenges in student readiness such as insufficient learning, loss of cognitive sharpness, behavioral and psychological challenges, inattentiveness, loss of study and interpersonal skills, and so forth. These problems may be further exacerbated if the developmental windows for learning certain skills and skillsets closed during the pandemic. Building upon a series of manuscripts published in Biochemistry and Molecular Biology Education [BAMBEd] that were submitted in response to a March 2020 editorial titled, "Teaching in the time of COVID," which called for educators to share their strategies for addressing the immediate challenges in biochemistry and molecular biology education caused by the pandemic, as well as a similarly titled workshop we led at the 2022 ASBMB Annual Meeting in April 2022, we will present a number of innovative techniques that employ alternate teaching modalities (such as asynchronous, synchronous or hybrid remote instruction, HyFlex, etc.). In addition, we will address challenges that were often exacerbated by the 'digital divide' and other socioeconomic factors. These effects remain evident as we enter the post-pandemic period.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

A New CURE in Molecular Biology: Testing a Novel Pyrrolidine Compound to Determine Mechanism of Action in an Upper-Division Molecular Biology Course

The COVID-19 pandemic shut down forced introductory biology and chemistry laboratory courses online at DePauw University from March 2020-June 2021, leaving multiple classes of students without the opportunity to learn basic laboratory skills that are essential for the molecular biology laboratory. In an effort to provide students with both basic laboratory skills and advanced molecular biology skills, a new course-based undergraduate research experience (CURE) was developed for the 2022-23 academic year. In collaboration with Dr. Jeff Hansen in the Chemistry and Biochemistry department, novel compounds with potential anti-tumor properties were identified. The CURE in Molecular Biology was designed to have students use Saccharomyces cerevisiae as a model system to evaluate possible cellular pathways affected by the compound, including: cytoskeleton and cell migration, nucleotide biosynthesis, glucose metabolism, apoptosis, and cell cycle regulation. Students learned techniques DNA isolation and PCR, transformation, RNA isolation, cDNA synthesis, qPCR, and Western Blotting, while contributing to an active research project. At the conclusion of the project, students were surveyed about their comfort with molecular techniques and data analysis.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Fructus aurantii extracts ameliorate intestinal inflammation and regulate the intestinal barrier through Fc epsilon RI signaling pathway

Fructus Aurantii (FA) is the dry and immature fruit of Citrus aurantium L. and its rutaceous cultivars. FA has been widely used to treat digestive system diseases since ancient China, and it promotes gastrointestinal (GI) motility in functional dyspepsia (FD), but its potential therapeutic mechanisms remain unclear. We examined the effects of FA ethanol extracts in an iodoacetamide (IA)-induced FD rat model. Firstly, key FA therapy targets for FD were gathered using systematic pharmacology. Combined with systemic pharmacological analyses, plasma metabolomics based on UPLC-QTOF-MS were conducted. Then, MetaboAnalyst was used to jointly analyze systemic pharmacology targets and metabolomic metabolites to select key metabolic pathways. Finally, the key path is verified by experiments. FA exerted distinct therapeutic effects in anti-inflammation and promoting gastrointestinal motility in our IA-induced FD rat model. When compared with the model group, FA down-regulated the inflammatory factors interleukin 1beta and tumor necrosis factor-a. At the same time, FA up-regulated tight junction proteins in the intestinal epithelial barrier. Through the integrated analysis of metabolomics and systemic pharmacology, we conducted experimental verification on Fc epsilon RI signaling pathway. When compared with the model group, FA down-regulatedphospho-mitogen activated protein kinase, phospho-extracellular signal regulated kinase1/2, myosin light chain kinase, and phospho-myosin regulatory light chain protein levels. Thus, FA ameliorated FD by regulating the Fc epsilon RI signaling pathway. Our integrated strategy identified underlying FA mechanisms toward FD treatment and provided a foundation for FA development as a clinical agent for FD.

Isolation, identification, and S1 gene sequencing of two strains of Avian Infectious bronchitis virus

Objective: Epidemiology and genetic variations of the infectious bronchitis virus(IBV) in Fujian province were studied. Method: Two strains of virus isolated from the diseased chickens in Fujian in 2021 were identified by chicken embryo pathogenicity test, electron microscope observation, and RT-PCR. S1 genes of the isolates were cloned, sequenced, and analyzed using biological software. Result: The two IBV strains were code named FJ-NP01 and FJ-FZ01. The full length of S1 of FJ-NP01 was 1 629 nt encoding 543 amino acids, and that of FJ-FZ01, 1 620 nt encoding 540 amino acids. The S1 gene cleavage site of FJ-FZ01 was HRRRR, same as all reference strains of genotype I branch;while that of FJ-NP01 HRRKR differed from the reported site of IBV isolated from genotype IV but same as that of TC07-2 reference strain of genotype VI. The homology of nucleotide and amino acid between the two isolates was 83.2% and 79.6%, respectively, but merely 75.7%-76.3%and 77.1%-83.5% with the Mass-type conventional vaccines H120 and H52, respectively. Further analysis showed that FJ-NP01was from a recombination event between CK CH GD LZ12-4 and L-1148, the homology of nucleotide acid between 1438-1506 nt of FJ-NP01 with CK CH GD LZ12-4 was 97%, and 95.9% between the other nucleotide acid of S1 gene with L-1148. Conclusion: It appeared that the IBV epidemic experienced in the province was complex in nature and that the existing Mass vaccines would not provide sufficient immune protection to deter the spread.

Interaction between target peptide and complementary peptide by bimolecular fluorescence complementary technology

[Objective] Using the bimolecular fluorescence complementation (BiFC) technology, the present experiment aimed to study the interaction relationship and localization of the target peptide and the complementary peptide based on the porcine epidemic diarrhea virus (PEDV) S protein receptor binding site peptide in living cells, so as to provide the foundation and theoretical support for the further use of the peptide in the detection of porcine epidemic diarrhea virus. [Method] The target peptide was designed according to the physical and chemical characteristics of the target protein, such as the amino acid composition, the type of charge, the ability to form intennolecular hydrogen bonds, the strength of polarity, and hydrophobicity;According to the amino acid composition of the target protein, a complementary peptide that interacted with it in theory was designed, and the target peptide and complementary peptide were predicted and analyzed by using bioinfonnatics tools;The target peptide and complementary peptide were inserted into the pBiFC-VC155 and pBiFC-VN173 vector, which was double digested by the EcoRI/XhoI and NotI/SalI, respectively, verified by enzyme digestion and sequencing, and then transfected into Vero cells to study the interaction between the target peptide and the complementary peptide, and the precise localization of BiFC complex in cells. [Result] Bioinfonnatics analysis showed that the target peptide and complementary peptide had hydrophilic and hydrophobic domains, respectively, and the hydrophilic domains were both positively and negatively charged, which could generate electrostatic attraction. The results of enzyme digestion and sequencing showed that the pBiFC-VC155-target peptide and pBiFC-VNI73-complementary peptide plasmids were successfully constructed;Cell transfection experiments showed that the target peptide and complementary peptide could form BiFC complexes in Vcro cells after co-transfection of recombinant plasmids, indicating that they could interact with each other;Indirect immuttolluorescence assay confirmed that the BiFC complex was mainly distributed in the nucleus. [Conclusion] It was confirmed that the peptide designed based on the PEW/ S protein receptor binding site can interact with each other in living cells, demonstrating the feasibility of the peptide for detection.

Antiviral activitiy of curcumin, demethoxycurcumin, bisdemethoxycurcumin and cyclocurcumin compounds of Curcuma longa against NSP3 on SARS-CoV-2

SARS-CoV-2 genome encodes two large polyproteins (pp), pp1a and pp1ab which are cleaved and transformed into a mature form by a protease, non-structural protein 3 (NSP3). NSP3 is encoded by open reading frame (ORF) 1a/b. Curcuma longa (C. longa) or turmeric has been documented to have antiviral effects. The aim of this study was to assess the viral activities of C. longa against SARS-CoV-2 focusing on its potency to inhibit viral replication by targeting NSP3. PubChem databases were used to obtain the metabolic profile of C. longa. The compound's interaction with nucleocapsid was analyzed using molecular docking with Molegro Virtual Docker. Bioinformatics analysis based on rerank score presents all compounds of C. longa have higher binding affinity than the native ligand with cyclocurcumin as the lowest score (-128.38 kcal/mol). This anti-viral activity was hypothesized from the similarity of hydrogen bonds with amino acid residues Ser 128 and Asn 40 as key residues present in Ribavirin. This study reveals that C. longa is the potential to be developed as an antiviral agent through replication inhibition in SARS-CoV-2 targeting its replication mediated by NSP3.