Gene Editing/Gene Therapies: BIOREACTOR RECOMBINANT PRODUCTION OF SARS-COV-2 VIRUS ANTIGENS IN CHO CELL CLONES BY USING NEW UCOE LENTIVIRAL VECTOR SYSTEM

Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy

Phenotypic Characteristics and Clonal Relationships of Stenotrophomonas maltophilia Isolates in Hospitalized Adults from a Private Center in Lima, Peru.

Stenotrophomonas maltophilia is an opportunistic pathogen, often associated with nosocomial infections. Ten S. maltophilia were isolated from clinical samples during the period January 2021 and June 2022. Eight (80%) patients had cancer as a background disease and 2 patients had coronavirus disease 2019. A fatal outcome was recorded in 4 cases (40% of patients). All the isolates were susceptible to minocycline and levofloxacin. Trimethoprim/sulfamethoxazole and ceftazidime resistance rates were 20% and 40% respectively. Eight different patterns were observed by Pulsed-Field Gel Electrophoresis, only two isolates being clonally identical. The isolation of S. maltophilia in clinical settings requires the implementation of infection prevention measures.

Production of Sars-Cov-2 Membrane Protein as Better Target Protein for Diagnostic Application

Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023

Hiv Detection in Wastewater as a New Epidemiological Tool

Background: Wastewater represents a broad, immediate, and unbiased accounting of the pathgens in the population. We aimed to develop methods to track HIV in wastewater utilizing a viral detection pipeline adapted from platforms developed to track SARS-COV-2. Method(s): We used samples from 6 wastewater treatment plants in the Houston area. We focused on regions of higher prevalence and lower prevalence. First, employing wastewater processing and nucleic acid extraction methods described by our group to detect SARS-COV-2, we tested a single high and low prevalence site in triplicate with all 3 primer sets. nucleic acid extracts from HIV and SIV cell culture supernatants were used as controls. Next, in subsequent samples, RT-PCR reactions with detections were subjected to gel electrophoresis to determine the amplified product sizes. To further confirm HIV detection, we sequenced the RT-PCR products and compared the proportion of reads which mapped to the expected amplified product. In a later set of studies, we fractionated samples into supernatant and pellet. We further tested HIV presence by performing whole virome sequencing on the extracts from some samples that produced detections and mapped reads to published genomes. A crAssphage genome was used as a negative control. Result(s): Samples from all sites resulted in signal detection at least once. Only reactions with gag and pol primers appeared to amplify the expected product. Products from the HIV positive control mapped almost exclusively to the HIV genome (97-100% of reads), with a fraction of reads from the SIV negative control doing the same (16-18% of reads). The ltr and pol products did not map the HIV genome while gag products did (34-44% of reads). Among the fractionated sample, in total, 6 supernatant fractions produced no detection compared to 7 of 8 pellet fractions. The whole virome sequencing produced reads that mapped to the HIV genome with at least 8X depth coverage. The sample with the lowest Ct detection (26) yielded HIV coverage several logs greater than those samples with higher Ct detection (37). Reads from all samples mapped to at least 20% of the HIV genome. Conclusion(s): This work provides the first evidence that HIV can be detected in municipal wastewater systems and has the potential to be developed into a new public health tool.

Isolation and Characterization of Protein Complexes of Overexpressed Human Serum Amyloid A from Cytokines-Induced Hepg2 Cells

Serum Amyloid A (SAA) is an apolipoprotein found in the serum of many vertebrate species and is associated with the acute-phase reaction in the body with expression levels reaching up to a 1000-fold increase. The loss of its alpha-helix conformation during its expression peak is directly linked to secondary amyloidosis. Recent studies have been suggested to play a role in cholesterol and HDL metabolism, retinol transport and tumor pathogenesis. Moreover, high SAA concentration in blood have been correlated with severe symptoms or death in patients with COVID-19. However, how this protein is involved in so many diseases is uncertain or not completely understood. Therefore, the purpose of this research is to determine which protein-protein interactions with SAA occur in human cells, and to predict its biochemical role based on new discovered complexes. Two major isoforms overexpressed during an acutephase reaction, human SAA1 and SAA2, are the focus of this study. Both are primarily produced in hepatocytes. HepG2 cells were cultured and induced with interleukin-1b, interleukin-6, LPS and retinol. Protein complexes associated with SAA will be isolated through a co-Immunoprecipitation technique, resolved by SDS-PAGE, and characterized by mass spectrometry. Our hypothesis focus on those protein complexes with SAA to explain how this protein lead other undiscovered metabolic pathways involved in both cellular and survival regulation. Special thanks to The Science and Technology Competency & Education Core (Stce) for Undergraduate and Graduate Junior Research Associates Working Program from the Puerto Rico IDeA Network Biomedical Research Excellence for funding part of this research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Plasma markers of Covid-19 severity: A pilot study

SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect certain disease presentations. Our cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins by nephelometry, full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection. When compared to healthy controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, alpha-1-acid glycoprotein, alpha1-antitrypsin (AAT), ceruloplasmin, haptoglobin, and highsensitivity C-reactive protein. The concentrations of alpha1-antichymotrypsin, alpha2-macroglobulin and serum amyloid A proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose and sialylated di-antennary glycans, while the non-sialylated di-antennary glycan A2G2 significantly decreased in COVID-19 patients compared to controls. COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight involvement of different pathophysiological mechanisms and grant further investigations.

Abnormal echocardiographic findings after COVID-19 infection: A multicenter registry

Background: The Coronavirus Disease 2019 (COVID-19) pandemic has transformed health systems worldwide. There is conflicting data regarding the degree of cardiovascular involvement following infection, generating uncertainty in patients and an additional healthcare burden with increased diagnostic testing. A registry was designed to evaluate the prevalence of echocardiographic abnormalities in Latin American adults recovered from COVID-19. Method(s): We prospectively evaluated 595 participants (mean age 45.5+/-14.9 years;50.8% female) from 10 institutions in Argentina and Brazil. Echocardiographic studies were conducted with General Electric equipment;2DE imaging and global longitudinal strain (GLS) of both ventricles were performed. Comparisons between groups were made with Chisquare, Fisher and Student's t-test. Logistic regression was performed to determine variables associated with abnormal echocardiogram findings. Result(s): A total of 61.7% of the participants denied any relevant cardiovascular medical history. Table 1 summarizes the comorbidities of the included patients. The majority of patients (82.5%) had the disease at home or in an out-of-hospital center. Of the patients who required hospitalization, 15.3% were in a general ward, 1.9% in intensive care and 0.3% required mechanical ventilation during the disease. The median time between infection and performance of the echocardiographic study was two months (IQR 1- 3 months). Among patients who reported symptoms following COVID-19 recovery (41.8%), the most frequently reported was dyspnea (47.4%), followed by mild symptoms such as asthenia, arterial hypertension or palpitations (32.9%), 12.9% referred chest pain, 6% of patients reported dyspnea and chest pain, and 0.8% reported various other symptoms. The mean left ventricular ejection fraction (LVEF) was 61.0+/-5.5% and the mean left atrial volume was 33.1+/-13.2 ml/m2. In patients without prior comorbidities, 8.2% had some echocardiographic abnormality (Figure 1). We found no significant differences in LVEF between symptomatic and asymptomatic patients (61.4% versus 60.6% respectively, p=0.104). Symptomatic patients showed slightly reduced GLS (-20.3% versus -20.9%, p=0.012) with a trend in the same direction in the RV free wall GLS (-25.6% versus -26.3%, p=0.103). Male patients were more likely to have any new echocardiographic abnormalities (OR 2.82, p=0.002). Time elapsed since infection resolution (p=0.245), the presence of symptoms (p=0.927), or history of hospitalization during infection (p=0.671) did not have any correlation with echocardiographic abnormalities. The difference between sexes remains unchanged after adjusting for left atrial volume, wall thicknesses, diastolic function and abnormal wall motion. Conclusion(s): Our results suggest that cardiovascular abnormalities after COVID-19 infection are rare and usually mild, especially in cases of mild disease. These abnormalities may be more frequent among males.

Protease Activity in the Excretory-Secretory Products of Nymphal Stage of Linguatula Serrata: First Detection of a Metalloprotease

The aim of this study was to evaluate the presence of proteases and determine the main protease present in the excretory-secretory products (ESPs) from nymphal stage of Linguatula serrata. Infected mesenteric lymph nodes of goats were collected from Tabriz slaughterhouse, northwestern Iran. Recovered Linguatula serrata nymphs were immersed in culture medium (MEM), then ESPs were collected and protease activity in presence of specific inhibitors was assayed. Protease enzyme was fur-ther characterised by SDS-PAGE. The results of this study showed that the main protease in the ESPs from the nymphal stage of L. serrata was a metalloprotease that was resistant to heat. In conclusion, these data show that a major protease secreted by the larval stage of L. serrata exhibited properties that may play a role in the pathogenesis of L. serrata nymphs.Copyright © 2023, Trakia University. All rights reserved.

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean red ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean red ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

INO-4800, a DNA Plasmid Vaccine Encoding the Ancestral SARS-CoV-2 Spike Protein, Is Stable Over a Range of Temperatures Not Requiring Ultra-Cold Chain Storage

Background. Effective vaccines deployable globally are needed to control the COVID-19 pandemic. INO-4800, a plasmid DNA vaccine encoding the ancestral SARS-CoV-2 spike (S) protein, has demonstrated safety and immunogenicity. We report the stability of multiple INO-4800 lots used in Phase 3 clinical trials. Methods. Lots of INO-4800 stored at the test temperatures (storage temperature [2-8degreeC] and accelerated temperature [25 +/- 2degreeC]) were sampled every 3 months. Sample stability at each timepoint was assessed in terms of 2 parameters: i) purity as measured by DNA isoform homogeneity by capillary gel electrophoresis (CGE) to quantify relative proportions of different plasmid topologies (supercoiled, open circular, and linear), and ii) potency as measured by a cell-based assay for flow cytometric detection of fluorescently labeled S protein. Results. At 2-8degreeC, all tested lots of INO-4800 retained structural homogeneity for >=12 months (Fig. 1A). Proportions of circular and supercoiled plasmid isoforms were >=96% and >=89%, respectively, remaining above minimum regulatory authority-approved specifications (>=85% for circular and >=80% for supercoiled isoforms). At 25 +/- 2degreeC, all tested lots retained structural homogeneity within specifications for >=6 months (Fig. 1B), with proportions of circular and supercoiled plasmid isoforms being >=96% and >=81%, respectively. Moreover, structural homogeneity of 1/ 2 (50%) lots was within specification at 9 months at 25 +/- 2degreeC. Potency of all tested lots was >=84% for >=6 months at both 2-8degreeC and 25 +/- 2degreeC, remaining above the regulatory authority-approved minimum of 77% (Figs. 1C and 1D). Moreover, 3/3 (100%) of tested lots were within potency specification at 12 months at 2-8degreeC and 2 lots and 1 lot, respectively, were within specification at 9 and 12 months at 25 +/- 2degreeC. Data from additional timepoints are accruing and will be presented. Conclusion. INO-4800 is stable across temperatures ranging from 2degreeC to 25 +/- 2degree C and does not require ultra-cold storage. This demonstrated stability confers upon INO-4800 the potential to be deployed globally even in resource-constrained settings lacking cold chain infrastructure, thereby contributing to pandemic preparedness and control. (Figure Presented).

RNA Transfection with Hypoxia Inducible Factor-1alpha

Introduction: Ischemic necrosis of dermal flaps is a devastating complication of reconstructive surgery. The increasing prevalence of diabetes, obesity, and an aging population adds to this concern. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of the adaptive response to hypoxia, controls the expression of angiogenic growth factors. The development of biologically active, gene-specific mRNAs, especially in COVID-19 vaccines, has shown the ability for intracellular protein expression. We sought to express HIF-1alpha through mRNA transfection and determined its biological activity by measuring the upregulation of selected downstream targets. Method(s): 5'-methyl-capped poly-A tailed mRNA was generated using T7 RNA polymerase and verified by gel electrophoresis. Predominant and variant HIF-1alpha mRNA were transfected into primary human dermal fibroblasts via Lipofectamine in triplicate, and RNA levels were assessed using RT-qPCR. All gene expression levels were normalized to beta-actin expression levels Results: At one day after transfection, the levels of HIF-1alpha transcript were significantly higher in the cells transfected with predominant (p = 0.0104) and variant (p = 0.0007) HIF-1alpha transcripts relative to the control. Additionally, the expression of HIF-1alpha transcription product genes VEGF (p = 0.0274) and ANG-1 (p = 0.05) were significantly higher in the cells transfected with the HIF-1alpha transcripts than the control. Conclusion(s): Our approach led to the successful transfection of HIF-1alpha mRNA into human fibroblasts, resulting in upregulation of HIF-1alpha downstream angiogenic targets. Thus, the use of biologically active HIF-1alpha mRNA transfection offers a promising approach to inhibit ischemic necrosis.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qamp-mini) with a pre-programmed chip and a simple-to-use Capillary Gel Electrophoresis system (Qsep Series Bio-Fragment Analyzer) with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces inaccurate test results with high false negative and false positive rates. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies), fast results (less than 120 minutes) and cost-effective results which should be suitable for decentralized testing application of COVID-19.

Insight about Echocardiographic manifestations in COVID-19

Background: COVID-19 infection is associated with many direct or indirect cardiovascular complications, including myocarditis, myocardial damage, arrhythmia, heart failure (HF), venous thromboembolism, myocardial ischemia and necrosis is associated with impaired ventricular function that causes an increased mortality risk in patients.Two-dimensional echocardiography (2DE) is an important, noninvasive test which helps to identify cardiac function and hemodynamic status. Two-dimensional echocardiography has gained importance in COVID-19 patients with multi-organ involvement, especially in cases involving hemodynamic instability. Due to risk of infection, routine 2DE is not recommended for each patient, and there are currently no comprehensive 2DE studies in COVID-19 patientsMyocardial injury has been shown to be associated with worse prognoses in patients with COVID-19 In these patients, myocardial involvement, segmental contraction defects or global hypokinesia may be observed in the left heart due to hypoxic damage, respiratory distress, and inflammation, and a recent report.

Prevalence, bio-serotype, antibiotic susceptibility and genotype of Yersinia enterocolitica and other Yersinia species isolated from retail and processed meats in Shaanxi Province, China

This study characterized Yersinia enterocolitica and other Yersinia spp. isolated from retail and processed meats sampled in Yangling, Shaanxi Province, China (2018–2019). The detection rate of Yersinia -positive samples was 14.30% (86/600). Yersinia was commonly prevalent in beefs (32.00%), frozen meats (25.00%), packaged meats (21.65%), and meats from supermarkets (17.23%). Y. enterocolitica was identified as the most prevalent species (50.00%, 62/124), followed by Y. frederiksenii (24.19%), Y. intermedia (16.94%), and Y. kristensenii (8.87%). Most Y. enterocolitica isolates were of bio-serotype 1A/nt (67.74%). Sixty-three isolates (50.81%) carried yst B and two isolates (1.61%) carried rfb C. Yersinia isolates were commonly resistant to ampicillin (91.94%), cefazolin (71.77%), amoxicillin/clavulanic acid (56.45%), cefoxitin (28.23%), and trimethoprim/sulfamethoxazole (1.61%). Sixty-six isolates (53.23%) were resistant to three or more antibiotics. Pulse field gel electrophoresis analysis revealed that the genetic homology of Yersinia isolates between pork, beef, and chicken was low, as was that of biotype 1A isolates. The results indicate that Y. enterocolitica and other Yersinia spp. (especially bio-serotype 1A/nt, ampicillin-resistant, and yst B-carrying strains) are prevalent in retail and processed meats in the study area, which provides valuable baseline data for food safety and public health safeguarding. • Yersinia spp. with different characteristics were prevalent in retail meat. • Yersinia enterocolitica was the most commonly detected species. • Yersinia enterocolitica biotype 1A isolates carried gene yst B and resisted multiple antibiotics. • The genetic relationship of Yersinia isolates between pork, beef, and chicken was diverse. [ FROM AUTHOR] Copyright of LWT - Food Science & Technology is the property of Academic Press Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Impact of lab experiences on student knowledge and confidence

Due to the COVID-19 pandemic, the sophomore class of 2021 spent very little time on campus as freshmen and missed the benefits and advantages of in-person learning. Three labs were added to a required sophomore biomedical engineering (BME) course, Biochemistry and Molecular Engineering. The objectives of these labs were three-fold: (i) reinforce the content presented in the online lecture, (ii) provide students with hands-on laboratory skills, and (iii) offer students engaged in an online course in-person experiences and the corresponding academic and social benefits. The objective of this course is to develop critical thinking, teamwork and collaborative skills, as well as the ability to use physical laboratory equipment to obtain and analyze data. We eliminated some of the homework problems to account for the time spent in preparation for lab, the lab sessions, and writing the post-lab reports. The labs accounted for 10% of the total grade and included a prelab quiz, a post-lab report, and one or two exam questions for each lab. The three labs were: 1. Introduction to Pipetting 2. DNA Extraction, PCR, and Gel Electrophoresis 3. Bacterial Transformation with a Plasmid After completing the labs, a student survey indicated that over 80% of students agreed or strongly agreed that they felt confident using all these techniques. Comparing confidence levels, 63% of the students were somewhat familiar with pipetting prior to Lab 1 which increased to over 90% post-lab. After Lab 2 (DNA extraction, PCR, and gel electrophoresis), confidence levels more than doubled, from 40% in pre-lab survey results to 86% afterwards. Student confidence in the final lab, which involved using a plasmid for genetic transfection of bacteria, went from 33% pre-lab to 81% post-lab. While the prelab census indicated that a significant percentage of the students had some prior lab proficiency in high school, a quarter of the students lacked experience in pipetting and a majority of the students were unfamiliar with PCR/gel electrophoresis and plasmids. As one student noted, “The labs were a good way to build basic lab skills and exposure for students who were previously unable to work in a college lab”. By the end of the course, 87% agreed or strongly agreed that “Gaining hands-on lab skills is an important part of this course. I believe it should be continued, possibly with more labs, in future years.” In addition to gaining hands-on laboratory experience, students enjoyed working with their classmates in-person and benefited from “learning by doing”. Student comments strongly indicated that the labs reinforced the lecture content. The labs “helped me retain the information better than simply reading about it.” The combination of replacing homework with labs, and reducing the total amount of time per week students spent on the course, did not result in significant differences in the quiz scores from pre-pandemic years. Given the success of this year's lab experiences, we plan to update the labs and include additional topics for future course offerings. © American Society for Engineering Education, 2022

First molecular confirmation of the fungus Leveillula taurica causing powdery mildew disease on sweet pepper in Iraq

A study was initiated to investigate powdery mildew disease on pepper during the growing season 2019/2020. Disease incidence scored 73, 85, 80 and 56% in Al-Mahmudiyah, Al-Yusufiyah and Al-Jadriya/Baghdad and Suwaira/Wasit, respectively. Morphological identification revealed L. taurica formed dimorphic conidia, lanceolate primary conidia and cylindrical to clavate secondary conidia. Conidiophores were straight, having short hyaline and unbranched baring single conidiospores. Sequence comparison of ITS region confirmed L. taurica shared 99% maximum nucleotide identity with sequences from South Korea (MH698492.1), Mexico (MG571545.1) and Australia (MT125857.1). To the best of our knowledge, this is the first molecular confirmation of L. taurica infecting pepper in Iraq. [ FROM AUTHOR] Copyright of Archives of Phytopathology & Plant Protection is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

PURIFICATION OF ANTI-RBD ANTIBODIES OBTAINED FROM LLAMA (Lama glama) FOR POTENTIAL COVID-19 THERAPY

Despite worldwide efforts to develop different treatments for SARS-CoV-2 COVID-19, the situation remains critical, requiring rapid and effective strategies. In this regard, antibodies (Ab) have demonstrated clinical potential. Among them, camelid nanoAb (VHH) arise as a possible alternative, as they recognize epitopes which are inaccessible to conventional Ab. Further advantages of VHH are their small size, high solubility, high stability, and resistance to low pH. The aim of this work is to describe a purification scheme of different isotypes of anti-SARS-CoV-2 immunoglobulin G (IgG) produced after immunizing two llamas (Lama glama). To achieve this, plasma was injected into an affinity chromatographic column (Protein G), and the resulting fractions were analyzed by SDS-PAGE under non-reducing conditions. The anti-RBD titers were determined by an “in house” ELISA, reaching titers of 52000 and 13000 for IgG1 and IgG3 fractions, respectively. Subsequently, an affinity column (HiTrap NHS-activated) was prepared to separate monospecific anti-RBD polyclonal Ab. RBD produced in our laboratory was covalently coupled to this column, achieving a coupling efficiency of 97%. Different isotypes of monospecific anti-RBD Ab (IgG1: 140 kDa and IgG3: 95 kDa) were obtained. IgG3 represent the starting point for obtaining VHH and/or evaluating their potential use as a therapeutic or preventive alternative, which represents a notable regional contribution in the fight against COVID-19.

Deregulation of the Host Cell Cycle Induced by SARS‐CoV‐2 Nucleocapsid Protein

Introduction: The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARSCoV2) is the causative agent responsible for the COVID-19 pandemic and has resulted in devastating impacts on global public health. The nucleocapsid (N) protein of other coronaviruses, such as SARS-CoV-1, have been previously implicated in the deregulation of the host cell cycle through interactions with cell cycle checkpoint proteins, Cyclin-Dependent Kinases (CDKs) or cyclins. In this study, we highlight the role of SARS-CoV-2 N-protein in modulating CDK expression, thereby, deregulating the host cell cycle. Methods: A549 cells were transfected with pCMV plasmids, harbouring the SARS-CoV-2 N-protein. Protein extracts of control and Nprotein transfected cells were electrophoresed on SDS-PAGE, transferred onto a nitrocellulose membrane and incubated with CDK2 and CDK4 antibodies. The blots were visualized and protein quantification was performed using ImageJ analysis. Results: Transfection of SARS-CoV-2 N resulted in differential expression of CDK2 and CDK4, which are key regulators that drive cell cycle progression through G0 or G1 phase into S phase. Notably, preliminary findings also demonstrate that N protein results in decreased CDK2 and CDK4 expression. Conclusion: The differential expression of CDKs caused by SARS-CoV-2 N-protein suggests its role in inducing cell cycle arrest at the S phase to facilitate SARS-CoV-2 replication. The results from this research may aid in future characterisation of the mechanisms for SARS-CoV-2-mediated cell cycle arrest, and contribute towards the development of novel antiviral strategies and therapies.

Studies on the Role of SARS-CoV-2 Nucleocapsid as an Interferon Antagonist

Introduction: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), responsible for the coronavirus disease 2019 (COVID-19) pandemic, has resulted in significantly disruptive global impacts. Cytokine storm syndrome (CSS) can accompany SARSCoV2 infection, and involves the excessive release of pro-inflammatory cytokines that lead to acute respiratory distress syndrome (ARDS) in infected patients. Given the correlation between ARDS and poor patient prognosis, inflammatory pathways (e.g., interferon-1 (IFN-1)) would be a target area for antiviral development. Our preliminary results have demonstrated a direct correlation between the SARS-CoV-2 nucleocapsid (N) protein and host intracellular IFN-1 pathway components IRF3 and STAT1. Methods: A549 cells were transfected with pCMV-GFP vectors expressing N protein and harvested. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting were performed. The membranes were then incubated with STAT-1, p-STAT1 and IRF3 antibodies and visualised. Protein content was quantified using ImageJ software. Results: Transfection with SARS-CoV-2 N was correlated with a decrease in intracellular IRF3 and reduced phosphorylation of STAT1, suggesting the involvement of N protein in the delayed IFN-1 response commonly observed in SARS-CoV-2 patients. These findings suggest that IRF3 and STAT1 may be part of the innate immune response affected by SARS-CoV-2 infection. Conclusion: Our results show that IRF3 and STAT1 are responsible for stimulating transcription of interferon signalling genes (ISGs). Future studies on SARS-CoV-2 N and its downstream effectors could provide further insight into the IFN-1 response during infection, and assist in future antiviral development strategies.