Hybrid Spaces for a New Dimension of Care

During the past few years, as a result of the Covid-19 pandemic, the contemporary definition andinterpretation of "care” has been updated and has also acquired new meanings for those who livein urban contexts.The paper explores how today design can codify tools to design systems of spaces-products-servicesthat promote a new way of living in which the concept of care, understood in its broadmeaning, of individuals in the contemporary urban context is central. In particular hybrid spacesfor living, working and travelling are investigated as an opportunity to increase processes ofattention and care that are not limited to the private sphere, but that contaminate theneighborhoods. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface-adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band-gap near-infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence-dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant noncanonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20- and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van't Hoff transition enthalpy of hybridization on SWCNT CP was −11.9 kJ mol-1, much lower than that of hybridization in solution (−707 kJ mol-1). We used SWCNT solvatochromism to calculate the solvent-exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with a (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNTs with an analyte at 37 °C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that the target sequence choice is important as secondary structure formation in the target is negatively correlated with hybridization. Best-performing CPs showed detection limits of 11 and 13 nM for DNA and RNA targets, respectively. Finally, we simulated sensing conditions using the saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence-specific hybridization on ssDNA-SWCNT CPs. © 2022 American Chemical Society.

The rapid diagnosis of Mycoplasma pneumonia using in situ hybridization on clinical samples.

The microbiological etiology of seasonal upper respiratory illnesses in the United States is dominated by viruses, including influenza A, B, respiratory syncytial virus, and SARS-CoV2. Mycoplasma pneumonia, treatable with antibiotics, can also cause upper respiratory symptoms and is typically associated with about 15 % of cases. There is no clinical or radiologic finding diagnostic of Mycoplasma pneumonia infection and PCR-based testing is not routinely used in the clinical setting. Further, the bacteria grows slowly in culture and the diagnostic IgM response will take days after the onset of infection. Thus, a rapid diagnostic test for Mycobacterium pneumonia infection is needed. This study documented two cases of Mycoplasma pneumonia infection of the upper respiratory system using in situ hybridization in a series of over 20 patients who were being tested for SARS-CoV2 infection. The respiratory secretions were placed on a glass slide, fixed in 10 % buffered formalin, and then tested using a Mycoplasma pneumonia probe. The high bacterial number associated with acute infection allowed for straightforward detection by in situ hybridization in a few hours. Antibiotic therapy led to rapid resolution of the symptoms. This highlights the ability of standard in situ hybridization as a rapid diagnostic test for Mycoplasma pneumonia in the clinical setting.

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Feature Selection and Parameter Optimization of SVM by Using a Hybridization Model

The feature selection approach (FSA) has lately emerged as a critical method for enhancing accuracy in several disciplines, including e-learning. Since the emergence of the COVID-19 pandemic, the education system in Iraq has diversified its methods to cope with the circumstances, and thus, the positive and negative aspects of e-learning in Iraq are revealed. In this study, we developed a hybrid model that predicts how to rely on a blended learning system during and after the COVID-19 pandemic. A hybrid model was implemented in MATLAB environment that suggested to optimize the support vector machine algorithm in blended e-learning. This hybrid model combines neighborhood component analysis and chi-square statistic. The proposed model demonstrated that platform influence is the most important factor in the e-learning field. It includes high-ranking features, such as adequacy of the virtual environment, a platform that provides sufficient tools for the user, user interest to learn new platforms, obligating students to attend, and having computer skills. The current work was compared with [11], which employed the SVM classification method, using the same datasets. The prediction accuracy of proposed work was better than that of the previous finding. The main goal of proposed work is to obtain a better optimization model than pervious finding by decreasing model error. The Error results was successfully reduced to 0.00012294. © 2022 IEEE.

[Digital adaptation of a therapeutic education program in the context of Covid-19]. / Adaptation numérique d'un programme d'éducation thérapeutique dans le contexte de la Covid-19.

The context of containment due to the Covid-19 epidemic forced professionals to suspend their face-to-face therapeutic education programs. For young patients with asthma, the situation was made even more complex by anxiety-provoking communications (which turned out to be inaccurate) about the possible aggravating role of corticosteroids in the event of Covid-19, which led to untimely discontinuations and sometimes to a decrease in their therapeutic adherence, exposing them to an increased risk of poor control of their disease. Faced with the feeling of abandonment felt by some families in this singular context, a team at Trousseau Hospital in Paris decided to rethink and adapt its distance therapeutic education workshops.

In silico analysis reveals hypoxia-induced miR-210-3p specifically targets SARS-CoV-2 RNA.

Human coronaviruses (HCoVs) until the emergence of SARS in 2003 were associated with mild cold and upper respiratory tract infections. The ongoing pandemic caused by SARS-CoV-2 has enhanced the potential for infection and transmission as compared to other known members of this family. MicroRNAs (miRNA) are 21-25 nucleotides long non-coding RNA that bind to 3' UTR of genes and regulate almost every aspect of cellular function. Several human miRNAs have been known to target viral genomes, mostly to downregulate their expression and sometimes to upregulate also. In some cases, host miRNAs could be sequestered by the viral genome to create a condition for favourable virus existence. The ongoing SARS CoV-2 pandemic is unique based on its transmissibility and severity and we hypothesised that there could be a unique mechanism for its pathogenesis. In this study, we exploited in silico approach to identify human respiratory system-specific miRNAs targeting the viral genome of three highly pathogenic HCoVs (SARS-CoV-2 Wuhan strain, SARS-CoV, and MERS-CoV) and three low pathogenic HCoVs (OC43, NL63, and HKU1). We identified ten common microRNAs that target all HCoVs studied here. In addition, we identified unique miRNAs which targeted specifically one particular HCoV. miR-210-3p was the single unique lung-specific miRNA, which was found to target the NSP3, NSP4, and NSP13 genes of SARS-CoV-2. Further miR-210-NSP3, miR-210-NSP4, and miR-210-NSP13 SARS-CoV-2 duplexes were docked with the hAGO2 protein (PDB ID 4F3T) which showed Z-score values of -1.9, -1.7, and -1.6, respectively. The role of miR-210-3p as master hypoxia regulator and inflammation regulation may be important for SARS-CoV-2 pathogenesis. Overall, this analysis advocates that miR-210-3p be investigated experimentally in SARS-CoV-2 infection.Communicated by Ramaswamy H. Sarma.

Hybridization Chain Reaction Lateral Flow Assays for Amplified Instrument-Free At-Home SARS-CoV-2 Testing.

The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 min. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/µL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/µL, 1000 copies/µL, 2000 copies/µL, 2000 copies/µL, and 20,000 copies/µL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection.

A microchamber-free and enzyme-free digital assay based on ultrabright fluorescent microspheres

Digital analysis is an effective single-molecule detection method and has attracted extensive attention in the field of bioassays. However, most digital assays require microchambers for signal compartmentalization. Herein, we developed a microchamber-free and enzyme-free digital assay by labeling paramagnetic beads directly with ultrabright fluorescent microspheres. In this assay, a DNA sandwich analysis was firstly performed on the bead to label a fluorescent microsphere. Then, the beads were loaded on the glass slide to form a monolayer film for signal readout. The whole analysis process does not require the participation of enzymes and the preparation of microchambers, which greatly simplifies the experimental steps and saves the costs. Furthermore, by introducing non-enzymatic hybridization chain reaction (HCR) and biotinylated DNA-conjugated gold nanoparticles (Au NPs-Bio), the capture efficiency and analytical sensitivity were improved. As a proof of concept, single-stranded DNA (ssDNA) of SARS-CoV-2 fragment was chosen as a model, and a detection limit of 1.5 fM was achieved. Spiked and recovery experiments on human serum and saliva samples validated the good performance of the proposed digital assay in real biological samples. The proposed assay provides a facile way of signal generation and readout for digital analysis. © 2023 Elsevier B.V.

Hemocytometric Characteristic of Covid-19 Patients

Introduction: Coronavirus disease 2019 is caused by severe acute respiratory syndrome. Primarily an infection of the lower respiratory tract, it is now well known to cause multisystem abnormalities. Hematologic manifestations constitute a significant area of concern. Coronavirus disease 2019 causes lymphopenia, neutrophilia, and thrombocytopenia. Method(s): We explored the hemocytometric characteristics of Covid-19 pazients, using the Sysmex XN-1000 (Sysmex Corporation - Japan) hematology analyser and EDTA-anticoagulated peripheral blood sample. The analyzer utilizes fluorescence flow cytometry for the leukocyte differential count, allowing for enhanced subset differentiation based on size (forward scatter;FSC), internal structure/granularity (side scatter;SSC) and DNA/RNA content (fluorescence expression;SFL). Result(s): Despite a decrease in lymphocyte count, an increase in certain subpopulations of lymphocytes was observed in COVID-19. Indeed, reactive lymphocytes antibody-synthesizing and high fluorescence lymphocyte cells were higher as compared with controls. The so-called high fluorescence cells represent lymphoplasmacytoid B cells and plasma cells. These lymphocytes are responsible for the adaptive humoral immune response. In the present study, we observed that in addition to lower absolute count, lynfocytes of patients with COVID-19 were slightly larger in size. The fraction of reactive lynfocytes within the whole population were significantly increase in patiens with COVID-19 and showed a tipical pattern in hemocytometric plot where the reactive lynfocytes appare as isolated population, different from those observed in patients with mononucleosis. A perifheral blood film assesment on light microscope showed a distint population with the caracteristic morphology of plasma cells with abundant basophilic cytoplasm and an eccentric nucleus. Cytoplasmic and nuclear morphological anomalies, from hyposegmented nuclei to apopto-sis, have been observed in circulating granulocytes, associated with an increase in relative counts of immature cells. Conclusion(s): Patients withCOVID-19 showed both quantitative and qualitative differences in leucocyte populations. This study is an analysis of a typical pattern observed in Covid-19 patients using the Sysmex XN -1000 (Sysmex Corporation - Japan) hematology analyzer. The on going study will be the correlation with immunophenotipic and clinical characteristic of these patients to understand the morphological changes observed.

Liver pathology in COVID-19 related death and leading role of autopsy in the pandemic.

BACKGROUND: Information on liver involvement in patients with coronavirus disease 2019 is currently fragmented. AIM: To highlight the pathological changes found during the autopsy of severe acute respiratory syndrome coronavirus 2 positive patients. METHODS: A systematic literature search on PubMed was carried out until June 21, 2022. RESULTS: A literature review reveals that pre-existing liver disease and elevation of liver enzyme in these patients are not common; liver enzyme elevations tend to be seen in those in critical conditions. Despite the poor expression of viral receptors in the liver, it seems that the virus is able to infect this organ and therefore cause liver damage. Unfortunately, to date, the search for the virus inside the liver is not frequent (16% of the cases) and only a small number show the presence of the virus. In most of the autopsy cases, macroscopic assessment is lacking, while microscopic evaluation of livers has revealed the frequent presence of congestion (42.7%) and steatosis (41.6%). Less frequent is the finding of hepatic inflammation or necrosis (19%) and portal inflammation (18%). The presence of microthrombi, frequently found in the lungs, is infrequent in the liver, with only 12% of cases presenting thrombotic formations within the vascular tree. CONCLUSION: To date, the greatest problem in interpreting these modifications remains the association of the damage with the direct action of the virus, rather than with the inflammation or alterations induced by hypoxia and hypovolemia in patients undergoing oxygen therapy and decompensated patients.

Comparative Analysis of Library Preparation Approaches for SARS-CoV-2 Genome Sequencing on the Illumina MiSeq Platform.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for over two years of the COVID-19 pandemic and a global health emergency. Genomic surveillance plays a key role in overcoming the ongoing COVID-19 pandemic despite its relative successive waves and the continuous emergence of new variants. Many technological approaches are currently applied for the whole genome sequencing (WGS) of SARS-CoV-2. They differ in key stages of the process, and they feature some differences in genomic coverage, sequencing depth, and in the accuracy of variant-calling options. In this study, three different protocols for SARS-CoV-2 WGS library construction are compared: an amplicon-based protocol with a commercial primer panel; an amplicon-based protocol with a custom panel; and a hybridization capture protocol. Specific differences in sequencing depth and genomic coverage as well as differences in SNP number were found. The custom panel showed suitable results and a predictable output applicable for the epidemiological surveillance of SARS-CoV-2 variants.

The 'New' New Normal-Digitalization and Hybridization of Work and Education Before, during and after the Covid-19 Pandemic

Before the COVID-19 coronavirus pandemic, work and education on the university campus were considered superior to equivalent activities conducted digitally or remotely. Despite being significantly digitally mature, many organizations did not consider or even permit digital or hybrid participation in meetings and education. In March 2020, the lockdown following the pandemic caused the transition of many organizations and most universities to online-only operation in record time. Often, this occurred while maintaining quality and production, even if some aspects relating to the user experience were lost. The purpose of this paper is to describe and analyze how digitalization following the pandemic influenced and transformed the digital work environment in higher education. KTH Royal Institute of Technology, Stockholm, Sweden, provided the experiences and data for this article. In conclusion we distinguished different phases of digital work and education before, during and after the pandemic: 1. The Pre-Pandemic Phase, 2. The Emergency Remote Phase, 3. The New Normal, 4. The Slow Acceptance Phase, and 5. The Dividing Phase, or what we refer to as The 'New' New Normal. In this phase it looks like it will be a battle between three perspectives;those who are looking to get back to the pre-pandemic conditions, those living in The New Normal, and those actively analyzing the lessons learned and aiming for The Thoughtful Blended Phase. Analyzing these five phases we discern that the implications for the future will be heavily dependent on the following aspects: management commitment and support;well-functioning technology and support organization;understanding the effects digitalization will have on culture, organization and well-being;increasing interest in participating in pedagogical development;new designs needed for hybrid work and education;the need to rethink structure and contents of meetings;and paying extra care and attention to the psychological well-being of staff and students.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band-gap near infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence-dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant noncanonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20-and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van't Hoff transition enthalpy of hybridization on SWCNT CP was -11.9 kJ mol-1, much lower than that of hybridization in solution (-707 kJ mol-1). We used SWCNT solvatochromism to calculate the solvent-exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with a (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNTs with an analyte at 37 degrees C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that the target sequence choice is important as secondary structure formation in the target is negatively correlated with hybridization. Best performing CPs showed detection limits of 11 and 13 nM for DNA and RNA targets, respectively. Finally, we simulated sensing conditions using the saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence-specific hybridization on ssDNA-SWCNT CPs.

Synthesis of 1,2,3-Triazole-Containing 2,3-Dihydrofuran Derivatives, Evaluation of Anticancer Activity and Molecular Docking Studies

A simple and convenient one-pot three-component method to synthesize 1,2,3-triazole-based 2,3-dihydrofuran derivatives from the condensation of 5,5-dimethylcyclohexane-1,3-dione, substituted phenacyl bromide, triethylamine and 1,2,3-triazole based benzaldehyde using a mixture of CH3CN/water as a reaction medium is described. The current protocol delivers numerous advantages such as good yield, short reaction time, easy work-up and simplicity in the procedure as it uses a green method and an eco-friendly catalyst. The target compounds are screened for their in-vitro anticancer activity and most of the compounds are found to exhibit promising activity compared to standard drug Doxorubicin. Molecular docking studies performed on caspase-3 and COVID-19 main protease revealed well defined binding interactions and docking scores.

Blood cytometry parameters in Sars Cov-2 patients: potential markers of disease characterization

IntroductionThe usefulness of leukocyte cell population data (CPD) derived from optical signals of new hematology analyzers is currenctly being investigated. In Covid-19 pandemic several reports showed the clinical importance of functional and quantitative blood parameters. Our study aimed to assess CPDs in positive Sars Cov-2 patients as potential disease markers.MethodsFrom February to April 2020 (1st wave), 540 patients (490 negative and 50 SARS CoV-2 positive) were enrolled in this retrospective study, as well as 2821 patients from September to December 2020 (2nd wave) (2762 negative and 59 SARS CoV-2 positive). SARS CoV-2 infection diagnosis was carried out by Multiplex rRT-PCR from nasopharyngeal swabs and clinical information collected in cardiology emergency department (ED). CPDs were detected by XN 2000 hematology analyzer (Sysmex Corporation) considering a single determination on whole blood. Comparisons between disease waves and SARS CoV-2 negative and positive patients were performed. Additionally, C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assayed. Statistical analysis using the univariate and multivariate general linear regressions were made.ResultsLeukocyte CPDs were classified into: cell complextity (NE, LY, MO X-axis), DNA/RNA content (NE, LY, MO Y-axis) and abnormal sized cells (NE, LY, MO Z-axis). We detected cytometric parameters increased from the reference population for all cell types for both 1st and 2nd wave (p<= 0.03). However, smaller quantitative alterations were found in the 2nd vs 1st wave: 5 CPDs vs 9 CPDs. In addition we found higher CPD values of the 1st compared to 2nd wave: (NESFL) (p=00004), (LY-Y) (p<=0.0001), (LY-Z) (p<=0.0001), (MO-X) (p<=0.0001), (MO-Y) (p<=0.0001). These findings were confirmed by the higher concentrations of CRP and LDH in the 1st vs 2nd wave: 17.3mgdL (8.5-59.3) vs 6.3 mg/dL (2.3-17.6) (p=0.0003) and 241.5 U/L (201-345) vs 195 U/L (174-228) (p=0.0005) (median, interquartile range) respectively.Conclusions Leukocyte CPDs showed increased cell activation in patients of 1st wave confirmed by biochemical data, correlated with worse clinical conditions of hospitalized patients. Our results highlighted the CPDs as disease characterization markers or useful for a predictive risk model.

Impact of the Flipped Classroom on the Motivation of Undergraduate Students of the Higher Institute of Nursing Professions and Health Techniques of Fez-Morocco

Motivation is the cornerstone of building success. If it is strong, achieving a greater number of objectives will be assured, and therefore a more glorious success can be met. Thus, to promote motivation, it is advisable to pay particular attention to the pedagogical approach adopted by the teacher since it has been proven that there is a positive relationship between the choice of an active approach and enhancing motivation. The situation generated by COVID-19 has been a profitable opportunity to experiment with "Flipped Classroom” pedagogy, to invert the class and adapt learning activities that have been traditionally offered to students through distance (online) and in-person learning alternately. Thus, the present study aims to evaluate this Flipped Pedagogy's impact on the motivation to learn of undergraduate students of the Higher Institute of Nursing Professions and Healthcare Techniques in Fez. Data have been collected from 410 students who were questioned before and after the adoption of the Flipped Classroom, based on a motivation scale validated by Rolland Viau in 1994, translating the determinants and indicators of motivation according to his socio-cognitive model of motivational dynamics. The answering percentage was 90.73%. Data analysis demonstrated a statistically significant association between the Flipped Classroom and students' motivation;the motivational profile, which was 18.54%, became 89.25% after flipping the class. The Flipped Classroom can significantly bring a surplus value to pedagogy, hence the importance of broadening its spectrum and integrating it as a new approach to teaching within health sciences educational institutions. © 2022, International Journal of Emerging Technologies in Learning. All Rights Reserved.

Hybridization Approach Toward Novel Antituberculars: Design, Synthesis, and Biological Evaluation of Compounds Combining Pyrazinamide and 4-Aminosalicylic Acid.

Apart from the SARS-CoV-2 virus, tuberculosis remains the leading cause of death from a single infectious agent according to the World Health Organization. As part of our long-term research, we prepared a series of hybrid compounds combining pyrazinamide, a first-line antitubercular agent, and 4-aminosalicylic acid (PAS), a second-line agent. Compound 11 was found to be the most potent, with a broad spectrum of antimycobacterial activity and selectivity toward mycobacterial strains over other pathogens. It also retained its in vitro activity against multiple-drug-resistant mycobacterial strains. Several structural modifications were attempted to improve the in vitro antimycobacterial activity. The δ-lactone form of compound 11 (11') had more potent in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compound 11 was advanced for in vivo studies, where it was proved to be nontoxic in Galleria mellonella and zebrafish models, and it reduced the number of colony-forming units in spleens in the murine model of tuberculosis. Biochemical studies showed that compound 11 targets mycobacterial dihydrofolate reductases (DHFR). An in silico docking study combined with molecular dynamics identified a viable binding mode of compound 11 in mycobacterial DHFR. The lactone 11' opens in human plasma to its parent compound 11 (t1/2 = 21.4 min). Compound 11 was metabolized by human liver fraction by slow hydrolysis of the amidic bond (t1/2 = 187 min) to yield PAS and its starting 6-chloropyrazinoic acid. The long t1/2 of compound 11 overcomes the main drawback of PAS (short t1/2 necessitating frequent administration of high doses of PAS).

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies.

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

Development of a high-sensitivity and short-duration fluorescence in situ hybridization method for viral mRNA detection in HEK 293T cells.

Coronavirus disease 2019 (COVID-19) is an extremely contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early disease recognition of COVID-19 is crucial not only for prompt diagnosis and treatment of the patients, but also for effective public health surveillance and response. The reverse transcription-polymerase chain reaction (RT-PCR) is the most common method for the detection of SARS-CoV-2 viral mRNA and is regarded as the gold standard test for COVID-19. However, this test and those for antibodies (IgM and IgG) and antigens have certain limitations (e.g., by yielding false-negative and false-positive results). We have developed an RNA fluorescence in situ hybridization (FISH) method for high-sensitivity detection of SARS-CoV-2 mRNAs in HEK 293T cell cultures as a model. After transfection of HEK 293T cells with plasmids, Spike (S)/envelope (E) proteins and their mRNAs were clearly detected inside the cells. In addition, hybridization time could be reduced to 2 hours for faster detection when probe concentration was increased. Our approach might thus significantly improve the sensitivity and specificity of SARS-CoV-2 detection and be widely applied for the high-sensitivity single-molecular detection of other RNA viruses (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Hepatitis A virus, all influenza viruses, and human immunodeficiency virus (HIV)) in various types of samples including tissue, body fluid, blood, and water. RNA FISH can also be utilized for the detection of DNA viruses (e.g., Monkeypox virus, human papillomavirus (HPV), and cytomegalovirus (CMV)) by detection of their mRNAs inside cells or body fluid.