Reverse Transcription Polymerase Chain Reaction Pattern of SARS-CoV-2 Beta Variant

N/a.

Histopathological study of cutaneous non-neoplastic lesions in punch biopsies

Skin being the largest organ of the body, is subjected to constant environmental insults through direct and indirect targets. Non-neoplastic lesions of skin comprise a vast array of diseases, which are usually approached by pattern based method in histopathology for microscopic diagnosis. This study was undertaken with an intention to learn such diseases by a simple minimally invasive punch biopsy procedure. A 2 year retro-prospective study of 82 punch biopsies which were documented as non-neoplastic lesions of skin were studied with respect to their demographical and histopathological profile. Simple descriptive statistics was applied in Microsoft Excel software. Out of the 82 cases studied, 46(56%) were males and 36(44%) were females. Maximum number of cases (n=23) were seen in 21-30 years of age. Most prominent site of lesions biopsied were from lower limb (23 cases) with legs being the commonest among them. Cutaneous infections (n=25) was the most common clinical category, with Mycobacterial lesions as the prominent subcategory (n=16). Granulomatous reaction constituted the most common major tissue reaction pattern among other patterns with a total number of 17 cases out of 82. Panniculitis was the most common minor reaction pattern observed. The pattern based approach in routine histopathological analysis proves a valuable descriptive tool for reaching an accurate diagnosis in cutaneous non-neoplastic lesions.

Construction of isoquinolinone framework from carboxylic ester directed umpolung ring opening of methylenecyclopropanes

An unprecedented reaction pattern of functionalized methylenecyclopropanes (MCPs) has been disclosed in this paper, in which the nucleophilic attack of anionic species onto partially polarity-reversed MCP unit can be realized...

Predicting the Right Mechanism for Hypervalent Iodine Reagents by Applying Two Types of Hypervalent Twist Models:Apical Twist and Equatorial Twist

Since the hypervalent twist followed by reductive elimination is a general reaction pattern for hypervalent iodine reagents, mechanistic studies about the hypervalent twist step could provide significant guidance for experiments....

Polymerization and isomerization cyclic amplification for nucleic acid detection with attomolar sensitivity

DNA amplification is one of the most valuable tools for the clinical diagnosis of nucleic acid-related diseases, but current techniques for DNA amplification are based on intermolecular polymerization reactions, resulting in the risk of errors in the intermolecular reaction pattern.

Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs

Phospholipidosis (PLD), the intracellular accumulation of phospholipids, is an adaptive response to toxic stimuli and serves as an important parameter in the biological assessment of compounds. Cationic amphiphilic drugs are the main inducers of PLD and may impair the function of alveolar macrophages. In vivo and in vitro models are used for PLD screening but the choice of the cellular model may be important because PLD develops in a cell- and species-specific manner. In this study, a panel of different staining (LysoSensor, Acridine Orange, Nile Red, HCS LipidTOX, LysoID) was evaluated in murine (DMBM-2, J774, RAW264.7) and human (THP-1, monocyte-derived macrophages from peripheral blood) cells to identify the most sensitive and easy to analyze staining method and to detect species-specific differences in the reaction pattern. Amiodarone and chloroquine served as inducers of PLD. High content screening was used to compare number, area, and intensity of the staining. Due to the fast staining protocol and the sensitivity of the detection, LysoID proved to be the most suitable dye of the testing. The lower induction of PLD by chloroquine reported in vivo was also seen in this study. THP-1 macrophages, followed by DMBM-2 cells, produced the most similar reaction pattern to human monocyte-derived macrophages.

An immunodominance hierarchy exists in CD8+ T cell responses to HLA-A*02:01-restricted epitopes identified from the non-structural polyprotein 1a of SARS-CoV-2

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-γ-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.ImportanceFor the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.

Vascular Obliteration Due To Endothelial And Myointimal Growth In COVID-19

Background: Severe coronavirus disease 2019 (Covid-19) is a systemic multi-organ viral invasion. Previous studies found that many patients had a procoagulant state and/or severe hypoxemia with relatively well-preserved lung mechanics. Mechanisms underlying the vascular and its surrounding tissue are not well known yet. Histological data in Covid-19 tissues´ patients are still limited and mainly focused on post-mortem analysis. Since SARS-CoV-2 largely affects cutaneous tissue, we aim to examine in depth skin lesions related to Covid-19 in order to understand better how the disease might affect living tissue.Methods: Five skin lesions from Covid-19 adult patients were selected for histological tissue examination. Vast amount of data of immunohistochemistry (IHC) and direct immunofluorescent (DIF) were part of the assessment. Results: A common strong vasculopathic reaction pattern based on prominent vascular endothelial and myointimal cellgrowth was identified. Endothelial cell distortion generated vascular lumen obliteration and a strike erythrocyte and serum extravasation. Extensive significant vascular C4d and C3 deposition throughout vascular cell wall was also identified. A regenerative epidermal hyperplasia with tissue structure preservation was found. Conclusions: Covid-19 could comprise an obliterative micro-angiopathy consisting on endothelial and myointimal intensive growth with complement activation. This mechanism, together with increased vascular permeability identified, could contribute to obliterative vascular lumen and hemorrhage in Covid-19. Activation of the complement and angiogenic pathways could have an important role in inducing and maintaining this vasculopathic reaction pattern. Thus, anticoagulation by itself could not completely reverse vascular lumen obliteration, with consequent hemorrhagic increased risk associated. Skin is the largest organ in the body, the most accessible one and can mirror other organs of the body. Findings of this study could contribute to a better understanding of physio-pathological mechanisms underlying SARS-CoV-2 infection on living tissue and could help further studies find potential targets for specific therapeutic interventions in Covid-19 severe patients.

Vascular Obliteration Due To Endothelial And Myointimal Growth In COVID-19

Background: Severe coronavirus disease 2019 (Covid-19) is a systemic multi-organ viral invasion. Previous studies found that many patients had a procoagulant state and/or severe hypoxemia with relatively well-preserved lung mechanics. Mechanisms underlying the vascular and its surrounding tissue are not well known yet. Histological data in Covid-19 tissues´ patients are still limited and mainly focused on post-mortem analysis. Since SARS-CoV-2 largely affects cutaneous tissue, we aim to assess the pathophysiologic mechanisms in depth in living skin tissue related to Covid-19.Methods: Five skin lesions from caucasian Covid-19 adult patients were selected for cutaneous tissue histological examination including immunohistochemistry (IHC) and direct immunofluorescent (DIF) vast amount of data. Results: A common strong vasculopathic reaction pattern based on prominent vascular endothelial and myointimal cellgrowth was identified. Endothelial cell distortion generated vascular lumen obliteration and a strike erythrocyte and serum extravasation. Extensive significant vascular C4d and C3 deposition throughout vascular cell wall was also identified. A regenerative epidermal hyperplasia with tissue structure preservation was also found. Conclusions: Covid-19 could comprise an obliterative micro-angiopathy consisting on endothelial and myointimal intensive growth with complement activation. This mechanism, together with increased vascular permeability identified, could contribute to obliterative vascular lumen and hemorrhage in Covid-19 disease. Activation of the complement and angiogenic pathways could have an important role in inducing and maintaining this vasculopathic reaction pattern. Thus, anticoagulation by itself could not completely reverse vascular lumen obliteration, with consequent hemorrhagic increased risk associated. Skin is the largest organ in the body, the most accessible one and can mirror other organs of the body. Findings of this study could contribute to a better understanding of physio-pathological mechanisms underlying Covid-19 infection on living tissue and could help further studies find potential targets for specific therapeutic interventions in Covid-19 severe patients.