TRIM32 inhibits Venezuelan Equine Encephalitis Virus Infection by targeting a late step in viral entry.

Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like arthritis, and encephalitis. The host ubiquitin proteasome system (UPS) plays critical roles in regulating cellular processes to control the infections with various viruses, including alphaviruses. Previous studies suggest alphaviruses hijack UPS for virus infection, but the molecular mechanisms remain poorly characterized. In addition, whether certain E3 ubiquitin ligases or deubiquitinases act as alphavirus restriction factors remains poorly understood. Here, we employed a cDNA expression screen to identify E3 ubiquitin ligase TRIM32 as a novel intrinsic restriction factor against alphavirus infection, including VEEV-TC83, SINV, and ONNV. Ectopic expression of TRIM32 reduces alphavirus infection, whereas depletion of TRIM32 with CRISPR-Cas9 increases infection. We demonstrate that TRIM32 inhibits alphaviruses through a mechanism that is independent of the TRIM32-STING-IFN axis. Combining reverse genetics and biochemical assays, we found that TRIM32 interferes with genome translation after membrane fusion, prior to replication of the incoming viral genome. Furthermore, our data indicate that the monoubiquitination of TRIM32 is important for its antiviral activity. Notably, we also show two TRIM32 pathogenic mutants R394H and D487N, related to Limb-girdle muscular dystrophy (LGMD), have a loss of antiviral activity against VEEV-TC83. Collectively, these results reveal that TRIM32 acts as a novel intrinsic restriction factor suppressing alphavirus infection and provides insights into the interaction between alphaviruses and the host UPS.

Alternative splicing of its 5'-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies.

Aberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5' untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.

Dermoscopy of cutaneous sarcoidosis: a cross-sectional study.

BACKGROUND: Although traditionally used for the diagnosis of skin tumors, in the past few years dermoscopy as a clinical diagnostic aid for inflammatory and infectious skin manifestations has also received more and more attention. The clinical variability of cutaneous sarcoidosis (CS) often makes its correct diagnosis challenging. Dermoscopy can be used as an auxiliary examination method. OBJECTIVE: Our aim was to evaluate the role of dermoscopy in the diagnosis and differential diagnosis of CS. METHODS: This was a retrospective analysis of 39 CS clinical and dermoscopic images collected in the Department of Dermatology, Huashan Hospital Affiliated with Fudan University from August 2013 to February 2021. RESULTS: Retrospective dermoscopic evaluation revealed small grouped, translucent orange globular structures in all 39 cases. Variable diameter linear vessels were found in 38 cases. A central scar-like area was seen in 26 cases. Bright white streaks were seen in 30 cases. The follicular plugs were seen in 15 cases. STUDY LIMITATIONS: First, the number of cutaneous sarcoidosis cases the authors collected is small. Second, due to the lack of a control group, the sensitivity and specificity of the proposed criteria were not calculated. Finally, since our study mainly includes suspicious lesions that were biopsied for diagnostic purposes, there may be a selection bias. CONCLUSION: Lesions showing on dermoscopy grouped translucent orange ovoid structures associated with linear vessels should raise the suspicion of CS. Central scar-like areas and bright white streaks are also helpful in the diagnosis of CS.

Rbc-determining biosensors in athletes' urine / Biossensores determinantes de hemácias na urina de atletas / Biosensores determinantes de glóbulos rojos en la orina de deportistas

ABSTRACT Introduction: There is a lack of electrochemical biosensors that allow finding hemoglobin (Hb), a protein found within red blood cells, available in athletes' urine samples. Objective: This work is focused on the production of dsDNA immobilized on an Au-modified glassy carbon electrode (dsDNA/Au/GCE) and its use as a sensor for the presence of urinary hemoglobin. Methods: The elements were deposited in spherical form and tested as a porosity electrode surface for DNA immobilization according to the surface scan of the functionalized dsDNA/Au/GCE using SEM analysis. DPV and amperometry were used to conduct electrochemical studies. Results: Amperometric analyses showed that Hb determination on dsDNA/Au/GCE showed better stability and sensitivity. In the existence of multiple interfering species and clinical urine samples produced, the selectivity and the actual ability of dsDNA/Au/GCE for hemoglobin determination were investigated. Conclusion: The results showed that dsDNA/Au/GCE is effective, reliable, and selective as an electrochemical sensor of Hb. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: Há uma carência de biossensores eletroquímicos que permitam encontrar a hemoglobina (Hb), uma proteína encontrada dentro dos glóbulos vermelhos do sangue, disponível em amostras de urina dos atletas. Objetivo: Este trabalho é focado na produção de dsDNA imobilizado em um eletrodo de carbono vítreo Au-modificado (dsDNA/Au/GCE) e seu uso como sensor para a presença de hemoglobina urinária. Métodos: Os elementos foram depositados em forma esférica e testados como superfície de eletrodo de porosidade para imobilização do DNA, de acordo com o exame de superfície do dsDNA/Au/GCE funcionalizado, utilizando análise SEM. DPV e amperometria foram usados para conduzir estudos eletroquímicos. Resultados: As análises amperométricas demonstraram que a determinação de Hb em dsDNA/Au/GCE apresentou um melhor grau de estabilidade e sensibilidade. Na existência de múltiplas espécies interferentes e amostras clínicas de urina produzidas, a seletividade e capacidade real do dsDNA/Au/GCE para a determinação da hemoglobina foram investigadas. Conclusão: Os resultados mostraram que o dsDNA/Au/GCE é efetivo, confiável e seletivo como sensor eletroquímico de Hb. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: Se carece de biosensores electroquímicos que permitan encontrar la hemoglobina (Hb), una proteína que se encuentra dentro de los glóbulos rojos, disponible en las muestras de orina de los deportistas. Objetivo: Este trabajo se centra en la producción de dsDNA inmovilizado en un electrodo de carbono vítreo modificado con Au (dsDNA/Au/GCE) y su uso como sensor de la presencia de hemoglobina urinaria. Métodos: Los elementos fueron depositados en forma esférica y probados como una superficie de electrodo porosa para la inmovilización de ADN, según el escaneo de la superficie del dsDNA/Au/GCE funcionalizado, utilizando el análisis SEM. Se utilizó la DPV y la amperometría para realizar estudios electroquímicos. Resultados: Los análisis amperométricos demostraron que la determinación de Hb en dsDNA/Au/GCE mostraba un mayor grado de estabilidad y sensibilidad. En la existencia de múltiples especies interferentes y muestras clínicas de orina producidas, se investigó la selectividad y la capacidad real del dsDNA/Au/GCE para la determinación de Hb. Conclusión: Los resultados mostraron que el dsDNA/Au/GCE es eficaz, fiable y selectivo como sensor electroquímico de Hb. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Dermoscopy of cutaneous sarcoidosis: a cross-sectional study

Abstract Background Although traditionally used for the diagnosis of skin tumors, in the past few years dermoscopy as a clinical diagnostic aid for inflammatory and infectious skin manifestations has also received more and more attention. The clinical variability of cutaneous sarcoidosis (CS) often makes its correct diagnosis challenging. Dermoscopy can be used as an auxiliary examination method. Objective Our aim was to evaluate the role of dermoscopy in the diagnosis and differential diagnosis of CS. Methods This was a retrospective analysis of 39 CS clinical and dermoscopic images collected in the Department of Dermatology, Huashan Hospital Affiliated with Fudan University from August 2013 to February 2021. Results Retrospective dermoscopic evaluation revealed small grouped, translucent orange globular structures in all 39 cases. Variable diameter linear vessels were found in 38 cases. A central scar-like area was seen in 26 cases. Bright white streaks were seen in 30 cases. The follicular plugs were seen in 15 cases. Study limitations First, the number of cutaneous sarcoidosis cases the authors collected is small. Second, due to the lack of a control group, the sensitivity and specificity of the proposed criteria were not calculated. Finally, since our study mainly includes suspicious lesions that were biopsied for diagnostic purposes, there may be a selection bias. Conclusion Lesions showing on dermoscopy grouped translucent orange ovoid structures associated with linear vessels should raise the suspicion of CS. Central scar-like areas and bright white streaks are also helpful in the diagnosis of CS.

Naringenin promotes cell autophagy to improve high-fat-diet-induced atherosclerosis in ApoE-/- mice.

Naringenin (NAR) is a major flavanone in citrus fruits that has multiple pharmacological attributes such as anticancer and antiatherogenic. This study aims to investigate the mechanism of NAR in high-fat-diet (HFD)-induced atherosclerosis (AS) in apolipoprotein E-knockout (ApoE-/-) mice. A HFD-induced AS ApoE-/- mouse model was established. The mice were treated with HFD, different doses of NAR and simvastatin (Simv). After drug treatment, the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), superoxide dismutase (SOD), and alanine aminotransferase (ALT) were determined. The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected using qRT-PCR and enzyme-linked immunosorbent assay. The plaque area of the aorta of AS mice was determined using oil red O staining. Western blot analysis was applied to measure the levels of autophagy-related proteins [protein 1 light chain 3B (LC3B), beclin 1, and p62]. The TC, TG, LDL-C, TNF-α, ALT, and MDA levels were significantly increased while the HDL-C, SOD, and GSH-Px levels were decreased in the HFD-induced AS ApoE-/- mice. NAR treatment reversed the expression of the above indicators in mice. After they were treated with different doses of NAR, the LC3B and beclin 1 levels were improved while the p62 protein level was decreased. This study suggested that NAR could promote cell autophagy to improve HFD-induced AS in ApoE-/- mice.

Naringenin promotes cell autophagy to improve high-fat-diet-induced atherosclerosis in ApoE-/- mice

Naringenin (NAR) is a major flavanone in citrus fruits that has multiple pharmacological attributes such as anticancer and antiatherogenic. This study aims to investigate the mechanism of NAR in high-fat-diet (HFD)-induced atherosclerosis (AS) in apolipoprotein E-knockout (ApoE-/-) mice. A HFD-induced AS ApoE-/- mouse model was established. The mice were treated with HFD, different doses of NAR and simvastatin (Simv). After drug treatment, the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), superoxide dismutase (SOD), and alanine aminotransferase (ALT) were determined. The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected using qRT-PCR and enzyme-linked immunosorbent assay. The plaque area of the aorta of AS mice was determined using oil red O staining. Western blot analysis was applied to measure the levels of autophagy-related proteins [protein 1 light chain 3B (LC3B), beclin 1, and p62]. The TC, TG, LDL-C, TNF-α, ALT, and MDA levels were significantly increased while the HDL-C, SOD, and GSH-Px levels were decreased in the HFD-induced AS ApoE-/- mice. NAR treatment reversed the expression of the above indicators in mice. After they were treated with different doses of NAR, the LC3B and beclin 1 levels were improved while the p62 protein level was decreased. This study suggested that NAR could promote cell autophagy to improve HFD-induced AS in ApoE-/- mice.

An infant with a mild SARS-CoV-2 infection detected only by anal swabs: a case report

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China and has spread rapidly worldwide. We present a mild SARS-CoV-2 infection in a baby with non-productive cough and normal chest computed tomography, in whom only anal swabs tested positive by real-time PCR testing for SARS-CoV-2. She was given atomization inhalation therapy with recombinant human interferon alfa-1b for 10 days. Her anal swabs remained positive for eight days, whereas her throat swabs were persistently negative by real-time PCR testing. Mild and asymptomatic cases, especially in children, might present with PCR negative pharyngeal/nasal swabs and PCR positive anal swabs. Those patients are potential sources of infection via fecal-oral transmission for COVID-19.

An infant with a mild SARS-CoV-2 infection detected only by anal swabs: a case report.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China and has spread rapidly worldwide. We present a mild SARS-CoV-2 infection in a baby with non-productive cough and normal chest computed tomography, in whom only anal swabs tested positive by real-time PCR testing for SARS-CoV-2. She was given atomization inhalation therapy with recombinant human interferon alfa-1b for 10 days. Her anal swabs remained positive for eight days, whereas her throat swabs were persistently negative by real-time PCR testing. Mild and asymptomatic cases, especially in children, might present with PCR negative pharyngeal/nasal swabs and PCR positive anal swabs. Those patients are potential sources of infection via fecal-oral transmission for COVID-19.

Decreased expression of MicroRNA-200 family in human breast cancer is associated with lymph node metastasis.

OBJECTIVES: MicroRNA-200 family (miR-200f) has been consistently reported to be deregulated and modulate the metastatic process in multiple cancers. In the present study, we detected the expression of miR-200f in breast cancer (BC) tissue and explored its relationships with clinicopathological characteristics, especially with lymph node metastasis. METHODS: Expression levels of miR-200a, miR-200b, miR-200c, miR-141, and miR-429 in 99 pairs of BC tissues and adjacent normal tissues were measured by real-time quantitative PCR. The correlation between miR-200f level and multiple clinicopathological factors was then examined by Mann-Whitney test, ANOVA, and operating characteristic (ROC) analysis. RESULTS: All members of the miR-200f were down-regulated in BC tissue compared with that in normal adjacent tissue; miR-200a, miR-200b, and miR-200c were highly decreased (p < 0.05), while the differences of miR-141 and miR-429 between patients and the control group were not statistically significant. Furthermore, all five members were found to be distinctly decreased with the incidence of lymph node metastasis (p < 0.05); When the patients were divided into three groups according to the number of lymph node metastasis (0; 1-3; ≥4), a gradual decrease of miR-200f expression was observed with the increasing number of lymph node metastasis; ROC revealed that miR-200b can differentiate patients with lymph node metastasis from those without lymph node metastasis. CONCLUSION: These observations imply that the down-regulation of miR-200f in human BC is associated with an invasive phenotype, and miR-200b may be useful to estimate the likelihood of the presence of pathologically positive lymph nodes.

Identification of candidate genes for lung cancer somatic mutation test kits.

Over the past three decades, mortality from lung cancer has sharply and continuously increased in China, ascending to the first cause of death among all types of cancer. The ability to identify the actual sequence of gene mutations may help doctors determine which mutations lead to precancerous lesions and which produce invasive carcinomas, especially using next-generation sequencing (NGS) technology. In this study, we analyzed the latest lung cancer data in the COSMIC database, in order to find genomic "hotspots" that are frequently mutated in human lung cancer genomes. The results revealed that the most frequently mutated lung cancer genes are EGFR, KRAS and TP53. In recent years, EGFR and KRAS lung cancer test kits have been utilized for detecting lung cancer patients, but they presented many disadvantages, as they proved to be of low sensitivity, labor-intensive and time-consuming. In this study, we constructed a more complete catalogue of lung cancer mutation events including 145 mutated genes. With the genes of this list it may be feasible to develop a NGS kit for lung cancer mutation detection.

Molecular cloning and in silico analysis of the duck (Anas platyrhynchos) MEF2A gene cDNA and its expression profile in muscle tissues during fetal development.

The role of myogenic enhancer transcription factor 2a (MEF2A) in avian muscle during fetal development is unknown. In this work, we cloned the duck MEF2A cDNA sequence (GenBank accession no. HM460752) and examined its developmental expression profiles in cardiac muscle, non-vascular smooth muscle and skeletal muscle. Duck MEF2A cDNA comprised 1479 bp encoding 492 amino acid residues. In silico analysis showed that MEF2A contained MADS (MCM1, AGAMOUS, DEFICIENS and SRF - serum response factor), MEF2 and mitogen-activated protein kinase (MAPK) transcription domains with high homology to related proteins in other species. Modified sites in these domains were conserved among species and several variants were found. Quantitative PCR showed that MEF2A was expressed in all three muscles at each developmental stage examined, with the expression in smooth muscle being higher than in the other muscles. These results indicate that the conserved domains of duck MEF2A, including the MADS and MEF2 domains, are important for MEF2A transcription factor function. The expression of MEF2A in duck smooth muscle and cardiac muscle suggests that MEF2A plays a role in these two tissues.

Molecular cloning and in silico analysis of the duck (Anas platyrhynchos) MEF2A gene cDNA and its expression profile in muscle tissues during fetal development

The role of myogenic enhancer transcription factor 2a (MEF2A) in avian muscle during fetal development is unknown. In this work, we cloned the duck MEF2A cDNA sequence (GenBank accession no. HM460752) and examined its developmental expression profiles in cardiac muscle, non-vascular smooth muscle and skeletal muscle. Duck MEF2A cDNA comprised 1479 bp encoding 492 amino acid residues. In silico analysis showed that MEF2A contained MADS (MCM1, AGAMOUS, DEFICIENS and SRF -serum response factor), MEF2 and mitogen-activated protein kinase (MAPK) transcription domains with high homology to related proteins in other species. Modified sites in these domains were conserved among species and several variants were found. Quantitative PCR showed that MEF2A was expressed in all three muscles at each developmental stage examined, with the expression in smooth muscle being higher than in the other muscles. These results indicate that the conserved domains of duck MEF2A, including the MADS and MEF2 domains, are important for MEF2A transcription factor function. The expression of MEF2A in duck smooth muscle and cardiac muscle suggests that MEF2A plays a role in these two tissues.