Systematic alanine and stapling mutational analysis of antimicrobial peptide Chem-KVL.

Chem-KVL is a tandem repeating peptide, with 14 amino acids that was modified based on a short peptide from a fragment of the human host defense protein chemerin. Chem-KVL increases cationicity and hydrophobicity and shows broad-spectrum antibacterial activity. To determine the molecular determinants of Chem-KVL and whether staple-modified Chem-KVL would improve antibacterial activity and protease stability or decrease cytotoxicity, we combined alanine and stapling scanning, and designed a series of alanine and staple-derived Chem-KVL peptides, termed Chem-A1 to Chem-A14 and SCL-1 to SCL-7. We next examined their antibacterial activity against several gram-positive and gram-negative bacteria, their proteolytic stability, and their cytotoxicity. Ala scanning of Chem-KVL suggested that both the positively charged residues (Lys and Arg) and the hydrophobic residues (Lue and Val) were critical for the antibacterial activities of Chem-KVL peptide. Of note, Chem-A4 was able to remarkably inhibit the growth of gram-positive and gram-negative bacteria when compared to the original peptide. And the antibacterial activities of stapled SCL-4 and SCL-7 were several times higher than those of the linear peptide against gram-positive and gram-negative bacteria. Stapling modification of peptides resulted in increased helicity and protein stability when compared with the linear peptide. These stapled peptides, especially SCL-4 and SCL-7, may serve as the leading compounds for further optimization and antimicrobial therapy.

Surveillance of SARS-CoV-2 antibodies of patients in the local affected area during Wuhan lockdown.

BACKGROUND: Serosurveillance is crucial in estimating the range of SARS-CoV-2 infections, predicting the possibility of another wave, and deciding on a vaccination strategy. To understand the herd immunity after the COVID-19 pandemic, the seroprevalence was measured in 3062 individuals with or without COVID-19 from the clinic. METHODS: The levels of SARS-CoV-2 antibody IgM and IgG were measured by the immuno-colloidal gold method. A fusion fragment of nucleocapsid and spike protein was detected by a qualitative test kit with sensitivity (89%) and specificity (98%). RESULTS: The seroprevalence rate for IgM and IgG in all outpatients was 2.81% and 7.51%, respectively. The sex-related prevalence rate of IgG was significantly higher (P < 0.05) in women than men. The highest positive rate of IgM was observed in individuals < 20 years of age (3.57%), while the highest seroprevalence for IgG was observed in persons > 60 years of age (8.61%). Positive rates of IgM and IgG in the convalescent patients were 31.82% and 77.27%, respectively, which was significantly higher than individuals with suspected syndromes or individuals without any clinical signs (P < 0.01). Seroprevalence for IgG in medical staff was markedly higher than those in residents. No significant difference of seroprevalence was found among patients with different comorbidities (P > 0.05). CONCLUSIONS: The low positive rate of the SARS-CoV-2 IgM and nucleic acid (NA) test indicated that the SARS-CoV-2 outbreak is subsiding after 3 months, and the possibility of reintroduction of the virus from an unidentified natural reservoir is low. Seroprevalence provides information for humoral immunity and vaccine in the future.

Evaluation of sex-related hormones and semen characteristics in reproductive-aged male COVID-19 patients.

In the past several months, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated infection (coronavirus disease 2019 [COVID-19]) developed rapidly and has turned into a global pandemic. Although SARS-CoV-2 mainly attacks respiratory systems, manifestations of multiple organs have been observed. A great concern was raised about whether COVID-19 may affect male reproductive functions. In this study, we collected semen specimens from 12 male COVID-19 patients for virus detection and semen characteristics analysis. No SARS-CoV-2 was found in semen specimens. Eight out of 12 patients had normal semen quality. We also compared the sex-related hormone levels between 119 reproductive-aged men with SARS-CoV-2 infection and 273 age-matched control men. A higher serum luteinizing hormone (LH) and a lower ratio of testosterone (T) to LH were observed in the COVID-19 group. Multiple regression analysis indicated that serum T: LH ratio was negatively associated with white blood cell counts and C-reactive protein levels in COVID-19 patients. It's the first report about semen assessment and sex-hormone evaluation in reproductive-aged male COVID-19 patients. Although further study is needed to clarify the reasons and underlying mechanisms, our study presents an abnormal sex hormone secretion among COVID-19 patients, suggesting that attention should be paid to reproductive function evaluation in the follow-up.

Aberrant cytokine expression in COVID-19 patients: Associations between cytokines and disease severity.

Cytokines play pleiotropic, antagonistic, and collaborative in viral disease. The high morbidity and mortality of coronavirus disease 2019 (COVID-19) make it a significant threat to global public health. Elucidating its pathogenesis is essential to finding effective therapy. A retrospective study was conducted on 71 patients hospitalized with COVID-19. Data on cytokines, T lymphocytes, and other clinical and laboratory characteristics were collected from patients with variable disease severity. The effects of cytokines on the overall survival (OS) and event-free survival (EFS) of patients were analyzed. The critically severe and severe patients had higher infection indexes and significant multiple organ function abnormalities than the mild patients (P < 0.05). IL-6 and IL-10 were significantly higher in the critically severe patients than in the severe and mild patients (P < 0.05). IL-6 and IL-10 were closely associated with white blood cells, neutrophils, T lymphocyte subsets, D-D dimer, blood urea nitrogen, complement C1q, procalcitonin C-reactive protein. Moreover, the IL-6 and IL-10 levels were closely correlated to dyspnea and dizziness (P < 0.05). The patients with higher IL-10 levels had shorter OS than the group with lower levels (P < 0.05). The older patients with higher levels of single IL-6 or IL-10 tended to have shorter EFS (P < 0.05), while the patients who had more elevated IL-6 and IL-10 had shorter OS (P < 0.05). The Cox proportional hazard model revealed that IL-6 was the independent factor affecting EFS. IL-6 and IL-10 play crucial roles in COVID-19 prognosis.

Circularly Polarized Luminescence of Achiral Metal-Organic Colloids and Guest Molecules in a Vortex Field.

Recently, scientists have reported a range of chiral fluorescence materials or chiral composites that can emit circularly polarized luminescence. Herein, two achiral metal-organic colloidal solutions were studied, showing active circularly polarized luminescence, which is observed in vortex stirring. The absolute values for glum are 0.05 and 0.03 and the plus or minus sign of glum depends on the colloidal structure and stirring direction, which make the property easy to manipulate. Further, the host-guest interaction study suggests both electrostatic interactions and coordination bonding may influence the chiroptical property from the colloidal solution to the guest molecule. Rhodamine 6G and its carboxylic acid derivative exhibit good quantum yields and acceptable glum values in the colloidal solution.

Asymptomatic SARS-CoV-2 Infection in Household Contacts of a Healthcare Provider, Wuhan, China.

We found that all 5 asymptomatic household contacts of a Wuhan, China, physician with coronavirus disease had severe acute respiratory syndrome coronavirus 2 detected by PCR. The index patient and 2 contacts also had abnormal chest computed tomography scans. Asymptomatic infected household contacts of healthcare workers with coronavirus disease might be underrecognized.

The presence of SARS-CoV-2 RNA in the feces of COVID-19 patients.

In December 2019, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China, and has spread globally. However, the transmission route of SARS-CoV-2 has not been fully understood. In this study, we aimed to investigate SARS-CoV-2 shedding in the excreta of COVID-19 patients. Electronical medical records, including demographics, clinical characteristics, laboratory and radiological findings of enrolled patients were extracted and analyzed. Pharyngeal swab, stool, and urine specimens were collected and tested for SARS-CoV-2 RNA by real-time reverse transcription polymerase chain reaction. Viral shedding at multiple time points in specimens was recorded, and its correlation analyzed with clinical manifestations and the severity of illness. A total of 42 laboratory-confirmed patients were enrolled, 8 (19.05%) of whom had gastrointestinal symptoms. A total of 28 (66.67%) patients tested positive for SARS-CoV-2 RNA in stool specimens, and this was not associated with the presence of gastrointestinal symptoms and the severity of illness. Among them, 18 (64.29%) patients remained positive for viral RNA in the feces after the pharyngeal swabs turned negative. The duration of viral shedding from the feces after negative conversion in pharyngeal swabs was 7 (6-10) days, regardless of COVID-19 severity. The demographics, clinical characteristics, laboratory and radiologic findings did not differ between patients who tested positive and negative for SARS-CoV-2 RNA in the feces. Viral RNA was not detectable in urine specimens from 10 patients. Our results demonstrated the presence of SARS-CoV-2 RNA in the feces of COVID-19 patients and suggested the possibility of SARS-CoV-2 transmission via the fecal-oral route.

Highly sensitive and specific graphene oxide-based FRET aptasensor for quantitative detection of human soluble growth stimulating gene protein 2.

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1-100 µg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established.

The clinical outcome of COVID-19 is strongly associated with microbiome dynamics in the upper respiratory tract.

OBJECTIVES: The respiratory tract is the portal of entry for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a variety of respiratory pathogens other than SARS-CoV-2 have been associated with severe cases of COVID-19 disease, the dynamics of the upper respiratory microbiota during disease the course of disease, and how they impact disease manifestation, remain uncertain. METHODS: We collected 349 longitudinal upper respiratory samples from a cohort of 65 COVID-19 patients (cohort 1), 28 samples from 28 recovered COVID-19 patients (cohort 2), and 59 samples from 59 healthy controls (cohort 3). All COVID-19 patients originated from the earliest stage of the epidemic in Wuhan. Based on a modified clinical scale, the disease course was divided into five clinical disease phases (pseudotimes): "Healthy" (pseudotime 0), "Incremental" (pseudotime 1), "Critical" (pseudotime 2), "Complicated" (pseudotime 3), "Convalescent" (pseudotime 4), and "Long-term follow-up" (pseudotime 5). Using meta-transcriptomics, we investigated the features and dynamics of transcriptionally active microbes in the upper respiratory tract (URT) over the course of COVID-19 disease, as well as its association with disease progression and clinical outcomes. RESULTS: Our results revealed that the URT microbiome exhibits substantial heterogeneity during disease course. Two clusters of microbial communities characterized by low alpha diversity and enrichment for multiple pathogens or potential pathobionts (including Acinetobacter and Candida) were associated with disease progression and a worse clinical outcome. We also identified a series of microbial indicators that classified disease progression into more severe stages. Longitudinal analysis revealed that although the microbiome exhibited complex and changing patterns during COVID-19, a restoration of URT microbiomes from early dysbiosis toward more diverse status in later disease stages was observed in most patients. In addition, a group of potential pathobionts were strongly associated with the concentration of inflammatory indicators and mortality. CONCLUSION: This study revealed strong links between URT microbiome dynamics and disease progression and clinical outcomes in COVID-19, implying that the treatment of severe disease should consider the full spectrum of microbial pathogens present.

Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion through activation of p21-activated kinase (PAK1) in pancreatic ß-Cells.

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet ß-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet ß-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet ß-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.

Percutaneous valved stent implantation in the ascending aorta for the treatment of very high-risk aortic regurgitation: an animal study.

PURPOSE: We investigated the effects of percutaneous valved stent implantation in the ascending aorta as an alternative treatment for aortic regurgitation in a canine model. MATERIALS AND METHODS: A total of 16 healthy dogs weighing an average of 18.3 ± 2.1 kg were used for the establishment of animal models of chronic aortic regurgitation by percutaneous aortic valve perforation and balloon dilation. At 2 mo after successful model establishment, all experimental animals underwent valved stent implantation in the ascending aorta and then were followed up for 3 mo. RESULTS: Experimental models of chronic aortic regurgitation were successfully established in 10 dogs. Surviving dogs underwent successful valved stent implantation in the ascending aorta and were subsequently followed up for 3 mo. The level of instantaneous aortic regurgitation at 3-mo follow-up was significantly reduced compared with that before valved stent implantation (2.4 ± 0.9 versus 10.6 ± 2.1 mL/s, P < 0.05). The left ventricular ejection fraction was significantly increased (53.8 ± 4.2% versus 37.8 ± 3.7%, P < 0.05), and the left ventricular end-diastolic volume was also significantly reduced (30.3 ± 2.2 versus 40.1 ± 3.6 mL, P < 0.05). No paravalvular leak, stroke, atrioventricular block, or other complications occurred in dogs undergoing valved stent implantation. CONCLUSIONS: Percutaneous valved stent implantation in the ascending aorta is feasible, effective, and safe as an alternative treatment for very high-risk aortic regurgitation in a canine model.

IL-6 drives T cell death to participate in lymphopenia in COVID-19.

Lymphopenia is a common observation in patients with COVID-19. To explore the cause of T cell lymphopenia in the disease, laboratory results of 64 hospitalized COVID-19 patients were retrospectively analyzed and six patients were randomly selected to trace their changes of T lymphocytes and plasma concentration of IL-6 for the course of disease. Results confirmed that the T-cell lymphopenia, especially CD4+ T cell reduction in COVID-19 patients, was a reliable indicator of severity and hospitalization in infected patients. And CD4+ T cell count below 200 cells/µL predicts critical illness in COVID-19 patients. In vitro assay supported that exposure to key contributors (IL-1ß, IL-6, TNF-α and IFN-γ) of COVID-19 cytokine storm caused substantial death of activated T cells. Among these contributors, IL-6 level was found to probably reversely correlate with T cell counts in patients. And IL-6 alone was potent to induce T cell reduction by gasderminE-mediated pyroptosis, inferring IL-6 took a part in affecting the function and status of T cells in COVID-19 patients. Intervention of IL-6 mediated T cell pryprotosis may effectively delay disease progression, maintain normal immune status at an early stage of infection.

Synthesis and Biological Evaluation of the Matrine Derivatives as a Novel Family of Potential Anticancer Agents.

BACKGROUND: FufangKushen injection' was a Chinese Traditional anticancer drug, which has been widely used to treat cancer in combination with other anticancer drugs. OBJECTIVE: Our goal is to synthesize a series of novel 13-dithiocarbamates matrine derivatives using matrine (1) as the lead compound, and evaluate the biological activities of the obtained compounds. METHODS: The in vitro cytotoxicity of the target compounds against three human cancer cell lines (Hep3B, LM3 and BeL-7404) was evaluated. To investigate the mechanism of biological activity, cell cycle analysis was performed. RESULTS: The results revealed that compounds 6o and 6v displayed the most significant anticancer activity against three cancer cell lines with IC50 values in the range of 3.42-8.05 µM, which showed better activity than the parent compound (Matrine). SAR analysis indicated that the introduction of a substituted amino dithiocarbamate might significantly enhance the antiproliferative activity. CONCLUSION: During the newly synthesized compounds, matrine analog 6v exhibited a potent effect against three human tumor cell lines. The mode of action of 6v was to inhibit the G0/G1 phase arrest. Therefore, compound 6v has been selected as a novel-scaffold lead to further structural optimizations or as a chemical probe for exploring anticancer pathways of this kind of compounds.

High Performance of SARS-Cov-2N Protein Antigen Chemiluminescence Immunoassay as Frontline Testing for Acute Phase COVID-19 Diagnosis: A Retrospective Cohort Study.

Objectives: COVID-19 emerged and rapidly spread throughout the world. Testing strategies focussing on patients with COVID-19 require assays that are high-throughput, low-risk of infection, and with small sample volumes. Antigen surveillance can be used to identify exposure to pathogens and measure acute infections. Methods: A total of 914 serum samples, collected from 309 currently infected COVID-19 patients, 48 recovered ones, and 410 non-COVID-19 patients, were used to measure N protein antigen levels by a chemilumineseent immunoassay. Diagnostic performances were analyzed in different periods after onset. Results: There was a high level of N protein antigen in COVID-19 patients (0.56 COI), comparing to the recovered patients (0.12 COI) and controls (0.19 COI). In receiver-operating characteristic curve analysis, the area under the curve of serum N protein antigen was 0.911 in the first week after onset. In this period, Sensitivity and specificity of serologic N protein antigen testing was 76.27 and 98.78%. Diagnosis performance of specific antibodies became better from the third week after onset. Subgroup analysis suggested that severe patients had higher levels of antigens than mild patients. Conclusions: High level of serum antigen suggested early infection and serious illness. Serum N protein antigen testing by chemiluminescence immunoassay is considered as a viable assay used to improve diagnostic sensitivity for current patients.

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis.

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal alpha-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.

Characterization of a novel human CDK5 splicing variant that inhibits Wnt/beta-catenin signaling.

The cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases, playing an essential role in regulating cell-cycle progression. In our present work, human CDK5 and a novel CDK5 splicing variant, named as CDK5-SV, were cloned from the cDNA library of human testis. CDK5-SV lacking the exon 7 of CDK5 encodes a protein of 260 amino acids. Through RT-PCR analysis in different human tissues, CDK5-SV was found to be expressed in testis, skeletal muscle, colon, bone marrow and ovary, while CDK5 was ubiquitously expressed. Immunofluorescence experiment in HeLa cells showed that the subcellular localizations of CDK5-SV and CDK5 were totally different. CDK5 mainly located in the cytoplasm, while CDK5-SV accumulated in nucleus. Reporter gene assay showed that when co-transfected with beta-catenin, CDK5 and CDK5-SV could both strongly inhibit the Wnt/beta-catenin signaling pathway. Consistently, CDK5-SV could also interact with beta-catenin as CDK5 does. Taken together, our findings suggest that CDK5-SV might also be a negative regulator of Wnt/beta-catenin signaling pathway.

Family cluster of three recovered cases of pneumonia due to severe acute respiratory syndrome coronavirus 2 infection.

The coronavirus disease (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, in late 2019 and has affected more than 1 270 000 people worldwide. The numbers of reported cases continue to rise and threaten global health. Transmissions among family members are frequently observed, although the route of transmission is partially known. Here we report three cases of SARS-CoV-2 infection within one family. Sequencing of the S gene of the viral genome showed 100% identity among samples, suggesting that the same strain caused the infection. Following treatment with oseltamivir and short-term methylprednisolone combined with symptomatic management, all three patients recovered within 3 weeks, as evidenced by the disappearance of their symptoms, clearance of pulmonary infiltrates and consecutive negative molecular diagnostic test findings. Our observations suggest the importance of preventing family transmission and the efficacy of current integrated treatment for mild/moderate pneumonia in COVID-19 cases.

Letter to the Editor: Low-density lipoprotein is a potential predictor of poor prognosis in patients with coronavirus disease 2019.

BACKGROUND: The pandemic of coronavirus disease 2019 (COVID-19) has become a global threat to public health. The lipid pathophysiology in COVID-19 is unknown. METHODS: In this retrospective longitudinal study, we monitored the serum lipids in 17 surviving and 4 non-surviving COVID-19 cases prior to their viral infections and duration the entire disease courses. RESULTS: In surviving cases, the low-density lipoprotein (LDL) levels decreased significantly on admission as compared with the levels before infection; the LDL levels remained constantly low during the disease progression and resumed to the original levels when patients recovered (pre-infection: 3.5 (3.0-4.4); on admission: 2.8 (2.3-3.1), p < 0.01; progression: 2.5 (2.3-3.0); discharge: 3.6 (2.7-4.1); median (IQR), in mmol/L). In non-surviving patients, LDL levels showed an irreversible and continuous decrease until death (1.1 (0.9-1.2), p = 0.02 versus the levels on admission). The ratio changes of LDL levels inversely correlated with ratio changes of high-sensitivity C-reactive protein levels. Logistic regression analysis showed increasing odds of lowered LDL levels associated with disease progression (odds ratio: 4.48, 95% IC: 1.55-12.92, p = 0.006) and in-hospital death (odds ratio: 21.72, 95% IC: 1.40-337.54, p = 0.028). CONCLUSIONS: LDL levels inversely correlated to disease severities, which could be a predictor for disease progress and poor prognosis.

Serum-soluble ST2 as a novel biomarker reflecting inflammatory status and illness severity in patients with COVID-19.

Aim: The authors studied the role of soluble ST2 (sST2) in COVID-19 and its relationship with inflammatory status and disease severity. Materials & methods: Serum levels of sST2 and interleukin (IL)-33, C-reactive protein (CRP), serum amyloid protein (SAA), IL-6 and procalcitonin (PCT), and T lymphocyte subsets from 80 subjects diagnosed with COVID-19 including 36 mild, 41 severe and three asymptomatic cases were tested. Results: Serum sST2 levels were significantly increased in COVID-19 patients, which were positively correlated with CRP, but negatively correlated with CD4+ and CD8+ T lymphocyte counts. Serum sST2 levels in nonsurviving severe cases were persistently high during disease progression. Conclusion: Serum sST2 level test is helpful for reflecting inflammatory status and illness severity of COVID-19.