Publications by authors named "Min Zheng"

869 Publications

Multiple basic amino acids in the cleavage site of H7N9 hemagglutinin contribute to high virulence in mice.

J Thorac Dis 2021 Aug;13(8):4650-4660

State Key Laboratory of Respiratory Disease, Institute of Integration of Traditional and Western Medicine, First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Background: Avian influenza A (H7N9) virus has caused more than 1,500 cases of human infection since its emergence in early 2013. Displaying little or no pathogenicity in poultry, but a 40% case-fatality rate in humans, five waves of H7N9 human infections occurred in China during 2013-2017, caused solely by a low pathogenicity strain. However, avian isolates possessing a polybasic connecting peptide in the hemagglutinin (HA) protein were detected in mid-2016, indicating that a highly pathogenic virus had emerged and was co-circulating with the low pathogenicity strains.

Methods: Here we characterize the pathogenicity of a newly emerged human H7N9 variant with a PEVPKRKRTAR/GLF insertion motif at the cleavage site of the HA protein and .

Results: This variant replicates in MDCK cells independently of TPCK-trypsin, which is indicative of high pathogenicity in chickens. The 50% mouse lethal dose (MLD) of this novel isolate was less than 10 plaque forming units (PFU), compared with 3.16×10 for an identical virus lacking the polybasic insertion, indicating a high virulence phenotype.

Conclusions: Our results demonstrate that the multiple basic amino acid insertion in the HA protein of the H7N9 variant confers high virulence in mammals, highlighting a potential risk to humans. Continuous viral surveillance is therefore necessary in the China region to improve pandemic preparedness.
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http://dx.doi.org/10.21037/jtd-21-226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8411188PMC
August 2021

Combined usage of trimetazidine with 3-bromopyruvate may lead to cardiotoxicity by activating oxidative stress and apoptosis in rats.

J Cardiovasc Pharmacol 2021 Sep 6. Epub 2021 Sep 6.

Department of Cardiology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China. Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China.

Abstract: The energy used by the heart is generated mainly by the metabolism of fatty acids and glucose. Trimetazidine (TMZ) inhibits fatty acid metabolism and is used for the treatment of heart diseases such as heart failure. 3-Bromopyruvate (3-BrPA) can suppress glucose metabolism, and it is considered a promising candidate agent for tumour therapy. As TMZ and 3-BrPA can separately inhibit the two main cardiac energy sources, it is necessary to investigate the effects of 3-BrPA combined with TMZ on the heart.Forty male Wistar rats were randomly divided into four groups: a control group, a TMZ group, a 3-BrPA group, and a 3-BrPA+TMZ group. Weight was recorded every day, and echocardiography was performed 14 days later. Heart function, the levels of ATP, oxidative stress-related factors (ROS, GSH, GSSG, MDA, SOD, and TAC) and apoptosis in heart tissues were assessed to evaluate the effects of 3-BrPA and TMZ on the heart. In our study, no obvious changes occurred in the 3-BrPA group or the TMZ group compared with the control group. The combination of 3-BrPA and TMZ worsened heart function, decreased ATP levels and increased oxidative stress and myocardial apoptosis. In conclusion, 3-BrPA and TMZ are not recommended for concurrent use.
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http://dx.doi.org/10.1097/FJC.0000000000001136DOI Listing
September 2021

Elastography ultrasound with machine learning improves the diagnostic performance of traditional ultrasound in predicting kidney fibrosis.

J Formos Med Assoc 2021 Aug 24. Epub 2021 Aug 24.

Department of Nephrology, Molecular Cell Lab for Kidney Disease, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, PR China. Electronic address:

Background: Noninvasively predicting kidney tubulointerstitial fibrosis is important because it's closely correlated with the development and prognosis of chronic kidney disease (CKD). Most studies of shear wave elastography (SWE) in CKD were limited to non-linear statistical dependencies and didn't fully consider variables' interactions. Therefore, support vector machine (SVM) of machine learning was used to assess the prediction value of SWE and traditional ultrasound techniques in kidney fibrosis.

Methods: We consecutively recruited 117 CKD patients with kidney biopsy. SWE, B-mode, color Doppler flow imaging ultrasound and hematological exams were performed on the day of kidney biopsy. Kidney tubulointerstitial fibrosis was graded by semi-quantification of Masson staining. The diagnostic performances were accessed by ROC analysis.

Results: Tubulointerstitial fibrosis area was significantly correlated with eGFR among CKD patients (R = 0.450, P < 0.001). AUC of SWE, combined with B-mode and blood flow ultrasound by SVM, was 0.8303 (sensitivity, 77.19%; specificity, 71.67%) for diagnosing tubulointerstitial fibrosis (>10%), higher than either traditional ultrasound, or SWE (AUC, 0.6735 [sensitivity, 67.74%; specificity, 65.45%]; 0.5391 [sensitivity, 55.56%; specificity, 53.33%] respectively. Delong test, p < 0.05); For diagnosing different grades of tubulointerstitial fibrosis, SWE combined with traditional ultrasound by SVM, had AUCs of 0.6429 for mild tubulointerstitial fibrosis (11%-25%), and 0.9431 for moderate to severe tubulointerstitial fibrosis (>50%), higher than other methods (Delong test, p < 0.05).

Conclusion: SWE with SVM modeling could improve the diagnostic performance of traditional kidney ultrasound in predicting different kidney tubulointerstitial fibrosis grades among CKD patients.
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http://dx.doi.org/10.1016/j.jfma.2021.08.011DOI Listing
August 2021

Assessing the stability of one-stage PN/A process through experimental and modelling investigations.

Sci Total Environ 2021 Aug 18;801:149740. Epub 2021 Aug 18.

Advanced Water Management Centre, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address:

The mainstream partial nitritation/anammox (PN/A) process has been intensively studied but its stability remains a key challenge. It is shown here that biofilm thickness can exhibit a critical role in controlling the process stability of mainstream PN/A against dissolved oxygen (DO) variation. In a laboratory moving bed biofilm reactor (MBBR), PN/A performance was initially established in 200 days by controlling a low DO of 0.13 ± 0.07 mg O/L in the bulk liquid, which deteriorated with an increase of DO (0.35 ± 0.13 mg O/L) for over two months, and then rapidly recovered in a month with the initial low DO level re-applied. Biofilm thickness of PN/A carriers was measured during the experiment, which became significantly thinner (367 ± 146 μm) at mainstream conditions. The thin thickness primarily decreased the in-situ consumption rate of nitrite, rather than ammonium, when DO increased from 0.1 to 0.4 mg O/L, due to that the thin thickness can only restrict anammox capacity. These results illustrated the role of biofilm thickness in regulating PN/A performance and microbial activities. Further investigation using an established model revealed the joint contribution of biofilm thickness and DO concentration to PN/A process, while particularly, the biofilm thickness can determine the optimal DO level for maximizing the nitrogen removal efficiency and system robustness against DO variation. These results highlight the need of considering biofilm thickness in PN/A process optimization and stability improvement in low-strength wastewater treatment.
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http://dx.doi.org/10.1016/j.scitotenv.2021.149740DOI Listing
August 2021

Clinical Features and Survival Outcome of Early-Stage Primary Pulmonary MALT Lymphoma After Surgical Treatment.

Front Surg 2021 4;8:713748. Epub 2021 Aug 4.

Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China.

We aimed to study the clinical features and survival outcomes of patients with early-stage primary pulmonary mucosa-associated tissue (MALT) lymphoma who underwent surgery. This is a retrospective, single-center study including 32 patients with early-stage primary pulmonary MALT lymphoma. Univariate and multivariate Cox analyses were performed to select independent prognostic factors. The overall survival (OS) was analyzed by the Kaplan-Meier method and was compared with the log-rank test. Among the 32 patients included, there were 16 men (50.0%) and 16 women (50.0%). The average age was 59.2 years old. Ten patients had non-specific clinical symptoms including cough, expectoration, and chest pain, and four patients had B symptoms. CT images are not specific and can be shown as peripheral, central, solid, and ground glass but more peripheral (93.8%) and solid (75.0%). In prognostic analysis, univariate analysis showed that tumor stage and size were associated with relapse-free survival (RFS) and OS [hazard ratio (HR) = 1.105, 95% CI: 1.021-1.197, = 0.011; HR = 1.211, 95% CI: 1.158-1.968, = 0.003, respectively]. It seems to indicate that higher stage and larger size indicate a worse prognosis, but we could not find statistically significant predictors in multivariate analysis. Sublobectomy was performed in 21 (65.6) cases, lobectomy was performed in the other 11 (34.4) cases, both of them can achieve good prognosis (5-year RFS and OS are both 100%), and there is no significant difference between them. The clinical manifestation of early-stage primary pulmonary MALT lymphoma is not significantly specific, and surgical resection is an effective treatment.
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http://dx.doi.org/10.3389/fsurg.2021.713748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371471PMC
August 2021

Metal-to-Ligand Ratio-Dependent Chemodivergent Asymmetric Synthesis.

Angew Chem Int Ed Engl 2021 Aug 18. Epub 2021 Aug 18.

Institute of Chemistry and BioMedical Sciences, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China.

Chemodivergent asymmetric synthesis was achieved by tuning the metal-to-ligand ratio in an organometallic catalytic system. Using N-(aroyloxy)phthalimide as the precursor of either an oxygen-centered aroyloxy radical or a nitrogen-centered phthalimidyl radical, enantioselective oxocyanation or aminocyanation of alkenes was achieved separately through a dual photoredox and copper catalysis. The metal-to-ligand ratio can exert chemoselective control while retaining the high enantiopurity of divergent products. Both reactions proceed efficiently with catalyst loading as low as 0.2 mol % and can be performed on a gram scale without loss of chemoselectivity or enantioselectivity. Chemodivergent asymmetric 1,5-aminocyanation or 1,5-oxocyanation of vinylcyclopropane can also be realized by this protocol. Mechanistic investigations involving electron paramagnetic resonance (EPR) experiments were performed to shed light on the stereochemical and chemodivergent results.
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http://dx.doi.org/10.1002/anie.202108617DOI Listing
August 2021

Centralized iron-dosing into returned sludge brings multifaceted benefits to wastewater management.

Water Res 2021 Sep 11;203:117536. Epub 2021 Aug 11.

Advanced Water Management Centre, The University of Queensland, St Lucia QLD 4072, Australia.

Iron salts (i.e. FeCl) are the most used chemicals in the urban wastewater system. Iron is commonly dosed into sewage or the mainstream system, which provides multiple benefits such as enhanced phosphorus removal and improved sludge settleability/dewaterability. This study reported and demonstrated a new approach that dosed FeCl into returned sludge in order to bring two more benefits to wastewater management: short-cut nitrogen removal via the nitrite pathway and less biomass production. This approach is achieved based on our findings that with similar amount of FeCl, centralized iron dosing into a sidestream sludge unit generated iron concentration two orders of magnitude higher than the common mainstream dosing (e.g. 10-40 mg Fe/L-wastewater), leading to sludge acidification (pH = 2.1) with Fe (III) hydrolysis. Together with accumulated nitrite in the supernatant of the sludge, ppm-level of free nitrous acid was generated and thus enabled sludge disintegration, cell lysis, and selective elimination of nitrite-oxidizing bacteria (NOB). Long-term effects on nitrifying bacteria and overall reactor performance were evaluated using two laboratory reactor experiments for over one year. The experimental reactor showed stable nitrite accumulation with an average NO/(NO + NO) ratio above 80% and ∼30% observed biomass yield reduction compared to those in control reactors. In addition, the centralized sludge dosing strategy still provided benefits such as improved settleability and dewaterability of sludge and enhanced phosphorus removal.
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http://dx.doi.org/10.1016/j.watres.2021.117536DOI Listing
September 2021

Sprue-Like Enteropathy and Liver Injury: A Rare Emerging Association with Olmesartan.

J Investig Med High Impact Case Rep 2021 Jan-Dec;9:23247096211037463

Bridgeport Hospital, Yale New Haven Health, Bridgeport, CT, USA.

Olmesartan-induced enteropathy is an underreported phenomenon, first described in 2012. While olmesartan's antihypertensive properties were confirmed early on, its association with a sprue-like enteropathy was subsequently noted. Although this association has been reported with olmesartan, there have been few reports of this association with other angiotensin-receptor blockers. We present a case of a 79-year-old male who presented with diarrhea, weight loss, jaundice, and transaminitis. Further history revealed that he had been taking olmesartan 40 mg daily for hypertension. Workup of his diarrhea and jaundice included duodenal and liver biopsies revealed findings consistent with a sprue-like enteropathy and an autoimmune hepatitis-like pattern. On discontinuation of olmesartan, his 1-month follow-up revealed significant improvement in his clinical status as well as his liver function tests. Olmesartan is an effective antihypertensive medication; however, physicians must be mindful of its side effect of causing a sprue-like enteropathy and liver injury. Patients should be counseled on discontinuing olmesartan, and they should be started on an alternative therapy for hypertension.
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http://dx.doi.org/10.1177/23247096211037463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361549PMC
August 2021

Dynamic Characteristic Analysis of Antibodies in Patients With COVID-19: A 13-Month Study.

Front Immunol 2021 20;12:708184. Epub 2021 Jul 20.

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

There is a worldwide pandemic of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; yet our understanding remains limited on the characteristic of antibodies, especially for dynamic long-term tracking. Sequential serum samples were collected up to 416 days post onset of symptoms (POS) from 102 patients who were hospitalized with coronavirus disease 2019 (COVID-19). Immunoglobulin (Ig)G, IgM, and IgA levels targeting SARS-CoV-2 spike 1 receptor-binding domain (S1-RBD), spike 2 extracellular domain (S2-ECD), and nucleocapsid protein (N) were quantified as well as neutralizing activity. We were pleasantly surprised to find that the antibody remained detective and effective for more than a year POS. We also found the varied reactions of different antibodies as time passed: N-IgA rose most rapidly in the early stage of infection, while S2-IgG was present at a high level in the long time of observation. This study described the long traceable antibody response of the COVID-19 and offered hints about targets to screen for postinfectious immunity and for vaccination development of SARS-CoV-2.
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http://dx.doi.org/10.3389/fimmu.2021.708184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8330131PMC
August 2021

Insights into complete nitrate removal in one-stage nitritation-anammox by coupling heterotrophic denitrification.

J Environ Manage 2021 Nov 2;298:113431. Epub 2021 Aug 2.

Advanced Water Management Centre, The University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia.

Nitritation-anammox has been considered to be the most promising process for nitrogen (N) removal from wastewater. However, the anammox reaction still produces an amount of nitrate, which cannot be removed further. This study hypothesizes that heterotrophic denitrification can be an appealing option to remove the residual nitrate in the one-stage nitritation-anammox process. Through monitoring N-removal performance and microbial community succession of a laboratory microaerobic reactor, the effect of four different levels of oxygen supply on nitrate removal was investigated. The reactor was continuously fed with real manure-free piggery wastewater containing ~240 mg NH-N/L and chemical oxygen demand (COD)/total nitrogen (TN) ratio of less than 1 for 180 days. With a high influent loading rate of 0.7 kg N/(m·d), efficient total nitrogen removal (>80 %) was achieved during stable operation of dissolved oxygen (DO) concentrations between 0.3 and 0.6 mg O/L, indicating N-removal via the nitritation-anammox pathway in the low-carbon wastewater treatment. At the same time, the effluent nitrate reduced with decreased oxygen supply and completely depleted at DO of 0.3 ± 0.1 mg O/L. In addition to oxygen, preventing ammonia nitrogen from falling to very low levels (<10 mg/L) could be also useful for the complete nitrate removal and stable nitritation-anammox. 16S rRNA gene-based analyses confirmed a complex microbial community including nitrifiers, denitrifiers and anammox bacteria in the biomass of the reactor. Collectively, this study provides new insights into high-level N-removal of a nitritation-anammox process by complete nitrate depletion.
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http://dx.doi.org/10.1016/j.jenvman.2021.113431DOI Listing
November 2021

Sarracenia purpurea glycerol-3-phosphate acyltransferase 5 confers plant tolerance to high humidity in Arabidopsis thaliana.

Physiol Plant 2021 Aug 4. Epub 2021 Aug 4.

School of Life Sciences, Southwest University, Chongqing, China.

Suberin, as a lipid polyester barrier, limits the movement of gas, water, and solutes, and plays important roles in plant protection and growth. In this study, a CDS encoding glycerol-3-phosphate acyltransferase 5 (GPAT5) was cloned from Sarracenia purpurea to investigate the gene function. SpGPAT5 shares 72% identity and 80% similarity to AtGPAT5 that is required for suberin synthesis. Fluorol Yellow 088 staining showed that the S. purpurea pitcher (specific leaf) tube contained more suberin in the adaxial surface compared to the lid, and SpGPAT5 transcripts were detected in the pitcher. Previous reported Atgpat5-1 phenotypes were complemented with SpGPAT5 showing that the Atgpat5-1 seed coat had increased permeability of tetrazolium red and the mutant was sensitive to salt. We also found that SpGPAT5 was able to revert the hyperhydric phenotype of Atgpat5-1 under high humidity. Thus, this study suggests that SpGPAT5 can functionally replace AtGPAT5 and contributes to plant tolerance to high humidity, which maybe assist in understanding the role of suberin-associated waxes in S. purpurea pitchers for water retention.
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http://dx.doi.org/10.1111/ppl.13516DOI Listing
August 2021

Spatially-Aware Context Neural Networks.

IEEE Trans Image Process 2021 6;30:6906-6916. Epub 2021 Aug 6.

A variety of computer vision tasks benefit significantly from increasingly powerful deep convolutional neural networks. However, the inherently local property of convolution operations prevents most existing models from capturing long-range feature interactions for improved performances. In this paper, we propose a novel module, called Spatially-Aware Context (SAC) block, to learn spatially-aware contexts by capturing multi-mode global contextual semantics for sophisticated long-range dependencies modeling. We enable customized non-local feature interactions for each spatial position through re-weighted global context fusion in a non-normalized way. SAC is very lightweight and can be easily plugged into popular backbone models. Extensive experiments on COCO, ImageNet, and HICO-DET benchmarks show that our SAC block achieves significant performance improvements over existing baseline architectures while with a negligible computational burden increase. The results also demonstrate the exceptional effectiveness and scalability of the proposed approach on capturing long-range dependencies for object detection, segmentation, and image classification, outperforming a bank of state-of-the-art attention blocks.
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http://dx.doi.org/10.1109/TIP.2021.3097917DOI Listing
August 2021

Corrigendum: Overexpression Cathepsin D Contributes to Perineural Invasion of Salivary Adenoid Cystic Carcinoma.

Front Oncol 2021 23;11:693392. Epub 2021 Jun 23.

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.

[This corrects the article DOI: 10.3389/fonc.2018.00492.].
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http://dx.doi.org/10.3389/fonc.2021.693392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262266PMC
June 2021

Serum free light chain is associated with histological activity and cirrhosis in patients with chronic hepatitis B.

Int Immunopharmacol 2021 Oct 7;99:107881. Epub 2021 Jul 7.

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou City, Zhejiang Province 310003, China. Electronic address:

Background: The antiviral immune response is the main cause of hepatocyte damage and inflammatory necrosis. The serum free light chain, reflecting the immune function of B-cells, is strongly associated with inflammation and disease activity. We aimed to investigate the association of serum free light chain with the progression of chronic hepatitis B.

Methods: A total of 208 eligible chronic hepatitis B patients who had undergone a liver biopsy were studied. Serum free light chains of all patients were measured by turbidimetry using an immunoassay. Liver histology was assessed according to the METAVIR scoring system (which grades the stage of fibrosis on a five-point scale, F0 = no fibrosis to F4 = cirrhosis, and histological activity on a four-point scale, A0 = no activity to A3 = severe activity). The association of serum free light chains with histological activity and fibrosis progression was evaluated.

Results: The concentration of serum free light chains in CHB patients increased gradually with histological activity and fibrosis progression. The intensity of histological activity was significantly correlated with the serum free kappa chain (r = 0.658, P < 0.001) and the serum free lambda chain (0.675, P < 0.001). The stages of fibrosis were correlated with the serum free kappa chain (r = 0.683, P < 0.001) and serum free lambda chain (0.664, P < 0.001). After adjusting for age, sex and other synergic factors, the serum free kappa chain remained a potential risk factor, but the serum free lambda chain was no longer associated with liver cirrhosis. Similar to FIB-4 and RPR, the serum free kappa chain exhibited excellent performance in the prediction of liver cirrhosis. The AUCs of serum free Kappa chain, FIB-4 and RPR were 0.873, 0.880 and 0.895, respectively, which were significantly higher than those of the AAR and APRI (0.718 and 0.746).

Conclusion: Our work revealed that serum free light chains were associated with histological activity and cirrhosis in chronic hepatitis B, which could play a crucial role in the immunopathogenesis of HBV-associated cirrhosis. In addition, free kappa light chain could be a useful predictor of liver cirrhosis.
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http://dx.doi.org/10.1016/j.intimp.2021.107881DOI Listing
October 2021

An ultrasound image-based deep multi-scale texture network for liver fibrosis grading in patients with chronic HBV infection.

Liver Int 2021 Jul 5. Epub 2021 Jul 5.

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, P. R. China.

Background & Aims: The evaluation of the stage of liver fibrosis is essential in patients with chronic liver disease. However, due to the low quality of ultrasound images, the non-invasive diagnosis of liver fibrosis based on ultrasound images is still an outstanding question. This study aimed to investigate the diagnostic accuracy of a deep learning-based method in ultrasound images for liver fibrosis staging in multicentre patients.

Methods: In this study, we proposed a novel deep learning-based approach, named multi-scale texture network (MSTNet), to assess liver fibrosis, which extracted multi-scale texture features from constructed image pyramid patches. Its diagnostic accuracy was investigated by comparing it with APRI, FIB-4, Forns and sonographers. Data of 508 patients who underwent liver biopsy were included from 4 hospitals. The area-under-the ROC curve (AUC) was determined by receiver operating characteristics (ROC) curves for significant fibrosis (≥F2) and cirrhosis (F4).

Results: The AUCs (95% confidence interval) of MSTNet were 0.92 (0.87-0.96) for ≥F2 and 0.89 (0.83-0.95) for F4 on the validation group, which significantly outperformed APRI, FIB-4 and Forns. The sensitivity and specificity of MSTNet (85.1% (74.5%-92.0%) and 87.6% (78.0%-93.6%)) were better than those of three sonographers in assessing ≥F2.

Conclusions: The proposed MSTNet is a promising ultrasound image-based method for the non-invasive grading of liver fibrosis in patients with chronic HBV infection.
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http://dx.doi.org/10.1111/liv.14999DOI Listing
July 2021

Arabidopsis SSB1, a Mitochondrial Single-Stranded DNA-Binding Protein, is Involved in ABA Response And Mitochondrial RNA Splicing.

Plant Cell Physiol 2021 Jun 29. Epub 2021 Jun 29.

Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Southwest University, Chongqing, China.

A mitochondrion is a semiautonomous organelle that provides energy for life activities and balances plant growth and stress responses. Abscisic acid (ABA) regulates multiple physiological processes, including seed maturation, seed dormancy, stomatal closure, and various abiotic stress responses. However, the relationship between mitochondrial activity and the ABA response is unclear. In this study, an Arabidopsis mutant, ssb1-1, was isolated because of its hypersensitivity toward ABA. Assessment results showed that ABA negatively regulates the expression of Arabidopsis SSB1. Mutations in ABA insensitive 4 (ABI4) and ABI5, genes of key transcription factors involved in ABA-dependent seed dormancy, attenuated the ABA sensitivity of ssb1-1 during germination, suggesting that Arabidopsis SSB1 may act as a regulator in ABA response. Inhibition of endogenous ABA biosynthesis reversed the NaCl-sensitive phenotype of the ssb1-1 mutant, indicating that enhanced ABA biosynthesis is critical for the salinity stress response of ssb1-1. Moreover, compared to that of the wild type, ssb1-1 accumulated more ROS and exhibited increased sensitivity to the application of exogenous H2O2 during seed germination. SSB1 is also required for mitochondrial RNA splicing, as indicated by the result showing that SSB1 loss of function led to decreased splicing efficiency of nad1 intron1 and nad2 intron1. Taken together, our data reported here provide insights into a novel role of Arabidopsis SSB1 in ABA signaling and mitochondrial RNA splicing.
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http://dx.doi.org/10.1093/pcp/pcab097DOI Listing
June 2021

Cobalt induces neurodegenerative damages through Pin1 inactivation in mice and human neuroglioma cells.

J Hazard Mater 2021 10 12;419:126378. Epub 2021 Jun 12.

Department of Preventive Medicine, School of Public Health, Fujian Medical University, Fuzhou 350122, China; Fujian Provincial Key Laboratory of Environmental Factors and Cancer, School of Public Health, Fujian Medical University, Fuzhou 350122, China; The Key Laboratory of Environment and Health, School of Public Health, Fujian Medical University, Fuzhou 350122, China. Electronic address:

Cobalt is a hazardous material that has harmful effects on neurotoxicity. Excessive exposure to cobalt or inactivation of the unique proline isomerase Pin1 contributes to age-dependent neurodegeneration. However, nothing is known about the role of Pin1 in cobalt-induced neurodegeneration. Here we find that out of several hazardous materials, only cobalt dose-dependently decreased Pin1 expression and alterations in its substrates, including cis and trans phosphorylated Tau in human neuronal cells, concomitant with neurotoxicity. Cobalt-induced neurotoxicity was aggravated by Pin1 genetic or chemical inhibition, but rescued by Pin1 upregulation. Furthermore, less than 4 μg/l of blood cobalt induced dose- and age-dependent Pin1 downregulation in murine brains, ensuing neurodegenerative changes. These defects were corroborated by changes in Pin1 substrates, including cis and trans phosphorylated Tau, amyloid precursor protein, β amyloid and GSK3β. Moreover, blood Pin1 was downregulated in human hip replacement patients with median blood cobalt level of 2.514 μg/l, which is significantly less than the safety threshold of 10 μg/l, suggesting an early role Pin1 played in neurodegenerative damages. Thus, Pin1 inactivation by cobalt contributes to age-dependent neurodegeneration, revealing that cobalt is a hazardous material triggering AD-like neurodegenerative damages.
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http://dx.doi.org/10.1016/j.jhazmat.2021.126378DOI Listing
October 2021

CXCL12/CXCR4 facilitates perineural invasion via induction of the Twist/S100A4 axis in salivary adenoid cystic carcinoma.

J Cell Mol Med 2021 Aug 25;25(16):7901-7912. Epub 2021 Jun 25.

State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Department of Oral Pathology, West China Hospital of Stomatology (Sichuan University), Chengdu, China.

The activation of CXCL12/CXCR4 axis participated in the progression of multiple cancers, but potential effect in terms of perineural invasion (PNI) in SACC remained ambiguous. In this study, we identified that CXCL12 substantially expressed in nerve cells. CXCR4 strikingly expressed in tumour cells, and CXCR4 expression was closely associated with the level of EMT-associated proteins and Schwann cell hallmarks at nerve invasion frontier in SACC. Activation of CXCL12/CXCR4 axis could promote PNI and up-regulate relative genes of EMT and Schwann cell hallmarks both in vitro and in vivo, which could be inhibited by Twist silence. After overexpressing S100A4, the impaired PNI ability of SACC cells induced by Twist knockdown was significantly reversed, and pseudo foot was visualized frequently. Collectively, the results indicated that CXCL12/CXCR4 might promote PNI by provoking the tumour cell to differentiate towards Schwann-like cell through Twist/S100A4 axis in SACC.
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http://dx.doi.org/10.1111/jcmm.16713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358865PMC
August 2021

Mammalian cells use the autophagy process to restrict avian influenza virus replication.

Cell Rep 2021 Jun;35(10):109213

State Key Laboratory for Emerging Infectious Diseases, InnoHK Centre for Virology, Vaccinology, and Therapeutics, and Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China. Electronic address:

Host adaptive mutations in the influenza A virus (IAV) PB2 protein are critical for human infection, but their molecular action is not well understood. We observe that when IAV containing avian PB2 infects mammalian cells, viral ribonucleoprotein (vRNP) aggregates that localize to the microtubule-organizing center (MTOC) are formed. These vRNP aggregates resemble LC3B-associated autophagosome structures, with aggresome-like properties, in that they cause the re-distribution of vimentin. However, electron microscopy reveals that these aggregates represent an accumulation of autophagic vacuoles. Compared to mammalian-PB2 virus, avian-PB2 virus induces higher autophagic flux in infected cells, indicating an increased rate of autophagosomes containing avian vRNPs fusing with lysosomes. We found that p62 is essential for the formation of vRNP aggregates and that the Raptor-interacting region of p62 is required for interaction with vRNPs through the PB2 polymerase subunit. Selective autophagic sequestration during late-stage virus replication is thus an additional strategy for host restriction of avian-PB2 IAV.
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http://dx.doi.org/10.1016/j.celrep.2021.109213DOI Listing
June 2021

A Correlative Study Between Personality Traits and the Preference of Site Selection in Cosmetic Treatment.

Front Psychiatry 2021 19;12:648751. Epub 2021 May 19.

Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

Cosmetic treatment was closely associated with beauty seekers' psychological well-being. Patients who seek cosmetic surgery often show anxiety. Nevertheless, not much is known regarding how personality traits relate to the selection of body parts that receive cosmetic treatment. This study aims to investigate the correlation between personality traits and various selection sites for cosmetic treatment via Eysenck Personality Questionnaire (EPQ). A cross-sectional approach was adopted to randomly recruited patients from a general hospital planning to undergo cosmetic treatments. All respondents completed the EPQ and provided their demographic information. The EPQ involves four scales: the extraversion (E), neuroticism (N), psychoticism (P), and lying scales (L). Psychological scales were evaluated to verify that people who selected different body sites for cosmetic intervention possessed different personality portraits. A total of 426 patients with an average age of 32.14 ± 8.06 were enrolled. Among them, 384 were females, accounting for more than 90% of patients. Five treatment sites were analyzed, including the body, eye, face contour, nose, and skin. Comparatively, patients with neuroticism were more likely to undergo and demand rhinoplasty (OR 1.15, 95% CI 1.07-1.24, < 0.001). Face contour treatment was commonly associated with extraversion (OR 1.05, 95% CI 1.00-1.11, = 0.044), psychoticism (OR 1.13, CI 1.03-1.25, = 0.013), and neuroticism (OR 1.05, CI 1.01-1.10, = 0.019). This novel study attempted to determine the personality profiles of beauty seekers. The corresponding assessments may provide references for clinical treatment options and enhance postoperative satisfaction for both practitioners and patients.
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http://dx.doi.org/10.3389/fpsyt.2021.648751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169958PMC
May 2021

A multidisciplinary team for the diagnosis and management of psoriatic arthritis.

Chin Med J (Engl) 2021 06 4;134(12):1387-1389. Epub 2021 Jun 4.

Department of Dermatology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, China.

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http://dx.doi.org/10.1097/CM9.0000000000001588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213281PMC
June 2021

Proteomic Analysis Identifies Prolonged Disturbances in Pathways Related to Cholesterol Metabolism and Myocardium Function in the COVID-19 Recovery Stage.

J Proteome Res 2021 07 3;20(7):3463-3474. Epub 2021 Jun 3.

State Key Laboratory for Diagnosis and Treatment of Infectious Disease, National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.

The COVID-19 pandemic has become a worldwide health crisis. So far, most studies have focused on the epidemiology and pathogenesis of this infectious disease. Little attention has been given to the disease sequelae in patients recovering from COVID-19, and nothing is known about the mechanisms underlying these sequelae. Herein, we profiled the serum proteome of a cohort of COVID-19 patients in the disease onset and recovery stages. Based on the close integration of our proteomic analysis with clinical data, we propose that COVID-19 is associated with prolonged disorders in cholesterol metabolism and myocardium, even in the recovery stage. We identify potential biomarkers for these disorders. Moreover, severely affected patients presented more serious disturbances in these pathways. Our findings potentially support clinical decision-making to improve the prognosis and treatment of patients.
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http://dx.doi.org/10.1021/acs.jproteome.1c00054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189036PMC
July 2021

Microsatellite instability and Epstein-Barr virus combined with PD-L1 could serve as a potential strategy for predicting the prognosis and efficacy of postoperative chemotherapy in gastric cancer.

PeerJ 2021 18;9:e11481. Epub 2021 May 18.

Division of Clinical Research, First Hospital of Jilin University, Changchun, Jilin Province, China.

Background: Microsatellite instability (MSI) and Epstein-Barr virus (EBV)-positive molecular subtypes exhibit complex immune responses in gastric cancer (GC), and PD-L1 has emerged as a prognostic biomarker associated with the cancer immune microenvironment. This study aimed to determine the prognostic value of molecular subtypes and whether the addition of PD-L1 would accurately predict the prognosis and guide postoperative chemotherapy for GC patients.

Methods: We performed molecular subtyping of tissue microarray slides from 226 GC patients who were treated with radical gastrectomy. The MSI status and PD-L1 expression were evaluated through immunohistochemistry (IHC) and EBV status through situ hybridization. Multiplex polymerase chain reaction (PCR) was also performed on 50 cases to validate the accuracy of IHC in defining MSI status. Differences in overall survival (OS) were assessed using the Kaplan-Meier method, log-rank test and Cox proportional hazards regression model.

Results: Among the 226 GC patients, 52 (23.2%) patients were classified as the MSI subtype, 11 (4.9%) were EBV subtype, and 161 (71.9%) were MSS (Microsatellite stable) /EBV subtype according to TCGA analysis. Two patients were both positive for MSI and EBV infection. EBV cases showed higher PD-L1 positivity than MSI cases and MSS/EBV cases (81.8% 50.0% 35.4%, = 0.003). Compared with the non-MSS/EBV (MSI or EBV cases) subgroup, GC patients with MSS/EBV were associated with the worst outcomes (HR = 1.610, 95% CI [1.0462.479], = 0.031). MSS/EBV GCs alone could benefit from postoperative chemotherapy (HR = 0.452, 95% CI [0.2990.682], <0.001), and PD-L1-positive expression could also predict a better prognosis (HR = 0.612, 95% CI [0.3890.962], = 0.033) in this subgroup. Considering both chemotherapy efficacy and PD-L1 expression in the MSS/EBV subgroup, chemotherapy could improve the prognosis for PD-L1-negative MSS/EBV GCs (HR = 0.357, 95% CI [0.2170.587], <0.001) but not PD-L1-positive MSS/EBV GCs.

Conclusions: Molecular subtyping combined with PD-L1 expression could serve as a potential strategy to better predict prognosis and guide postoperative chemotherapy of GC patients.
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http://dx.doi.org/10.7717/peerj.11481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139270PMC
May 2021

Unravelling adaptation of nitrite-oxidizing bacteria in mainstream PN/A process: Mechanisms and counter-strategies.

Water Res 2021 Jul 13;200:117239. Epub 2021 May 13.

Advanced Water Management Centre, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address:

Stable suppression of nitrite-oxidizing bacteria (NOB) is still a major challenge for the implementation of partial nitritation and anammox (PN/A) in mainstream treatment. Despite numerous suppression strategies demonstrated, it is increasingly recognized that NOB could develop resistance to these strategies, threatening the long-term stability of the mainstream PN/A process. This study aims to understand adaption mechanisms and develop counter-strategies to overcome the adaptation. To this end, three previously-demonstrated suppression strategies, including NOB inactivation via side stream sludge treatment with free ammonia (FA), the use of low dissolved oxygen (DO), and the use of anammox to scavenge nitrite, were stepwise applied, over a period of 800 days, to a laboratory-scale reactor treating effluent from a high-rate activated sludge (HRAS) plant. FA sludge treatment alone sustained nitrite accumulation for about two months, after which NOB adaptation occurred causing PN to fail. The FA adaptation was induced by a shift in the NOB community from Nitrospira to Ca. Nitrotoga. The latter was found to have higher resistance to FA and also a higher maximum specific growth rate. Low DO at 0.2-0.4 mg O L was then applied, in conjunction with FA treatment, which successfully eliminated Ca. Nitrotoga and re-established PN. However, new and unidentified NOB with a higher apparent oxygen affinity emerged in three months, again leading to PN failure. Lastly, as the third strategy for NOB suppression, anammox was introduced as an in-situ nitrite-scavenger. The combo-strategy delivered reliable NOB suppression with no further adaptation in the remaining experimental period (eight months). The resulted one-stage PN/A reactor achieved a nitrogen removal efficiency of 84.2 ± 5.37%. A control reactor, operated in parallel under the same conditions but without FA treatment, only achieved 10.4 ± 4.6% nitrogen removal, with anammox completely outcompeted by NOB in the last phase.
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http://dx.doi.org/10.1016/j.watres.2021.117239DOI Listing
July 2021

Bioleaching of toxic metals from anaerobically digested sludge without external chemical addition.

Water Res 2021 Jul 7;200:117211. Epub 2021 May 7.

Advanced Water Management Centre, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address:

Anaerobically digested (AD) sludge is widely applied to agricultural land as fertilizer. However, heavy metals in AD sludge potentially pose a significant threat to environment. This study reports a novel bioleaching approach, with no need for externally added chemicals. Sludge acidification was achieved using the protons produced from microbial oxidation of the inherent ammonium in AD sludge. An acid-tolerant microbial consortium, dominated by ammonia-oxidizing bacteria from the genus Candidatus Nitrosoglobus (i.e. relative abundance of 72.5 ± 2.3% based on 16S rRNA gene sequencing), was enriched after 120 days incubation in a laboratory sequencing batch reactor. The consortium oxidizes ammonium even at pH 2.5, at approximately 30% of its maximum rate, measured at pH 5.5. Inoculating the consortium at a solid ratio of 1:20, caused the pH of the AD sludge to decrease from 7.5 to 2.0 over five days under aerobic conditions. As a result, metals in the AD sludge were efficiently extracted into the liquid phase. In particular, two of the most abundant toxic metals, Cu and Zn, were solubilized with high efficiencies of 88 ± 4% and 96 ± 3%, respectively. Overall, the results of this study enable the economical and safe reuse of excess sludge generated during biological wastewater treatment.
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http://dx.doi.org/10.1016/j.watres.2021.117211DOI Listing
July 2021

Effects of miR-132-3p on progress and epithelial mesenchymal transition of non-small cell lung cancer via regulating KLF7.

J Thorac Dis 2021 Apr;13(4):2426-2436

Thoracic Surgery Department, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Background: MicroRNAs (miRNAs) often appear as oncogenes or tumor suppressor genes. The aim of this research was to examine miR-132-3p and Kruppel-like factor 7 (KLF7) effects in the development of non-small cell lung cancer (NSCLC).

Methods: We used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine miR-132-3p expression in tissue specimens and 6 cells (A549, H1650, H292, H1299, H1944, BEAS-2b). Luciferase report forecasted the targeting relationship between miR-132-3p and KLF7. The expression of KLF7 and interstitial protein was determined by western blot. Proliferation test and Transwell assay were adopted for examining cell development. The Cell Counting Kit-8 (CCK-8) colorimetric method was used to observe the effects of miR-132-3p and KLF7 on the proliferation, metastasis, and invasion of NSCLC tumor cells. In order to determine whether the metastasis of NSCLC tumor cells was epithelial-mesenchymal transition (EMT)-mediated, supplementary experiments with E-cadherin and vimentin were performed.

Results: An increased expression of miR-132-3p was detected in NSCLC. Its mimic promoted the proliferation of tumor cells. As an immediate site of miR-132-3p, KLF7 was reversely adjusted via miR-132-3p and restrained the development of tumor cells in NSCLC, the effects of which were attenuated via KLF7 over-expression. Besides, the presence of EMT-related diversions was confirmed in NSCLC.

Conclusions: By targeting KLF7, miR-132-3p was capable of promoting the proceeding of NSCLC tumor cells. We discovered miR-132-3p/KLF7 route may exhibit curative target for NSCLC.
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http://dx.doi.org/10.21037/jtd-21-353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107552PMC
April 2021

Chinese guidelines on the clinical application of 5-aminolevulinic acid-based photodynamic therapy in dermatology (2021 edition).

Photodiagnosis Photodyn Ther 2021 Sep 12;35:102340. Epub 2021 May 12.

Institute of Photomedicine, Shanghai Skin Disease Hospital, School of Medicine, Tongji University, Shanghai, China. Electronic address:

Photodynamic Therapy with 5-aminolevulinic acid (ALA-PDT) has been widely applied in the treatment of skin diseases in China. To further standardize, guide, and promote the clinical applications of ALA-PDT in dermatology, the Chinese Society of Dermatology, Chinese Association of Rehabilitation Dermatology, Photomedicine Therapeutic Equipment Group of Committee on Skin Disease, and Cosmetic Dermatology of China Association of Medical Equipment invited relevant experts engaged in ALA-PDT to revise and update the first edition of "Clinical application of 5-aminolevulinic acid-based photodynamic therapy: an expert consensus statement" and establish a more current edition, to provide an updated reference for Chinese dermatologists in clinical practice. In the guideline, the expert group reached consensus opinions on ALA-PDT with regard to mechanisms of action, therapeutic protocol, clinical applications, adverse reactions and countermeasures, precautions, care, and evaluation of efficacy.
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http://dx.doi.org/10.1016/j.pdpdt.2021.102340DOI Listing
September 2021

Insights into Nitrous Oxide Mitigation Strategies in Wastewater Treatment and Challenges for Wider Implementation.

Environ Sci Technol 2021 06 12;55(11):7208-7224. Epub 2021 May 12.

School of Chemical Engineering, the University of Queensland, St. Lucia, Queensland 4072, Australia.

Nitrous oxide (NO) emissions account for the majority of the carbon footprint of wastewater treatment plants (WWTPs). Many NO mitigation strategies have since been developed while a holistic view is still missing. This article reviews the state-of-the-art of NO mitigation studies in wastewater treatment. Through analyzing existing studies, this article presents the essential knowledge to guide NO mitigations, and the logics behind mitigation strategies. In practice, mitigations are mainly carried out by aeration control, feed scheme optimization, and process optimization. Despite increasingly more studies, real implementation remains rare, which is a combined result of unclear climate change policies/incentives, as well as technical challenges. Five critical technical challenges, as well as opportunities, of NO mitigations were identified. It is proposed that (i) quantification methods for overall NO emissions and pathway contributions need improvement; (ii) a reliable while straightforward mathematical model is required to quantify benefits and compare mitigation strategies; (iii) tailored risk assessment needs to be conducted for WWTPs, in which more long-term full-scale trials of NO mitigation are urgently needed to enable robust assessments of the resulting operational costs and impact on nutrient removal performance; (iv) current mitigation strategies focus on centralized WWTPs, more investigations are warranted for decentralised systems, especially decentralized activated sludge WWTPs; and (v) NO may be mitigated by adopting novel strategies promoting NO reduction denitrification or microorganisms that emit less NO. Overall, we conclude NO mitigation research is reaching a maturity while challenges still exist for a wider implementation, especially in relation to the reliability of NO mitigation strategies and potential risks to nutrient removal performances of WWTPs.
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http://dx.doi.org/10.1021/acs.est.1c00840DOI Listing
June 2021

First detection and complete genome analysis of the Lyon IARC polyomavirus in China from samples of diarrheic cats.

Virus Genes 2021 Jun 10;57(3):284-288. Epub 2021 May 10.

Institute of Military Veterinary Medicine, The Academy of Military Medical Sciences, Changchun, 130122, China.

Lyon IARC polyomavirus (LIPyV), a newly discovered polyomavirus (PyV), was first identified in 2017 in human skin samples in the USA. Later, it was detected in several other countries in samples of human and feline origin. Our aim was to find out if the virus occurs in China. To this end, 100 fecal samples were collected from cats with diarrhea in Guangxi Province during 2016 and 2018 and tested with polymerase chain reaction (PCR). Only 2 samples that originated from two related individuals were found to be positive. Based on the sequence identity of the 240-bp PCR products, the two positive samples supposedly contained identical viruses. Therefore, only one of them, which was designated as LIPyV-GXNN01, was selected for full genome amplification, cloning, sequencing and analysis. LIPyV-GXNN01, which comprises 5,263 nucleotides, has an early region that consists of small T antigen (ST-Ag) and large T antigen (LT-Ag) and a late region coding for the VP1, VP2, and VP3 structural proteins. Moreover, the LIPyV-GXNN01 strain structural proteins share 95.9-99.4%, 97.6-99.2%, and 97.1-99.2% nucleic acid identity with the VP1, VP2, and VP3of other LIPyV reference strains, respectively. A phylogenetic analysis revealed that GXNN01 clustered together with previously reported LIPyV strain. This present study is the first report of LIPyV in China.
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http://dx.doi.org/10.1007/s11262-021-01840-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107205PMC
June 2021

TLR2 senses the SARS-CoV-2 envelope protein to produce inflammatory cytokines.

Nat Immunol 2021 07 7;22(7):829-838. Epub 2021 May 7.

Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA.

The innate immune response is critical for recognizing and controlling infections through the release of cytokines and chemokines. However, severe pathology during some infections, including SARS-CoV-2, is driven by hyperactive cytokine release, or a cytokine storm. The innate sensors that activate production of proinflammatory cytokines and chemokines during COVID-19 remain poorly characterized. In the present study, we show that both TLR2 and MYD88 expression were associated with COVID-19 disease severity. Mechanistically, TLR2 and Myd88 were required for β-coronavirus-induced inflammatory responses, and TLR2-dependent signaling induced the production of proinflammatory cytokines during coronavirus infection independent of viral entry. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand. In addition, blocking TLR2 signaling in vivo provided protection against the pathogenesis of SARS-CoV-2 infection. Overall, our study provides a critical understanding of the molecular mechanism of β-coronavirus sensing and inflammatory cytokine production, which opens new avenues for therapeutic strategies to counteract the ongoing COVID-19 pandemic.
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http://dx.doi.org/10.1038/s41590-021-00937-xDOI Listing
July 2021
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