Simultaneous identification of DNA and RNA pathogens using metagenomic sequencing in cases of severe acute respiratory infection.

Metagenomic next-generation sequencing (mNGS) is a valuable technique for identifying pathogens. However, conventional mNGS requires the separate processing of DNA and RNA genomes, which can be resource- and time-intensive. To mitigate these impediments, we propose a novel method called DNA/RNA cosequencing that aims to enhance the efficiency of pathogen detection. DNA/RNA cosequencing uses reverse transcription of total nucleic acids extracted from samples by using random primers, without removing DNA, and then employs mNGS. We applied this method to 85 cases of severe acute respiratory infections (SARI). Influenza virus was identified in 13 cases (H1N1: seven cases, H3N2: three cases, unclassified influenza type: three cases) and was not detected in the remaining 72 samples. Bacteria were present in all samples. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were detected in four influenza-positive samples, suggesting coinfections. The sensitivity and specificity for detecting influenza A virus were 73.33% and 95.92%, respectively. A κ value of 0.726 indicated a high level of concordance between the results of DNA/RNA cosequencing and SARI influenza virus monitoring. DNA/RNA cosequencing enhanced the efficiency of pathogen detection, providing a novel capability to strengthen surveillance and thereby prevent and control infectious disease outbreaks.

Prediction of compressive comfort of graded compression sleeves based on calf feature classification.

This study explored the relationship between the design of calf compression sleeves and the comfort of young women in long sitting and standing work environments. By studying the relationship between material elongation and comfort pressure, a prediction model was obtained. The characteristics of the calf models of 94 women were classified, and the influence of different calf characteristics on the pressure distribution and tensile value design was objectively analyzed through simulation tests. The samples were then produced based on the simulation results, and subjective and objective try-on tests and evaluations were carried out. The results show that the difference in the shape of the calf has a certain impact on the pressure distribution and comfort. The predicted value of the theoretical model is in good agreement with the actual test value. The sample can bring a suitable gradient pressure and meet the comfort requirements and safety standards.

Clinical Characteristics, Diagnosis, and Therapeutics of COVID-19: A Review.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that suddenly emerged at the end of December 2019 and caused coronavirus disease 2019 (COVID-19) continues to afflict humanity, not only seriously affecting healthcare systems but also leading to global social and economic imbalances. As of August 2022, there were approximately 580 million confirmed cases of COVID-19 and approximately 6.4 million confirmed deaths due to this disease. The data are sufficient to highlight the seriousness of SARS-CoV-2 infection. Although most patients with COVID-19 present primarily with respiratory symptoms, an increasing number of extrapulmonary systemic symptoms and manifestations have been associated with COVID-19. Since the outbreak of COVID-19, much has been learned about the disease and its causative agent. Therefore, great effort has been aimed at developing treatments and drug interventions to treat and reduce the incidence of COVID-19. In this narrative review, we provide a brief overview of the epidemiology, mechanisms, clinical manifestations, diagnosis, and therapeutics of COVID-19.

Dual-amplification colorimetric detection of bisphenol A based on catalytic hairpin assembly and DNAzyme-caused fragment self-assembly hybridization chain reaction.

An efficient and innovative strategy for colorimetric detection of bisphenol A (BPA) is shown here based on target-induced catalytic hairpin assembly (CHA) and DNAzyme-caused fragment self-assembly hybridization chain reaction (HCR). BPA can bind with its aptamer hairpin to trigger CHA, thus forming Y-shaped DNA nanostructures with an enzyme-strand (E-DNA) tail. Subsequently, the E-DNA can cyclically cleave the substrate hairpin, generating many fragments which can cause self-assembly HCR to form long strand DNA. Finally, the formed long strand DNA can hybridize with short single strand DNA on AuNPs, causing the color change of AuNPs from red to blue. Six important detection conditions of the proposed aptasensor were optimized. Under optimal conditions, the biosensor has high sensitivity for BPA detection at concentrations ranging from 0.8 pM to 500 pM and the detection limit is as low as 0.2 pM, providing a promising prospective ultrasensitive detection of BPA.

Differential diagnosis of lymphoma with 18F-FDG PET/CT in patients with fever of unknown origin accompanied by lymphadenopathy.

PURPOSE: To investigate the value of 18F-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) in the differential diagnosis of lymphoma in patients with fever of unknown origin (FUO) accompanied by lymphadenopathy and to develop a simple scoring system to distinguish lymphoma from other etiologies. METHODS: A prospective study was conducted on patients with classic FUO accompanied by lymphadenopathy. After standard diagnostic procedures, including PET/CT scan and lymph-node biopsy, 163 patients were enrolled and divided into lymphoma and benign groups according to the etiology. The diagnostic utility of PET/CT imaging was evaluated, and beneficial parameters that could improve diagnostic effectiveness were identified. RESULTS: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PET/CT in diagnosing lymphoma in patients with FUO accompanied by lymphadenopathy were 81.0, 47.6, 59.3, and 72.7%, respectively. The lymphoma prediction model combining high SUVmax of the "hottest" lesion, high SUVmax of the retroperitoneal lymph nodes, old age, low platelet count, and low ESR had an area under the curve of 0.93 (0.89-0.97), a sensitivity of 84.8%, a specificity of 92.9%, a PPV of 91.8%, and an NPV of 86.7%. There was a lower probability of lymphoma for patients with a score < 4 points. CONCLUSIONS: PET/CT scans show moderate sensitivity and low specificity in diagnosing lymphoma in patients with FUO accompanied by lymphadenopathy. The scoring system based on PET/CT and clinical parameters performs well in differentiating lymphoma and benign causes and can be used as a reliable noninvasive tool. REGISTRATION NUMBER: This study on FUO was registered on http://www. CLINICALTRIALS: gov on January 14, 2014, with registration number NCT02035670.

Early clinical efficacy of pegylated interferon treatment in patients with different phases of chronic HBV infection: A real-world analysis.

Although there are therapeutic advantages for hepatitis B virus (HBV) withpegylated interferon alpha (peg-IFNα) treatment compared with nucleos(t)ide analog (NAs) therapy, the effect difference in infected population at different phases has not been well established. We studied the clinical efficacy of peg-IFNα in two populations with HBV infection, including inactive HBsAg carrier (IHC) and chronic hepatitis B (CHB). A total of 328 HBV-infected patients were included in this real-world analysis. Patients were divided into two groups according to the infected stages. Peg-IFNα monotherapy or combination therapy with NAs were used in IHCs, and peg-IFNα added-on NAs therapy was applied to patients with CHB. The primary efficacy endpoint was HBsAg loss at Week 24. Results: The Kaplan-Meier cumulative rates of HBsAg loss were 39.50% (n = 47/119) in IHC group and 28.71% (n = 60/209) in CHB group at Week 24 (p < .05). After Propensity Score Matching (PSM), the HBsAg loss rates were 36.84% (n = 35/95) and 32.63% (n = 31/95), respectively (p > .05). Patients with baseline HBsAg level < 100 IU/ml achieved higher rates of HBsAg clearance in IHC and CHB group (before PSM: 47.44% vs. 42.86%, after PSM: 49.12% vs. 45.83%, all p values > .05). Baseline HBsAg level and its level decline from baseline to Week 12 can be as the predictors for HBsAg loss at Week 24 in both groups. Hence, the efficacy of HBsAg clearance was broadly similar between IHCs and NA-treated CHB patients during the early peg-IFNα therapy. A significant downward trend of HBsAg level was observed in both groups during peg-IFNα therapy.

Erosion Resistance of Valve Core Surface Combined with WC-10Co-4Cr Coating Process under Different Pretreatments.

The erosion of the valve core causes valve failure problems. Thus, a novel method to extend the erosion resistance of the valve was innovatively proposed, namely, nanosecond laser ablation micro-pits on the substrate surface and high velocity oxygen fuel (HVOF) spraying WC coating to extend the erosion resistance of the valve. The characterization and evaluation of the erosion resistance of the WC-sprayed coating after the pretreatment of the 3Cr13 substrate surface polishing/grit blasting/nanosecond laser ablation circular micro-dimple were conducted using the unit coupon erosion test of liquid-solid two-phase flow, followed by the test evaluation and analysis of the erosion resistance test of the WC coating after different pretreatments of the full-size valve core. Results showed that the micro-dimple pretreatment on the surface of the 3Cr13 substrate increased the contact area rate and bonding strength of the substrate and the WC coating. By taking erosion volume loss as the evaluation index, the erosion resistance of the micro-dimple pretreatment on the surface of the 3Cr13 substrate was increased by about 31.98% compared with that of the polishing pretreatment. Therefore, the new method of surface nanosecond laser texture pretreatment and HVOF-spraying WC coating can effectively improve the erosion resistance of the valve.

Diagnostic models for fever of unknown origin based on 18F-FDG PET/CT: a prospective study in China.

BACKGROUND: This study aims to analyze the 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) characteristics of different causes of fever of unknown origin (FUO) and identify independent predictors to develop a suitable diagnostic model for distinguishing between these causes. A total of 524 patients with classical FUO who underwent standard diagnostic procedures and PET/CT were prospectively studied. The diagnostic performance of PET/CT imaging was analyzed, and relevant clinical parameters that could improve diagnostic efficacy were identified. The model was established using the data of 369 patients and the other 155 patients comprised the validation cohort for verifying the diagnostic performance of the model. RESULTS: The metabolic characteristics of the "hottest" lesion, the spleen, bone marrow, and lymph nodes varied for various causes. PET/CT combined with clinical parameters achieved better discrimination in the differential diagnosis of FUO. The etiological diagnostic models included the following factors: multisite metabolic characteristics, blood cell counts, inflammatory indicators (erythrocyte sedimentation rate, C-reactive protein, serum ferritin, and lactate dehydrogenase), immunological indicators (interferon gamma release assay, antinuclear antibody, and anti-neutrophil cytoplasm antibody), specific signs (weight loss, rash, and splenomegaly), and age. In the testing cohort, the AUCs of the infection prediction model, the malignancy diagnostic model, and the noninfectious inflammatory disease prediction model were 0.89 (95% CI 0.86-0.92), 0.94 (95% CI 0.92-0.97), and 0.95 (95% CI 0.93-0.97), respectively. The corresponding AUCs for the validation cohort were 0.88 (95% CI 0.82-0.93), 0.93 (95% CI 0.89-0.98), and 0.95 (95% CI 0.92-0.99), respectively. CONCLUSIONS: 18F-FDG PET/CT has a certain level of sensitivity and accuracy in diagnosing FUO, which can be further improved by combining it with clinical parameters. Diagnostic models based on PET/CT show excellent performance and can be used as reliable tools to discriminate the cause of FUO. Trial registration This study (a two-step method apparently improved the physicians' level of diagnosis decision-making for adult patients with FUO) was registered on the website http://www.clinical-trials.gov on January 14, 2014, with registration number NCT02035670.

Overexpression of angiotensin-converting enzyme 2 contributes to the amelioration of Streptococcus uberis-induced inflammatory injury in mammary epithelial cells.

Streptococcus uberis (S. uberis) is an environmentally important pathogenic bacterium and is the main pathogenic microorganism responsible for mastitis, which causes significant economic losses worldwide. Currently, there is no particularly effective treatment other than antibiotic therapy. Angiotensin-converting enzyme 2 (ACE2) plays an anti-inflammatory as well as an anti-injury role in numerous inflammatory diseases. Therefore, this study aimed to assess the hypothesis that S. uberis-induced mammary epithelial cells injury associated with ACE2, angiotensin II (Ang II) as well as angiotensin 1-7 (Ang-(1-7)) imbalance and that overexpression of ACE2 can repair S. uberis-induced mammary epithelial cells injury. We observed that the expression level of ACE2 was significantly downregulated after treatment of EpH4-Ev cells with S. uberis. Next, this assay verified the role of ACE2 in S. uberis-induced inflammatory injury in EpH4-Ev cells by overexpressing the ACE2 gene as well as its silencing. The results showed that overexpression of the ACE2 gene significantly activated the interleukin-10/signal transducer and activator of transcription 3/suppressors-of-cytokine-signaling 3 (IL-10/STAT3/SOCS3) pathway, thereby inhibiting the nuclear factor-κB (NF-κB) as well as pyroptosis pathways. Furthermore, overexpression of the ACE2 gene reversed the downregulation of zonula occludens 1 (ZO-1), Occludin, Claudin-1, and Claudin-2 caused by S. uberis, suggesting that ACE2 could promote to repair the blood-milk barrier. However, siRNA silencing of the ACE2 gene produced the opposite effect. These results suggest that ACE2 ameliorates S. uberis-induced mammary epithelial cells injury. AVAILABILITY OF DATA: All data generated or analyzed during this study are included within the article and its additional information file.

Infection-associated Hemophagocytic Syndrome in Critically Ill Patients with COVID-19.

Infection-associated hemophagocytic syndrome (IAHS), a severe complication of various infections, is potentially fatal. This study aims to determine whether IAHS occurs in critically ill patients with coronavirus disease 2019 (COVID-19). We conducted a retrospective observational study on 268 critically ill patients with COVID-19 between February 1st, 2020 and February 26th, 2020. Demographics, clinical characteristics, laboratory results, information on concurrent treatments and outcomes were collected. A diagnosis of secondary hemophagocytic lymphohistiocytosis (sHLH) was made when the patients had an HScore greater than 169. Histopathological examinations were performed to confirm the presence of hemophagocytosis. Of 268 critically ill patients with confirmed SARS-CoV-2 infection, 17 (6.3%) patients had an HScore greater than 169. All the 17 patients with sHLH died. The interval from the onset of symptom of COVID-19 to the time of a diagnosis of sHLH made was 19 days and the interval from the diagnosis of sHLH to death was 4 days. Ten (59%) patients were infected with only SARS-CoV-2. Hemophagocytosis in the spleen and the liver, as well as lymphocyte infiltration in the liver on histopathological examinations, was found in 3 sHLH autopsy patients. Mortality in sHLH patients with COVID-19 is high. And SARS-CoV-2 is a potential trigger for sHLH. Prompt recognition of IAHS in critically ill patients with COVID-19 could be beneficial for improving clinical outcomes.

Mesenchymal Stem Cells Overexpressing ACE2 Favorably Ameliorate LPS-Induced Inflammatory Injury in Mammary Epithelial Cells.

Mesenchymal stem cells (MSCs) are capable of homing injury sites to exert anti-inflammatory as well as anti-damage effects and can be used as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2) plays an important role in numerous inflammatory diseases, but fewer studies have been reported in animal mastitis. We hypothesized that MSCs overexpressing ACE2 is more effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory injury in mammary epithelial cells compared to MSCs alone. The results showed that MSC-ACE2 inhibited the LPS induction by upregulation of TNF-α, IL-Iß, IL-6, and iNOS mRNA expression levels in EpH4-Ev cells compared with MSCs. Furthermore, results showed that both MSC and MSC-ACE2 were significantly activated IL-10/STAT3/SOCS3 signaling pathway as well as inhibited TLR4/NF-κB and MAPK signaling pathways, but MSC-ACE2 had more significant effects. Meanwhile, MSC-ACE2 promoted the expression of proliferation-associated proteins and inhibited the expression of the apoptosis-associated proteins in EpH4-Ev cells. In addition, MSC and MSC-ACE2 reversed the LPS-induced downregulation expression levels of the tight junction proteins in mammary epithelial cells, indicating that both MSC as well as MSC-ACE2 could promote blood-milk barrier repair, and MSC-ACE2 was more effective. These results suggested that MSCs overexpressing ACE2 were more anti-inflammatory as well as anti-injurious action into LPS-induced inflammatory injury in the EpH4-Ev cells. Thus, MSCs overexpressing ACE2 is expected to serve as a potential strategy for mastitis treatment.

Combined clinical parameters improve the diagnostic efficacy of 18F-FDG PET/CT in patients with fever of unknown origin (FUO) and inflammation of unknown origin (IUO): A prospective study in China.

OBJECTIVES: To improve the diagnostic efficacy of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) for Chinese patients with fever of unknown origin (FUO) and inflammation of unknown origin (IUO), with combined clinical parameters. MATERIALS AND METHODS: FUO/IUO patients who underwent a standard diagnostic work-up and 18F-FDG PET/CT scanning were enrolled and divided into a local uptake lesion subgroup and a non-specific abnormal uptake subgroup. Beneficial clinical parameters for improving the diagnostic efficacy of PET/CT were identified. RESULTS: From January 2014 to January 2019, 253 FUO/IUO patients were studied. In total, 147 patients had local uptake lesions and 106 patients had non-specific abnormal uptake. In the local uptake lesion group, the positioning accuracy of PET/CT was 37.2% in grades 1 and 2, and 66.3% in grades 3 and 4. With the following combination of clinical parameters, the positioning accuracy increased to 75.0% and 90.0%, respectively: time from admission to performing PET/CT scanning <6.5 days and C-reactive protein level >95 mg/l. In the non-specific abnormal uptake group, the combination of sex (male), bicytopenia, and lactic dehydrogenase improved the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for diagnosing malignancy from 64.3%, 69%, 60%, and 72.7%, respectively, to 83.3%, 81%, 81.4%, and 82.9%, respectively. With the combination of sex (male), white blood count, serum ferritin level, and hepatosplenomegaly, the infection prediction model had a sensitivity, specificity, PPV, and NPV of 78%, 76.2%, 76.6%, and 77.6%, respectively. CONCLUSIONS: Combined clinical parameters improved the localization diagnostic value of 18F-FDG PET/CT in the local uptake lesion subgroup and the etiological diagnostic value in the non-specific abnormal uptake subgroup.

Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi.

Monascus azaphilone pigments (MonAzPs) are very widely used as food colorants, but their biosynthetic pathway has remained poorly characterized for more than half a century. In this study, the individual steps of MonAzPs biosynthesis in Monascus ruber M7 were elucidated by a combination of targeted gene knockouts, heterologous gene expression, and in vitro chemical and enzymatic reactions. This study describes the first rational engineering of MonAzPs biosynthesis and provides a roadmap for future pathway engineering efforts directed towards the selective production of the most valuable pigments and serves as a model for the biosynthesis of fungal azaphilones in general.

Identification and role analysis of an intermediate produced by a polygenic mutant of Monascus pigments cluster in Monascus ruber M7.

Monascus pigments (Mps) are a group of azaphilonic secondary metabolites produced by Monascus spp. via a polyketide pathway. A mutant deleted an about 30 kb region of Mps gene cluster from Monascus ruber M7 was isolated previously, which produces a high amount of a light yellow pigment. The current study revealed that the mutant named ΔMpigJ-R lost proximate eight genes of the Mps gene cluster in M. ruber M7 through genetic analysis at DNA and RNA levels. The produced light yellow material was identified as a benzaldehyde derivative named as 6-(4-hydroxy-2-oxopentyl)-3-methyl-2, 4-dioxocyclohexane carb-aldehyde (M7PKS-1) by FT-IR, NMR, and MS. The sodium acetate-1-(13)C feeding experiment indicated that M7PKS-1 was a product produced from polyketide pathway. Finally, the feeding of M7PKS-1 helped to induce and regain Mps production of the mutants (ΔMpigA and ΔMpigE) which were previously unable to biosynthesize Mps and proved that M7PKS-1 was an initial intermediate of Mps. The results in this study provide a line of action to unveil Monascus pigments biosynthesis pathway.

[Identification of a pigment-polyketide synthase gene deleted mutant of Monascus ruber M7].

OBJECTIVE: To reveal the function of a polyketide synthase gene (pksPT), probably responsible for the synthesis of Monascus pigments in Monascus ruber M7. METHODS: The pksPT was analyzed using bioinformatics method; it was disrupted using Agrobacterium tumefaciens mediated transformation method, generating the pksPT-deleted mutant (ΔpksPT). Colonial morphology, conidial germination, pigment and citrinin production, and growth rate of ΔpksPT were analyzed. RESULTS: The pksPT with the length of 8687 bp encoded a putative protein of 2690 amino acids, which is a non-reduced type III polyketide synthase and has some active domains with the arrangement of KS (ß-ketosynthase)-AT (Acyltransferase)-ACP (Acyl carrier protein)-ACP-ME (Methyltransferase). The analysis of ΔpksPT displayed that it could generate cleistothecum and conidum normally and was unable to produce any kinds of Monascus pigments; compared to M7, the growth rate of ΔpksPT was increased obviously and the yield of citrinin in ΔpksPT was increased about 2. 8 times. CONCLUSION: pksPT is of extremely importance to the biosynthetic pathways of Monascus pigments in M7 and the synthesis of Monascus pigments gives a significant effect on the produce of citrinin as well as the growth of M7.

Exome sequencing revealed novel germline mutations in Chinese Peutz-Jeghers syndrome patients.

BACKGROUND AND AIMS: Peutz-Jeghers Syndrome (PJS) is an autosomal dominant disorder which predisposes to the development of various cancers. Germline mutation in the serine/threonine kinase 11 gene (STK11) is known as one of the major causes of PJS. However, a notable proportion of PJS samples do not carry any mutation in STK11, suggesting possible genetic heterogeneity in the disease and the existence of other causative variants. METHODS AND RESULTS: In order to identify other germline variants in the coding regions of the genome that are associated with PJS, we performed exome sequencing in three Chinese individuals with PJS and identified 16 common germline variants (12 protein-coding including STK11, 4 in pre-microRNAs). We further validated protein-coding variants in six PJS individuals (three with wild-type STK11) and predicted the functional impact. As result, we found that 7 coding variants are likely to have functional impacts. Especially, we identified 2 new germline variants which are represented in all six PJS samples and are independent of STK11 mutation. CONCLUSIONS: Our study provided an exomic view of PJS. The germline variants identified in our analysis may help to resolve the complex genetic background of the disease and thus lead to the discovery of novel causative variants of PJS.

MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7.

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries. However, MP biosynthesis pathway is still a controversy, and only few related genes have been reported. In this study, the function of MpigE, a gene involved in MP biosynthesis in Monascus ruber M7, was analyzed. The results revealed that the disruption, complementation, and overexpression of MpigE in M. ruber M7 had very little effects on the growth and phenotypes except MPs. The MpigE deletion strain (∆MpigE) just yielded four kinds of yellow MPs and very little red pigments, while the wild-type strain M. ruber M7 produced a MP complex mixture including three (orange, red, and yellow) categories of MP compounds. Two of the four yellow MPs produced by ∆MpigE were the same as those yielded by M. ruber M7. The MpigE complementation strain (∆MpigE::MpigE) recovered the ability to generate orange and red MPs as M. ruber M7. The MP types produced by the MpigE overexpression strain (M7::PtrpC-MpigE) were consistent with those of M. ruber M7, while the color value was about 1.3-fold as that of M. ruber M7 (3,129 U/g red kojic). For the production of citrinin, the disruption of MpigE almost had no influence on the strain, whereas the overexpression of MpigE made citrinin decrease drastically in YES fermentation. This work will make a contribution to the study on the biosynthesis pathway of MPs in M. ruber.

Deletion of pigR gene in Monascus ruber leads to loss of pigment production.

Pigments produced by Monascus are traditional food colorants and are widely used as dietary supplements. Since genes involving in pigment biosynthesis have not been reported, we describe the identification of a putative pigment-regulatory gene (pigR) obtained by molecular analysis of an albino strain of Monascus ruber M7. In the pigR-deleted strain (ΔpigR), neither the pigments nor pigR expression were detected by HPLC or reverse-transcription PCR, respectively, whereas the introduction of the pigR, together with a constitutive trpC promoter into ΔpigR, caused it to produce 5.4 U of red pigments/g dry mycelia, about 12-fold higher than Monascus ruber M7 (0.46 U/g dry mycelia). Thus pigR up-regulates pigment production in Monascus ruber M7.