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CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.

Severe COVID-19 disease caused by SARS-CoV-2 is frequently accompanied by dysfunction of the lungs and extrapulmonary organs. However, the organotropism of SARS-CoV-2 and the port of virus entry for systemic dissemination remain largely unknown. We profiled 26 COVID-19 autopsy cases from four cohorts in Wuhan, China, and determined the systemic distribution of SARS-CoV-2. SARS-CoV-2 was detected in the lungs and multiple extrapulmonary organs of critically ill COVID-19 patients up to 67 days after symptom onset. Based on organotropism and pathological features of the patients, COVID-19 was divided into viral intrapulmonary and systemic subtypes. In patients with systemic viral distribution, SARS-CoV-2 was detected in monocytes, macrophages, and vascular endothelia at blood–air barrier, blood–testis barrier, and filtration barrier. Critically ill patients with long disease duration showed decreased pulmonary cell proliferation, reduced viral RNA, and marked fibrosis in the lungs. Permanent SARS-CoV-2 presence and tissue injuries in the lungs and extrapulmonary organs suggest direct viral invasion as a mechanism of pathogenicity in critically ill patients. SARS-CoV-2 may hijack monocytes, macrophages, and vascular endothelia at physiological barriers as the ports of entry for systemic dissemination. Our study thus delineates systemic pathological features of SARS-CoV-2 infection, which sheds light on the development of novel COVID-19 treatment.

The COVID-19 pandemic has incurred tremendous costs worldwide and is still threatening public health in the “new normal.” The association between neutralizing antibody levels and metabolic alterations in convalescent patients with COVID-19 is still poorly understood. In the present work, we conducted absolutely quantitative profiling to compare the plasma cytokines and metabolome of ordinary convalescent patients with antibodies (CA), convalescents with rapidly faded antibodies (CO), and healthy subjects. As a result, we identified that cytokines such as M-CSF and IL-12p40 and plasma metabolites such as glycylproline (gly-pro) and long-chain acylcarnitines could be associated with antibody fading in COVID-19 convalescent patients. Following feature selection, we built machine-learning–based classification models using 17 features (six cytokines and 11 metabolites). Overall accuracies of more than 90% were attained in at least six machine-learning models. Of note, the dipeptide gly-pro, a product of enzymatic peptide cleavage catalyzed by dipeptidyl peptidase 4 (DPP4), strongly accumulated in CO individuals compared with the CA group. Furthermore, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination experiments in healthy mice demonstrated that supplementation of gly-pro down-regulates SARS-CoV-2–specific receptor-binding domain antibody levels and suppresses immune responses, whereas the DPP4 inhibitor sitagliptin can counteract the inhibitory effects of gly-pro upon SARS-CoV-2 vaccination. Our findings not only reveal the important role of gly-pro in the immune responses to SARS-CoV-2 infection but also indicate a possible mechanism underlying the beneficial outcomes of treatment with DPP4 inhibitors in convalescent COVID-19 patients, shedding light on therapeutic and vaccination strategies against COVID-19.

Summary 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.

Background: Anhydrous ethanol, for its part, has been successfully used to treat renal cyst, hepatocellular carcinoma and ovarian chocolate cyst et al. However, in spite of the high frequency of tuberculous purulent pleural effusion, we found that only a few very early studies that attempted to assess the use of intrapleural anhydrous ethanol in tuberculous effusions with signs of empyema. We report a patient who was injected anhydrous ethanol into pleural cavity to treat chronic tuberculous empyema. Case presentation: A 23-year old male was admitted in the hospital because of chronic tuberculous empyema. Ultra-sonography guided thoracentesis and thoracic close drainages were done, but had no effect. However, the patient refused Video-assisted Thoracoscopic Surgery (VATS) and traditional thoracotomy. Therefore, we injected anhydrous ethanol into the pleural cavity after getting the patient's consent. Pyothorax was quickly controlled and the patient finally recovered fully. Conclusion: Surgical operation is the main treatment of chronic tuberculous empyema, which has a high cost and large injury, and many patients do not accept this treatment. In this study, intrapleural injection of anhydrous ethanol could achieve the purpose of eliminating the pus cavity, which is particularly suitable for patients who cannot tolerate surgery or are unwilling to undergo surgical treatment. Keywords: Tuberculous, Chronic empyema, Anhydrous ethanol

The mediastinal lymph node tuberculous abscesses (MLNTAs) are secondary to mediastinal tuberculous lymphadenitis. Surgical excision is often required when cold abscesses form. This study was aimed to examine video-assisted thoracoscopic surgery (VATS) for the treatment of MLNTA. Clinical data of 16 MLNTA patients who were treated in our hospital between December 1, 2013 and December 1, 2015 were retrospectively analyzed. All of the patients underwent the radical debridement and drainage of abscesses, and intrathoracic lesions were removed by VATS. They were also administered the intensified anti-tuberculosis treatment (ATT), and engaged in normal physical activity and follow-up for 3 to 6 months. The results showed that VATS was successfully attempted in all of the 16 MLNTA patients and they all had good recovery. Two patients developed complications after surgery, with one patient developing recurrent laryngeal nerve injury, and the other reporting poor wound healing. It was concluded that VATS is easy to perform, and safe, and has high rates of success and relatively few side-effects when used to treat MLNTA.

Long noncoding RNAs (lncRNAs) are emerging as important regulators during tumorigenesis by serving as competing endogenous RNAs (ceRNAs). In this study, the qRT-PCR results indicated that the lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P) was overexpressed in oral squamous cell carcinoma (OSCC) and decreased the survival rate of OSCC patients. CCK-8 and clonal colony formation assays were used to detect the effects of PDIA3P on proliferation. Results revealed that silencing PDIA3P by small interfering RNA (siRNA) inhibited OSCC cell proliferation and repressed tumor growth and reduced the expression of proliferation antigen Ki-67 in vivo. Furthermore, the interaction between PDIA3P and miRNAs was then analyzed by qRT-PCR and luciferase reporter gene assay. We found that PDIA3P negatively regulated miR-185-5p in OSCC cells. Simultaneously, we found that silencing PDIA3P by siRNA suppressed proliferation via miR-185-5p in OSCC cells. Moreover, silencing PDIA3P by siRNA inhibited CCND2 protein (no influence on mRNA levels) expression via miR-185-5p in OSCC cells, and CCND2 facilitated cell proliferation of SCC4 and SCC15 cells induced by sh-PDIA3P#1. Therefore, our study demonstrated that PDIA3P may be a therapeutic target for the treatment of OSCC.

Understanding neuroanatomical sympathetic circuitry and neuronal connections from the caudal pedunculopontine tegmental nucleus to skeletal muscle is important to the study of possible mechanisms of pedunculopontine tegmental nucleus (PPTg) and cuneiform nucleus (CnF) that are involved in the regulation of skeletal muscle activity of the sympathetic pathway. The aim of this study was to use virus PRV-614 to trace the melanocortin-sympathetic neural pathways from PPTg and CnF to a hindlimb muscle (gastrocnemius) in spinally transected MC4R-GFP transgenic mice. PRV-614 was injected into the gastrocnemius muscle after receiving a complete spinal cord transection below the L2 level. PRV-614/MC4R-GFP and PRV-614/TPH dual-labeled neurons were found in the dissipated parts of PPTg (dpPPTg), but not between the compact parts of PPTg (cpPPTg) and CnF. It is proposed that a hierarchical pathway of neurons within the caudal pedunculopontine tegmental nucleus sends projections to the RVLM, which in turn projects onto the IML sympathetic preganglionic neurons that regulate muscle blood flow through melanocortin-sympathetic signals. Our results collectively indicate that MC4Rs expressed in caudal pedunculopontine tegmental nucleus may be involved in skeletal muscle activity of melanocortin-sympathetic pathways.

HLA-B*46:01:21 differs from HLA-B*46:01:01 by one nucleotide exchange at position 834(G>A) with no amino exchange.

Potential release of phosphorus in lake sediments depends largely on the fractions and distribution of organic phosphorus. MaoDu was chosen as the research area in the west of Lake Taihu. The chemical forms of the surface sediments were investigated by the Ivannoff extraction methods. The results showed that the main organic phosphorus form of sediments at different sampling places was humic acid-OP. It accounted for 35% of the total organic phosphorus. Horizontal distribution: Total organic phosphorus content from the shore to the middle of total showed a trend of rising after decline; vertical distribution: Total organic phosphorus content was the lowest in 2-6 cm and the highest in 4-8 cm. Various phosphorus content followed the order of humic acid-OPresidual-OPHCl-OPfulvic acid-OP; the relative content of NaHCO

MicroRNAs are small non-coding RNAs with regulatory biological activity, by modulating target genes on epigenetic, transcriptional, post-transciptional and translational levels. Hundreds of reports indicated that miRNAs play important roles in non-small cell lung cancer (NSCLC). Actually, microRNAs are both regulation targets and regulators targeting effector genes. This article reviewed multifaceted role of microRNAs associated to NSCLC, not only targeting to but also targeted by tumor related genes, to help us understand microRNAs related complex regulation networks. Aberrant expressed micoRNAs and their targets were summarized; the statistical results showed that several microRNAs may play key roles by targeting multiple tumor associated targets. On the other hand, Oncogenes and tumor repressors represented by PTEN were also shown to be the most popular targets of microRNAs. Additionally, ZEB1/2 may be a featured pathway in NSCLC, with significant frequency modulated by microRNAs.

Background Extracorporeal membrane oxygenation (ECMO) is performed as an acceptable life-saving bridging procedure in patients with severe acute respiratory distress syndrome (ARDS).To patients with avian influenza A (H7N9)-associated ARDS, ECMO could be adopted as a feasible therapeutic solution. We present our successful experience with ECMO utilized in a respiratory failure patient with H7N9 infection. Case presentation A 44 years-old female with H7N9-induced ARDS was admitted to intensive care unit (ICU) and was treated with veno-venous ECMO for six days, antiviral therapy, prolonged corticosteroid infusion and other therapies. She suffered significant hemorrhage requiring transfusion of platelets and multidrug-resistant Acinetobacter Baumannii infection during ECMO support. Bleeding and infection almost killed the patient's life. Fortunately, she was alive at last and completly recovered after 38 days of ICU stay. Conclusions ECMO was effective in this H7N9 patient with a fatal respiratory failure. Mechanical circulatory support was the only chance for our patient with H7N9-associated ARDS to survive until respiratory function recovery. Early detection and rapid response are essential to these serious ECMO-associated complications such as hemorrhage, thrombosis and infection.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Increasing evidence suggests that microRNAs (miRNAs) are associated with HCC tumorigenesis. The present study was designed to define the role of miR-141 in HCC. The expression of miR-141 was significantly decreased in four HCC cell lines. Overexpression of miR-141 suppressed both the growth and the motility of HCC cells. Furthermore, we identified zinc finger E-box binding homeobox 2 (ZEB2) as a target of miR-141 and miR-141 functioned as a tumor suppressor via ZEB2 targeting in HCC. These data provide a novel potential therapeutic target for HCC treatment.

The identity of higher-order neurons and circuits playing an associative role to control renal function is not well understood. We identified specific neural populations of rostral elements of brain regions that project multisynaptically to the kidneys in 3-6 days after injecting a retrograde tracer pseudorabies virus (PRV)-614 into kidney of 13 adult male C57BL/6J strain mice. PRV-614 infected neurons were detected in a number of mesencephalic (e.g. central amygdala nucleus), telencephalic regions and motor cortex. These divisions included the preoptic area (POA), dorsomedial hypothalamus (DMH), lateral hypothalamus, arcuate nucleus (Arc), suprachiasmatic nucleus (SCN), periventricular hypothalamus (PeH), and rostral and caudal subdivision of the paraventricular nucleus of the hypothalamus (PVN). PRV-614/Tyrosine hydroxylase (TH) double-labeled cells were found within DMH, Arc, SCN, PeH, PVN, the anterodorsal and medial POA. A subset of neurons in PVN that participated in regulating sympathetic outflow to kidney was catecholaminergic or serotonergic. PRV-614 infected neurons within the PVN also contained arginine vasopressin or oxytocin. These data demonstrate the rostral elements of brain innervate the kidney by the neuroanatomical circuitry.

Phospholipid fatty acids (PLFAs) method was applied to analyze the influence of submerged plants on sediment microbial community structure, in order to investigate the changes of sediment microbial community structure for different kinds of the submerged plants in different growth periods. Particularly, Potamogeton crispus L., Potamogeton pectinatus L and the mixed group were chosen as the typical submerged plants in Hongze Lake for investigation in this paper. The results indicated that the change of total PLFAs in different periods was significant, on the contrary, the PLFA change for different groups in the same period was insignificant. The values of G⁺ PLFA/G⁻ PLFA in the submerged plant group were also highly related to the different growth periods, which demonstrated that the root function of the submerged plant had a severe impact on the microbial community in sediment. Furthermore, some environmental factors, such as Temperature, pH, TOC and DO, were correlated to characteristic phospholipid of PLFAs in sediment, which means the environmental factors could also affect the microbial community structure.

To gain insights into the effect of MexB gene under the short interfering RNA (siRNA), we synthesized 21 bp siRNA duplexes against the MexB gene. RT-PCR was performed to determine whether the siRNA inhibited the expression of MexB mRNA. Changes in antibiotic susceptibility in response to siRNA were measured by the E-test method. The efficacy of siRNAs was determined in a murine model of chronic P. aeruginosa lung infection. MexB-siRNAs inhibited both mRNA expression and the activity of P. aeruginosa in vitro. In vivo, siRNA was effective in reducing the bacterial load in the model of chronic lung infection and the P. aeruginosa-induced pathological changes. MexB-siRNA treatment enhanced the production of inflammatory cytokines in the early infection stage (P ＜ 0.05). Our results suggest that targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections. [BMB Reports 2014; 47(4): 203-208]

To investigate the effects of duodenal-jejunal bypass(DJB) and sleeve gastrectomy(SG) on the expression of liver glucokinase(GCK) in diabetic rats.Animal models of Goto-Kakizaki rats and Sprague-Dawley rats were established by DJB and SG. Results of fasting glycemia and insulin were compared. Liver tissue was harvested 8 weeks postoperatively.Quantitative real-time PCR and Western blot were used to detect liver GCK mRNA and protein expression after operation.Fasting plasma glucose levels of DJB group and SG group in GK rats were markedly declined 3 day and 1, 2, 4, 6, 8 weeks postoperatively(all P0.01), while Sham group only dropped 3 day and 1 week postoperatively, and there were no significant differences 2 weeks postoperatively(P0.05). Fasting plasma glucose levels of each group in SD rats did not change after operation. In GK rats, GCK mRNA level (1.45 +/-0.29) and protein expression (494.25 +/-30.25) after DJB were higher than Sham group (1.05 +/-0.19 and 409.13 +/-26.86) and control group (1.04 +/-0.17 and 404.75 +/-30.90). GCK mRNA level and protein expression after SG were 0.65 +/-0.25 and 345.25 +/-28.13 respectively, which were significantly lower than those in control group(all P0.01). All the groups in SD rats experienced similar GCK expression change.Both DJB and SG can decrease the plasma glucose levels of GK rats, while there are different effects on the expression of liver GCK.

To explore the benefit of neoadjuvant chemotherapy in advanced gastric cancer patients treated by laparoscopy.Fifteen patients with histologically proved gastric adenocarcinomas (stages II(, III(, IIII(M(0)) were treated with FOLFOX7 neoadjuvant chemotherapy followed by laparoscopy between June 2005 and March 2007( trial group). Thirty patients were assigned to the control group with only laparoscopic treatment in the same period. The clinicopathological data were compared between two groups.All the patients in trial group accepted four cycles of preoperative chemotherapy and the toxicity was less than grade 3. Two of them achieved complete response, 10 achieved partial response and 3 kept stable disease. Ten patients of trial group underwent laparoscopic-assisted radical gastrectomy. The rates of R(0)-resection(80.0%) and pN(0) (60.0%) in trial group were significantly higher than those in control group(46.7% and 20.0%), while the rate of positive lymph node 11.0%(34/309) was significantly lower than that of control group 23.8%(142/596). The operation time and postoperative complication were similar in two groups.Advanced gastric cancer after neoadjuvant chemotherapy can be down-regulated in the stage, increase the rate of R(0)-resection, diminish the infiltration extent of tumor, decrease the metastasis of lymph node, and increase the possibility of laparoscopic radical gastrectomy.