The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins

Animals ; Monkeypox/drug therapy* ; Antiviral Agents/therapeutic use* ; Phosphoric Monoester Hydrolases/therapeutic use* ; Macaca fascicularis

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.
CRISPR-Associated Protein 9/metabolism* ; CRISPR-Cas Systems ; DNA ; Gene Editing/methods* ; Humans ; Streptococcus pyogenes/metabolism*

The demand for rehabilitation therapists is rising in response to social development, disease spectrum changes and population aging. Under the guidance of the integration of rehabilitation and clinical medicine, Beijing Rehabilitation Hospital has carried out a comprehensive scientific design and practice in the sub-professional training mode for new rehabilitation therapists according to the discipline development and clinical needs, strengthened their training of sub-professional skills, and provided an effective way to standardize the profession admission and specialty advancement.

Objective: To explore relationships between the enrichment of ETBF, Fn, Hp in feces, tissues and colorectal cancer. Methods: Feces, lesion tissue and adjacent tissue from 24 patients with colorectal cancer and 31 patients with adenomas were collected, and we collected Feces and tissue of 20 healthy control persons. Then the copy numbers of enterotoxigenic B. fragilis (ETBF), Fusobacterium nucleatum (Fn) and Helicobacter pylori (Hp) were determined by quantitative real-time PCR. Immunohistochemical method was used to examine the expression intensity of EGFR and p53, and the relationships between different expression intensity of EGFR, p53 and the numbers of three bacterias. Results: In the feces, copy numbers of ETBF and Fn were as follous: colorectal cancer group>adenomas group>healthy control group (P<0.05). Copy numbers of Hp were as follous: colorectal cancer group>healthy control group (P<0.01); adenomas group>healthy control group (P<0.01). In the tissue, copy numbers of ETBF, Fn were as follows: colorectal cancer group>adenomas group>healthy control group (P<0.05). Copy numbers of Hp were as follows: colorectal cancer group>healthy control group (P<0.01); adenomas group>healthy control group (P<0.01). Copy numbers of those three bacteria in the lesion tissue and the adjacent tissue had no significant difference. This happened both in colorectal cancer group and adenomas group. The different expression intensity of EGFR, p53 and the number of three bacteria showed no obviously statistical correlation(P>0.05). Conclusion Adenomatous polyp and colorectal cancer patients show high enrichment of ETBF, Fn and Hp in both feces and tissues. ETBF, Fn and Hp probably contribute to the development of adenomatous polyp and colorectal cancer. Trial registration Chinese Clinical Trial Registry, ChiCTR-BOC-17012509.

Objective: To investigate the changes of the intestinal mucosa-associated microbiota in the patients with ulcerative colitis (UC), and to explore their correlation with the clinical manifestations. Methods: From June to October 2016, at Gastrointestinal Endoscopy Center, the Second Affiliated Hospital of Xi′an Jiaotong University, 28 patients with UC and 16 healthy individuals who underwent colonoscopy examination were enrolled. The mucosa specimens of them were collected for fluorescent in situ hybridization (FISH). The bacterial flora were observed and counted, the correlation between the bacterial flora and the clinical manifestations were analyzed. Kruskal-Wallis test and Spearman rank correlation analysis were performed for statistical analysis. Results: Among 28 patients with UC, 16 were at active phase and 12 at remission phase. The number of total bacteria flora, Escherichia coli, Clostridium and Bacteroides of the active UC group and remission UC group were all more than those of healthy control group; however the number of Lactobacillus and Bifidobacteria were less than those of healthy control group, and the differences were statistically significant (χ²=23.34, 19.94; 23.40, 12.96; 23.39, 19.16; 23.32, 10.46; 23.19, 4.25; 18.94, 12.33; all P<0.05). The number of total bacteria flora, Escherichia coli and Bacteroides of the active UC group were more than those of the remission UC group, however the number of Lactobacillus and Bifidobacteria were less than those of the remission UC group, and the differences were statistically significant (χ²=7.32, 5.63, 5.62, 20.38 and 4.82; all P<0.05). In the patients with UC, the defecation frequency was positively correlated with the count of Bacteroides (r=0.459, P=0.014) and was negatively correlated with the count of Lactobacillus (r=-0.634, P<0.01). In UC patients, bloody stool, endoscopic appearance and total Mayo score were positively correlated with the counts of universal bacteria, Escherichia coli and Bacteroides (r=0.469, 0.403, 0.376; 0.604, 0.562, 0.475; 0.551, 0.463, 0.461; all P <0.05); and which were negatively correlated with Lactobacillus and Bifidobacteria (r=-0.570, -0.413; -0.899, -0.458; -0.862, -0.480; all P<0.05). The evaluation by the doctors was positively correlated with the counts of Escherichia coli (r=0.415, P=0.028), however was negatively correlated with Lactobacillus and Bifidobacteri (r=-0.841, -0.529; both P<0.01). Conclusion The intestinal microbiota have sigificantly changed in patients with UC which are correlated with some clinical manifestations of UC.

Objective To investigate the effects of p38 MAPK inhibitor on glutamate (Glu), glutamine (Gln), taurine amino acid (Tau), glycine (Gly) and gamma aminobutyric acid (GABA) in hippocampus of rats with vascular dementia (VD).Methods Twenty-four healthy male SD rats were randomly divided into sham operation group, VD model group and inhibitor group. VD model was established by permanent ligation of bilateral carotid artery method, and the sham operation group stripping bilateral carotid artery but not ligation. Rats of the inhibitor group were injected p38 MAPK inhibitor SB202190 after the establishment of VD model. Morris water maze was used to evaluate the learning and memory function of rats. The samples of DG region of hippocampus were collected by microdialysis, and the contents of amino acids were detected. Results The learning and memory abilities were significantly better in sham operation group and p38 MAPK inhibitor group than those of VD model group. The latency time was significantly shorter in p38 MAPK group than that of VD model group. The time of locating the platform quadrant and the number of crossing the original platform were significantly higher in sham operation group and p38 MAPK inhibitor group than those of VD model group (P <0.05). Compared with sham group, the levels of Glu, Gln and Tau were significantly lower, and Gly and GABA were significantly higher, in VD group and p38 MAPK inhibitor group (P<0.05). Compared with VD group, Glu, Gln and Tau were significantly increased, Gly and GABA were significantly decreased in p38 MAPK inhibitor group (P<0.05). Conclusion p38 MAPK inhibitor has protective effect on VD hippocampal injury, which may be related to its ability to inhibit VD-induced abnormal secretion of amino acids and regulate the secretion of various amino acids.

BACKGROUND:Transplantation of alogeneic intervertebral disc can be facilitated by the cryopreservation of the intervertebral disc. But the traditional cryopreservation methods always lead to the appearing of ice crystals inside and outside the cels which can cause celular injury. The vitrification method that can avoid the formation of ice crystals have been widely applied in the cryopreservation field. However, only a few reports have assessed the vitrified cryopreservation of the intervertebral disc, and the toxicity of cryoprotectants to the nucleus pulposus cels have not been fuly explored.
OBJECTIVE:To determine the order of toxicity of five commonly used cryoprotectants that are used alone or in combination to rabbit nucleus pulposus cels, and to select the optimal cryoprotectant for the vitrification of the intervertebral disc.
METHODS: We chose five most commonly used cryoprotectants including dimethyl sulphoxide, formamide, ethylene glycol, propylene glycol and glycerol. Then, 5 single commonly used cryoprotectants, 10 mixed agents containing any 2 commonly used cryoprotectants, and 10 mixed agents containing any 3 commonly used cryoprotectants were formulated. Cel viability of nucleus pulposus cels was determined using cel counting kit-8 and fluorescein diacetate/propidium iodide method. Al data obtained were analyzed statisticaly to choose the appropriate combining scheme with less toxicity.
RESULTS AND CONCLUSION: The order of the toxicity of these five commonly used cryoprotectants from low to high was ethylene glycol, glycerol, formamide, dimethyl sulphoxide, and propylene glycol. The toxicity of the combined agents containing two or three commonly used cryoprotectants was lower than that of any commonly used cryoprotectants that were used to formulate them. The toxicity of the mixed agents that contained ethylene glycol or glycerol was lower than that of any other mixed agents. So we can choose the mixed cryoprotectants that contain ethylene glycol and (or) glycerol for the vitrification of the intervertebral disc.

non-myeloablative BuCy+fludarabine conditioning regimen,and another one was treated with TBI+VP-1 6 +CTX+CCNU conditioning regimen.Only one case received short-term MTX,cyclosporin A and ATG regimen for prevention of graft-versus-host disease (GVHD).The GVHD prevention regimens of the other patients were based on short-term MTX,cyclosporin A,ATG and mycophenolate mofetil regimen.The hematopoietic reconstitution, complications and prognosis were observed.Results One patient died of intracranial hemorrhage,and hematopoi-etic reconstitution was achieved in the other 20 patients.The median time for hematopoietic reconstitution shortened by one day in large-dose group compared with that in low-dose group.Adverse reactions included high fever, shivering,gastrointestinal tract adverse reaction,liver injury,oral mucositis and other rare side effects.GVHD occurred more frequently in patients with HLA mismatched transplantation.Nine patients with aGVHD and 9 patients with cGVHD recovered after effective treatment.Within 100 days after transplantation,18 patients had bacterial or fungal infection,mainly upper respiratory tract infection;7 patients had cytomegalovirus infection;2 had EB viremia,and one had urinary BK virus infection.Only one patient died of VOD.Hemorrhagic cystitis occurred in 5 patients and improved after treatment.The median survival time was 24 months (ranging from 136 days to 9 years).One-year and 3-year overall survival rates were 85.2% and 63.9%,the disease free survival rates were 81% and 23.8%,recurrence free survival rates were 71.4% and 14.3%,respectively.Conclusion URD-HSCT was an effective method to treat leukemia.Conditioning regimen of BuCy and modified BuCy2 were safe and effective,the adverse reactions were reversible and well tolerated.Hematopoietic reconstitution time shortened in large-dose MNC and CD34 + cell number groups compared with that in low-dose group.The occurrence rate of GVHD with HLA mismatched transplantation was more than that of HLA matched transplantation.Low-dose heparin,prostaglandin E1 and Danshen injection can effectively prevent VOD.

The social and behavioral research in promoting human health and the impact on human social relations and structures has been confirmed .With the development of social and behavioral science , the importance of ethics review for the research is also increased .Despite its uniqueness , the basic principles of ethics in social and behavioral research are consistent with other biomedical studies .This paper introduces ethics review points of social and behavioral research from the research design , risk and benefits , informed consent , deception and debriefing , expedited review , exemption of ethics review, privacy and confidentiality .This paper also introduces the relevant experiences from Europe and America giving references for the domestic professionals .