Distribution of the COVID-19 epidemic and correlation with population emigration from Wuhan, China / 中华医学杂志(英文版)

BACKGROUND@#The ongoing new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) outbreak is spreading in China, but it has not yet reached its peak. Five million people emigrated from Wuhan before lockdown, potentially representing a source of virus infection. Determining case distribution and its correlation with population emigration from Wuhan in the early stage of the epidemic is of great importance for early warning and for the prevention of future outbreaks.@*METHODS@#The official case report on the COVID-19 epidemic was collected as of January 30, 2020. Time and location information on COVID-19 cases was extracted and analyzed using ArcGIS and WinBUGS software. Data on population migration from Wuhan city and Hubei province were extracted from Baidu Qianxi, and their correlation with the number of cases was analyzed.@*RESULTS@#The COVID-19 confirmed and death cases in Hubei province accounted for 59.91% (5806/9692) and 95.77% (204/213) of the total cases in China, respectively. Hot spot provinces included Sichuan and Yunnan, which are adjacent to Hubei. The time risk of Hubei province on the following day was 1.960 times that on the previous day. The number of cases in some cities was relatively low, but the time risk appeared to be continuously rising. The correlation coefficient between the provincial number of cases and emigration from Wuhan was up to 0.943. The lockdown of 17 cities in Hubei province and the implementation of nationwide control measures efficiently prevented an exponential growth in the number of cases.@*CONCLUSIONS@#The population that emigrated from Wuhan was the main infection source in other cities and provinces. Some cities with a low number of cases showed a rapid increase in case load. Owing to the upcoming Spring Festival return wave, understanding the risk trends in different regions is crucial to ensure preparedness at both the individual and organization levels and to prevent new outbreaks.

Mesenchymal stem cells do not differentiate into "quasi-sperm" / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine whether the surviving mesenchymal stem cells (MSCs) in the testis after transplantation can differentiate into quasi-sperm.</p><p><b>METHODS</b>(1) Making an animal model with sterilized testes. Forty 4-week old white male BASB/C mice were used to establish an animal model with sterilized testes and divided randomly into an experimental and a control group. (2) Cell preparation. The MSCs from 10 gray male 129-mice were isolated, cultured and purified by Percoll density gradient centrifugation combined with the adherent method. When the MSCs grew to an adequate number, they were made into a cell suspension with NS at a concentration of 1 million cells/ml. (3) Xenogeneic transplantation of the MSCs into the testis. The MSC suspension was blindly injected into the testes of the mice in the experimental group and NS into the testes of the controls. (4) Post-transplantation observation. Forty white female BASB/C mice were adopted, each put into a box with a male mouse from the experimental group or the control group, and then observed for pregnancy.</p><p><b>RESULTS</b>In the experimental group, 8 cases of pregnancy (40%) were observed at 31-46 d (38.5 d on average), the offspring all white. In the control group, only 1 case of pregnancy (5%) was seen at 45 d, the offspring all white, too. It was suggested that the MSCs of the 129-mice failed to differentiate into functional quasi-sperm and pass their genes to their offspring, as would expectedly have been presented by a mixture of black and white. The pregnancy rates of the two groups were significantly different (P < 0.05), which indicated that MSCs could promote the healing of the testis damage.</p><p><b>CONCLUSION</b>MSCs cannot differentiate into quasi-sperm after heterogeneity transplantation into the testis, but can promote the healing of the testis damage.</p>

Comparison of effectiveness of whole viral, N and N199 proteins by ELISA for the rapid diagnosis of severe acute respiratory syndrome coronavirus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA.</p><p><b>METHODS</b>SARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens.</p><p><b>RESULTS</b>The sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001).</p><p><b>CONCLUSION</b>N199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.</p>

Making of the animal model with sterilized testes / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the methods of making an animal model with sterilized testes.</p><p><b>METHODS</b>(1) X-ray local irradiation. Seventy 8-10-week-old male mice were equally divided into 6 experiment groups and a control group. The testes of the mice in the 6 experiment groups were irradiated sequentially by 1000, 1200, 1400, 1600, 1800 and 2000 cGy X-ray for 10 minutes, while those in the control group remained untreated. And then the pregnancy test was performed. (2) Cyclophosphamide injection. Forty 4-5-week-old male mice were divided into 3 experiment groups and a control group, the former treated with different doses of Cyclophosphamide via ip and the latter Natiichloridi Saline (N.S.) via i.p., followed by the pregnancy test. (3) Diphereline injection. Twenty 8-10-week-old male mice were equally divided into an experiment group and a control group, the former treated with Diphereline via ip and the latter N.S. via i.p., followed by the pregnancy test. (4) Identification by such pathologic examinations as TUNE1. technology, HE staining and immunohistochemical staining.</p><p><b>RESULTS</b>(1) X-ray local irradiation. The male mice of Group 1 and 2 made their female partners pregnant respectively 10 and 15 days after the X-ray irradiation, but not those of Group 3 and 4 in our 3-month observation, and those of Group 5 and 6 died respectively 2 and 5 days after the X-ray irradiation. By comparison, the controls got their female partners pregnant within 3 days after placed together. (2) Cyclophosphamide injection. The male mice of Group 1 gained weight about 7 g and achieved pregnancy 9-14 days after drug termination, those of Group 2 gained around 4 g but failed to effect pregnancy, and those in Group 3 lost weight and died respective at 3, 4 and 5 weeks during the medication, while the controls all got their female partners pregnant within 3 days after put together. (3) Diphereline injection. The 10 male mice of the experiment group effected pregnancy 3 weeks after drug termination, while the 10 controls achieved the same result with 3 days after placed together. (4) Pathologic identification: TUNEL technology showed that apoptotic cells were occasionally seen (0.71 +/- 0.12)% in the testis tissue of the control group and remarkably increased (10.36 +/- 1.48)% in the model group, with significant difference between the two groups (P < 0.05). HE staining revealed normal testis tissues and convoluted seminiferous tubules with large numbers of germ cells in the control group, but atrophied convoluted seminiferous tubules and estranged cell linkage with only Ledig's cells but no germ cells in the model group. Immunohistochemical staining showed that the positive expression rates of CD29, Hsp90alpha and CD117 were respectively (50.30 +/- 5.2)%, (41.6 +/- 3.5)% and (73.6 +/- 3.7)% in the control group, as compared with (1.3 +/- 0.2)%, 0% and (1.6 +/- 0.3)% in the model group, with significant difference (P < 0.01). The positive expression rate of p53 was (19.7 +/- 0.8)% in the control group, significantly different from that of the model group, which was (39.4 +/- 2.9)% (P < 0.01).</p><p><b>CONCLUSION</b>The animal model with sterilized testes can be made either by X-ray local irradiation of the testis or by Cyclophosphamide injection via i.p..</p>

Isolation and culture of mouse marrow mesenchymal stem cells (MSCs) and biological characteristics of MSCs cultured in conditions for spermatogonia in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To isolate, culture and purify mouse bone marrow mesenchymal stem cells (MSCs) and observe the main biological characteristics of MSCs cultured in conditions for spermatogonia in vitro.</p><p><b>METHODS</b>The tibias and femurs were dissected from 5 - 6-week old mice and the marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured, purified in vitro by Percoll density gradient centrifugation combined with adherent method and identified by dynamic observation of stem cell characteristics by transmission electron microscope, HE staining, and immunohistochemical detection of cell markers. The quantities of such cytokines as IL-6, IL-8, G-CSF and SCF in culture liquid with MSCs were measured by ELISA, and compared with those of the control group. MSCs of the third generation were divided into two groups to be induced and cultured. MSCs of the control group were cultured with basal medium, while those of the experimental group with conditional medium. The results were analysed by microscopic observation, HE staining and immunohistochemical methods.</p><p><b>RESULTS</b>Pure MSCs were obtained. The cultured cells, with stem cell characteristics, shuttle-shaped at HE staining, immature under the transmission electron microscope and CD44 and CD90 positive by immunohistochemical detection, could be identified as MSCs. Compared with the control group, the quantities of IL-6, IL-8, G-CSF and SCF in the experimental group increased significantly (P < 0.05). The shapes of MSCs changed and immunohistochemical staining for CD27, CD119 and Oct-4 was positive in the experimental group, but both were just the opposite in the control group.</p><p><b>CONCLUSION</b>Pure MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method and identified by dynamically observing stem cell characteristics, HE staining, observation under the transmission electron microscope and immunohistochemical detection of cell markers. MSCs can secrete cytokines such as IL-6, IL-8, G-CSF, SCF, and so on. MSCs cultured in conditions for spermatogonia may show some biological characteristics of spermatogonia.</p>

Transplantation of mouse bone marrow mesenchymal stem cells into the xenogeneic testis / 中华男科学杂志

<p><b>OBJECTIVE</b>To study transplantation of mouse bone marrow mesenchymal stem cells (MSCs) into the xenogeneic testis.</p><p><b>METHODS</b>(1) The tibias and femurs were dissected from 5-6-week-old mice. The marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured and purified in vitro by Percoll density gradient centrifugation combined with adherent method. (2) MSCs of the third generation were adopted and marked with Hoechest33342 for observation, and then made into cell-suspending fluid. (3) The marked MSCs were transplanted into the testis of the xenogeneic mouse by testis net injection. The biopsies of the testis tissues were carried out at different time and made into frost slices at three sites for observation.</p><p><b>RESULTS</b>(1) A lot of purified MSCs were obtained at the third generation. (2) The nucleoli of the marked MSCs showed light-yellow under the fluoroscope. (3) Xenogeneic transplantation of mouse bone marrow MSCs by testis net injection was successful, without immunoreaction. On the 1 st day after transplantation, MSCs only concentratively distributed in the medial slices, the nucleoli being light-yellow; On the 1 st and 3 rd day, MSCs dispersively distributed in the medial slices; On the 6th, 9th and 12th day, MSCs presented in all the slices of the three sites, some ranging tubally; On the 15th and 18th day, the fluorescence of MSCs weakened; On the 21 st day, the fluorescence of MSCs disappeared.</p><p><b>CONCLUSION</b>Transplantation of mouse bone marrow MSCs into the xenogeneic testis by testis net injection is effective and feasible, without immunoreaction. MSCs can survive after transplantation.</p>

Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain.</p><p><b>METHODS</b>The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.</p><p><b>RESULTS</b>The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed.</p><p><b>CONCLUSIONS</b>The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL(pro).</p>

E14 mouse embryonic stem cells differentiate into hepatocyt ESC / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate in vitro methods of inducing mouse embryonic stem cell(s) (ESC) into hepatocytes.</p><p><b>METHODS</b>E14 mouse ESC were cultivated in suspension and plated to form aggregates, the embryoid bodies. They were allowed to outgrow on the plated culture with the stepwise addition of growth factors-- acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and oncostatin M (OSM) into the culture medium. Morphology was investigated by phase contrast microscopy. Gene expressions of endodermal and liver specific mRNA were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Indocyanine green (ICG) uptake assay and periodic acid-Schiff reaction (PAS) were performed to assess the differentiation and function of the cells.</p><p><b>RESULTS</b>Morphology analysis revealed a difference between ESC-derived hepatic cells and original ESC in that the former showed distinct round or polygonal shapes with clear boundaries, some arranged tightly in cords, while the latter grew in clones without clear boundaries between cells. Those ESC-derived hepatic cells expressed endodermal and liver specific genes mRNA--TTR, AAT, AFP, ALB, G6P and TAT. ICG uptake assay and PAS reaction were positive for those ESC-derived hepatic cells. The ICG positive cells were about 85.1% in number.</p><p><b>CONCLUSION</b>ESC-derived hepatic cells possess characteristics of hepatocytes, which would promise the eventual clinical use of ESC in treating damaged liver tissues.</p>

Humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from F69 strain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The etiologic agent of severe acute respiratory syndrome (SARS) has been confirmed to be a novel coronavirus (CoV), namely SARS-CoV. Developing safe and effective SARS-CoV vaccines is essential for us to prevent the possible reemergence of its epidemic. Previous experiences indicate that inactivated vaccine is conventional and more hopeful to be successfully developed. Immunogenicity evaluation of an experimental inactivated SARS-CoV vaccine in rabbits was conducted and reported in this paper.</p><p><b>METHODS</b>The large-scale cultured SARS-CoV F69 strain was inactivated with 0.4% formaldehyde and purified, then used as the immunogen combined with Freund's adjuvant. Eight adult New Zealand rabbits were immunized four times with this experimental inactivated vaccine. Twelve sets of rabbit serum were sampled from the third day to the seventy-fourth day after the first vaccination. The titers of specific anti-SARS-CoV IgG antibody were determined by indirect enzyme-linked immunosorbent assay, and the neutralizing antibody titers were detected with micro-cytopathic effect neutralization test.</p><p><b>RESULTS</b>Rapid and potent humoral immune responses were induced by the inactivated SARS-CoV vaccine in all the eight test rabbits. Titers of both specific IgG antibody and neutralizing antibody peaked at about six weeks after first vaccination, with the maximum value of 1:81 920 and 1:20 480, respectively. After that, serum antibody levels remained at a plateau or had a slight decrease, though two boosters were given in the succedent 4 to 5 weeks. Cross neutralization response existed between SARS-CoV F69 strain and Z2-Y3 strain.</p><p><b>CONCLUSIONS</b>The inactivated SARS-CoV vaccine made from F69 strain owns strong immunogenicity, and the cross neutralization response between the two different SARS-CoV strains gives a hint of the similar neutralizing epitopes, which provide stable bases for the development of inactivated SARS-CoV vaccines.</p>