Interrupting TGF-ß1/CCN2/integrin-α5ß1 signaling alleviates high mechanical-stress caused chondrocyte fibrosis.

OBJECTIVE: Mechanical-stress has been reported to trigger cartilage fibrosis, in which transforming growth factor (TGF)-ß and connective tissue growth factor (CCN2) are involved. However, the function of integrin-α5ß1, a cytomembrane receptor, on mechanical-stress related fibrosis has not yet been elucidated. This study aims to reveal the interaction of TGF-ß1/CCN2/integrin-α5ß1 in the mechanical-stress induced chondrocyte (CH) fibrosis. PATIENTS AND METHODS: We used different levels (5% and 10%) of cyclic tension simulation (CTS) to stretch CHs and observed the gene expression of TGF-ß1/CCN2/integrin-α5ß1 as well as the fibrous related genes containing collagen I/II/III, Runx2, MMP13, and ADAMTS-5 by real-time polymerase chain reaction (RT-PCR) or immunofluorescence. We used the siRNA or the corresponding antagonist of TGF-ß1, CCN2, integrin-α5ß1 during the CTS to clear the effect of them in the fibrosis progress. In addition, to verify the crosstalk between TGF-ß1, CCN2, and integrin-α5ß1, we used the recombinant human (rh)-TGF-ß1 and CCN2 to culture CHs without CST. RESULTS: 24 hours-10% CTS was sufficient to induce a decrease of collagen II and increase the collagen I/III, Runx2, MMP13, and ADAMTS-5 gene expression. Under CTS, TGF-ß1 silencing resulted in a decline of CCN2, integrin-α5ß1, and alleviated the CHs fibrosis. Apart from this, blocking CCN2 or integrin-α5ß1 expression also contributed to the suppression of 10% CTS induced CHs fibrosis. Meanwhile, the exogenic protein supplement raised the cellular TGF-ß1 or CCN2 expression and increased the integrin-α5ß1 mRNA level. However, the downregulation of TGF-ß1 or CCN2 did not affect integrin-α5ß1 expression, whether the CTS exited or not. CONCLUSIONS: High mechanical-stress induces CHs fibrosis via the activation of TGF-ß1/CCN2/integrin-α5ß1 signaling, and interrupting the TGF-ß1, CCN2, or integrin-α5ß1 expression can alleviate the fibrous process.

Coronavirus disease 2019: a bibliometric analysis and review.

OBJECTIVE: On December 8, 2019, many cases of pneumonia with unknown etiology were first reported in Wuhan, China, subsequently identified as a novel coronavirus infection aroused worldwide concern. As the outbreak is ongoing, more and more researchers focused interest on the COVID-19. Therefore, we retrospectively analyzed the publications about COVID-19 to summarize the research hotspots and make a review, to provide reference for researchers in the world. MATERIALS AND METHODS: We conducted a search in PubMed using the keywords "COVID-19" from inception to March 1, 2020. Identified and analyzed the data included title, corresponding author, language, publication time, publication type, research focus. RESULTS: 183 publications published from 2020 January 14 to 2020 February 29 were included in the study. The first corresponding authors of the publications were from 20 different countries. Among them, 78 (42.6%) from the hospital, 64 (35%) from the university and 39 (21.3%) from the research institution. All the publications were published in 80 different journals. Journal of Medical Virology published most of them (n=25). 60 (32.8%) were original research, 29 (15.8%) were review, 20 (10.9%) were short communications. 68 (37.2%) epidemiology, 49 (26.8%) virology and 26 (14.2%) clinical features. CONCLUSIONS: According to our review, China has provided a large number of research data for various research fields, during the outbreak of COVID-19. Most of the findings play an important role in preventing and controlling the epidemic around the world. With research on the COVID-19 still booming, new vaccine and effective medicine for COVID-19 will be expected to come out in the near future with the joint efforts of researchers worldwide.

RBM5-AS1 participates in fracture healing and inhibits apoptosis of bone cells through the up-regulation of ß-catenin.

OBJECTIVE: The aim of this study was to detect the expression of RBM5-AS1 during fracture healing, and to explore its possible mechanism. MATERIALS AND METHODS: A mice tibia fracture model was constructed in this study. Mice in the control group and experimental group were sham-operated on the left tibia and were operated in the right tibia, respectively. The tibia bones of both groups were obtained at 4 d, 8 d, 12 d, 16 d, 20 d, and 24 d after the operation. Quantitative polymerase chain reaction (qPCR) was used to detect the expression of RBM5-AS1 in tibiae. After interfering with the expression of RBM5-AS1 in bone cells, Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation ability, and flow cytometry was applied to detect apoptosis. Western blot was used to measure the protein expression of beta-catenin and RBM5 after down-regulating RBM5-AS1. Finally, beta-catenin was interfered in osteoblasts to explore the relationship between RBM5-AS1 and beta-catenin. RESULTS: Compared with the control group, the expression of RBM5-AS1 in the experimental group was significantly increased on the 4 d, 8 d, 12 d, and 16 d after fracture surgery. However, no statistical difference was observed on the 20 d and 24 d between the two groups. After interfering with RBM5-AS1, the apoptosis of chondrocytes and osteoblasts was significantly increased in both mouse and human cells, while the expression of beta-catenin was strikingly decreased. Further up-regulation of beta-catenin could reduce the apoptosis of bone cells. The expression of RBM5, which was a natural antisense transcript of RBM5-AS1, was increased after down-regulating RBM5-AS1. CONCLUSIONS: RBM5-AS1 can inhibit the apoptosis of bone cells by promoting the expression of beta-catenin and can be used as a biomarker for fracture healing.

Vitrification of boer goat morulae and early blastocysts by straw and open-pulled straw method.

The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.

[Prediction and evaluation of heterosis of beef cattle and their application].

The genetic structure and genetic variation of eight beef cattle cross parents populations were analyzed by six microsatellite loci, and heterosis of beef cattle was predicted. On the basis of microsatellite analysis, the effect of 18 cross combinations was estimated by the method of individual animal model BLUP. A new method of molecular quantitative genetics that select best of all cross combination was submitted. The results showed that the combinations with Hereford Limousine and Charolais as paternal parent are better than others in Fengning and Longhua regions; the combinations with Limousine Angus and Hereford as paternal parent are better than others in Zanhuang regions; the combinations with Hereford Limousine and Piemontese as paternal parent are better than others in Funing regions. Effect of three breeds cross is better than two breeds.

[GABA inhibits progesterone production of luteal cells of rat ovary].

In the present work we report that GABA inhibits progesterone production and increases the amount of hydroxyl radical in cultured rat luteal cells. The inhibitory effect of GABA on progesterone production may be mediated by adenylate cyclase system and GABA(A) receptor, but not through protein synthesis pathway.

[Separation and determination of effective components in silymarin and preparations of silymarin by MECC].

AIM: To develop a micellar electrokinetic capillary chromatographic (MECC) method for the separation and determination of silybin, isosilybin, silydianin and silycristin in Legalon capsules, Yiganling tablets and silymarin. METHODS: A buffer solution containing 30 mmol.L-1 disodium tetraborate, 50 mmol.L-1 taurodeoxycholic acid sodium salt and 20 mmol.L-1 beta-cyclodextrin (pH 9.2) was found to be the most suitable electrolyte for this separation. The applied voltage was 20 kV and UV detection wavelength was 288 nm. Rutin was used as internal standard. RESULTS: The average recoveries of 99.9% for silycristin, 98.3% for silydianin, 99.0% for silybin and 98.2% for isosilybin were obtained. CONCLUSION: The method is sensitive and accurate for the analysis of diasteroisomers of silybin and isosilybin.

Determination of quinolizidine alkaloids in traditional Chinese herbal drugs by nonaqueous capillary electrophoresis.

A rapid method for the determination of quinolizidine alkaloids by nonaqueous capillary electrophoresis was developed. A total of 10 alkaloids (matrine, sophocarpine, oxymatrine, oxysophocarpine, sophoridine, cytisine, sophoramine, aloperine, lehmannine and dauricine) could be easily separated within 18 min. A running buffer composed of 50 mM ammonium acetate, 10% tetrahydrofuran and 0.5% acetic acid in methanol was found to be the most suitable for this separation. Five of these alkaloids were selected for further studies. The linear calibration ranges were 2.51-50.1 microg/ml for sophoridine and sophocarpine, 2.71-54.2 microg/ml for matrine, 3.30-65.9 microg/ml for oxymatrine, and 3.10-62.0 microg/ml for oxysophocarpine. The recovery of the five alkaloids was 98.0-101.3% with relative standard deviations from 1.03 to 2.68% (n=5). The limits of detection for all 10 alkaloids were over the range 0.93-2.31 microg/ml. The method was successfully applied to the phytochemical analysis of alkaloid extracts from three commonly used traditional Chinese herbal drugs: Sophora flavescens Ait. (Kushen), S. alopecuroides L. (Kudouzi or Kugancao) and S. tonkinensis Gapnep (Shandougen).

Determination of L-threonate in calcium L-threonate preparations by capillary electrophoresis with indirect UV detection.

A validated capillary electrophoresis method with indirect UV detection for the determination of L-threonate in a calcium L-threonate (Ca-Th) chewable tablet and dry syrup was described. The 2,6-naphthalenedicarboxylic acid (NDC) was used as the background carrier ion and the tetradecyltrimethylammonium bromide (TTAB) was used as electroosmotic flow modifier. The running buffer contained 2 mM NDC, 6 mM disodium carbonate and 0.2 mM TTAB. The detection wavelength was set at 240 nm. A linear calibration range of 50-500 microg/mL was obtained (r = 0.9996). The limit of detection and limit of quantitation were found to be 6 microg/mL and 20 microg/mL, respectively. The recovery was 99.45% (RSD = 0.57%, n = 5) and 99.12% (RSD = 0.48%, n = 5) for the tablet and dry syrup preparation, respectively. Repeatability tests on the migration times or peak areas proved that the results were of high precision. The RSD of peak area ratio was lower than 1% (0.74%, n = 5). The factors influencing quantitation were discussed and three batches of chewable tablet and also of dry syrup were assayed by the proposed method.

Assay for the determination of low dosage form of formoterol dry syrup by capillary electrophoresis with head-column field-amplified sample stacking.

The development of a capillary zone electrophoresis method with head-column field-amplified sample stacking injection for the determination of formoterol (FMTR) in a low dosage dry syrup form was described. To obtain the highest sensitivity, the sample solution was prepared by high content of organic solvent with the presence of a small amount of H+ (60-100 microM) and the capillary inlet end was dipped in water before electroinjection. This method was fully validated in terms of repeatability (RSDs for migration time, peak area of FMTR and peak area ratio between FMTR and I.S. at 1 microg/ml of FMTR was 0.76, 1.10 and 0.55% respectively), reproducibility (RSDs from different capillaries, analytes, days and instruments were 1.52%, 1.04%, 1.16% and 1.93% respectively), linearity (y = 0.827x - 0.085, r = 0.9993 (n = 6) over the range of 0.25-2.0 microg/ml), limits of quantitation, ruggedness and robustness. The method was applied to the determination of the drug in commercial dry syrup preparation (recovery was 100.9%, RSD = 1.5%, n = 5) and proved to be fast and reliable for the quantitation analysis of FMTR in the pharmaceutical form.

Determination of metformin in plasma by capillary electrophoresis using field-amplified sample stacking technique.

A capillary electrophoresis method was described for the determination of metformin in human plasma based on the extraction of the ion-pair with bromothymol blue into chloroform. Phenformin was used as internal standard. Field-amplified sample stacking injection was employed with an electrokinetic injection voltage of 10 kV for 10 s. The running buffer was 0.1 M phosphate buffer (pH 2.5), running voltage was 20 kV and the UV absorbance detection was set at 195 nm. The limit of quantitation was 0.25 microg/ml. Linearity range of calibration curve was 0.25 to 3.5 microg/ml. Recoveries for three levels (0.25, 1 and 2 microg/ml) were 80.24%, 67.44% and 58.97% (n = 5 for each level), respectively. The intra-day precisions for the three levels were 11.9%, 3.09% and 4.33% and the inter-day precisions were 12.4%, 4.57% and 4.94%, respectively. The concentrations of metformin hydrochloride in human plasma of eight volunteers were measured after orally administrating metformin enteric-capsule and tablet.

Determination of thiamphenicol in human plasma by micellar electrokinetic capillary chromatography.

A micellar electrokinetic chromatographic method is described for the determination of thiamphenicol in human plasma. The plasma sample was basified by adding K2HPO4 and was then extracted with ethyl acetate. After the solvent was evaporated, the residue was reconstituted in water. Approximately 40 nl of the solution were injected hydrodynamically. The running buffer was 20 mM borate (pH 9.2) containing 40 mM sodium dodecyl sulfate and 10% acetonitrile. The applied voltage was 18 kV and the detector wavelength was set at 195 nm. On-column sample stacking was achieved during the analysis to enhance the sensitivity; the limit of quantitation was 0.1 microg/ml. Linearity was over the range of 0.2 to 10 microg/ml. Recovery was 93.7+/-3.3%, the intra-day precision and accuracy was 99.6+/-2.8%; the inter-day precision and accuracy was 98.4+/-3.4%. The concentration of thiamphenicol in human plasma from eight volunteers was measured after administering thiamphenicol capsules orally.

[Effect of tyrosine on progesterone production by perifused rat large and small corpus luteal cells].

The effect of tyrosine on progesterone production by rat large and small luteal cells was studied by perifusion method. The large and small luteal cells were separated by sucrose density gradient centrifugation. After pre-perifusion for 1 h the effluent from the perifused large or small cells was separately collected at 10 min intervals by a fraction collector. Progesterone content of each sample was measured by RIA. Results showed that the basal progesterone production by large luteal cells was 2-fold more than that of the small ones. However, the suppressive effect of tyrosine on hCG- induced progesterone production was more pronounced in small luteal cells. These differences may be due to the fact that small luteal cells uptake more 3H- tyrosine.

[Variation in extracellular/intracellular Ca2+ does not affect tyrosine (Tyr) suppressive effect on hCG-induced progesterone production by rat corpus luteal cells in vitro].

Ovaries of immature female rats primed with PMSG-hCG were digested with collagenase-DNAase solution to obtain the corpus luteal cell suspensions. After pre-incubation for 1 h, luteal cell suspensions were then incubated with different factors for 2 h. The progesterone contents in the incubates were measured by RIA. It was demonstrated that high Ca2+/high K+/A23187 significantly enhanced both the basal or hCG-induced progesterone production by rat luteal cells. To the contrast, decreased Ca2+ concentration in medium/EGTA/verapamil had inhibitory effect on progesterone production in the presence of hCG. Tyr had suppressive effect on hCG-induced progesterone production. but not in the presence of high Ca2+/high K+/A23187. The present study suggested that progesterone production by luteal cells of rat is influenced by concentration. Yet, variation in extra/intra-cellular Ca2+ does not affect Tyr suppressive effect on hCG-induced progesterone production. It seems that progesterone production by calcium and hCG occurs through two different mechanisms.

[Anterior displacement of tibial tubercle in the treatment of patellofemoral arthritis].

This paper reports 41 cases (44 knees) with Patellofemoral arthritis treated by anterior displacement of tibial tubercle (modification of the classic maquet procedure of anterior displacement of tibial tubercle). Their average age was 47.5 years. 31 cases (34 knees) were followed-up for an average of 5 years and 7 months. Excellent results amounted to 64% and good results to 26%. Among theme, 12 cases were malalignment, 1 with lateral displacement of patella, 1 with subdislocation of patella and 7 with varus deformity of knee. Anterior displacement of tibial tubercle plus simultaneous proximal realignment operation was performed in 3 cases, plus distal realignment operation in 9 cases and plus coventry operation in 7 cases.