Chitin scaffold combined with autologous small nerve repairs sciatic nerve defects.

Although autologous nerve transplantation is the gold standard for treating peripheral nerve defects, it has many clinical limitations. As an alternative, various tissue-engineered nerve grafts have been developed to substitute for autologous nerves. In this study, a novel nerve graft composed of chitin scaffolds and a small autologous nerve was used to repair sciatic nerve defects in rats. The novel nerve graft greatly facilitated regeneration of the sciatic nerve and myelin sheath, reduced atrophy of the target muscle, and effectively restored neurological function. When the epineurium of the small autogenous nerve was removed, the degree of nerve regeneration was similar to that which occurs after autogenous nerve transplantation. These findings suggest that our novel nerve graft might eventually be a new option for the construction of tissue-engineered nerve scaffolds. The study was approved by the Research Ethics Committee of Peking University People's Hospital (approval No. 2019PHE27) on October 18, 2019.

Exosomal lncRNA KLF3-AS1 derived from bone marrow mesenchymal stem cells stimulates angiogenesis to promote diabetic cutaneous wound healing.

AIMS: We focused on BMSC-derived exosomal lncRNA KLF3-AS1 and its significance in diabetic cutaneous wound healing. METHODS: Potential interaction between KLF3-AS1 and miR-383, miR-383 and VEGFA were predicted using bioinformatic analysis and validated by luciferase reporter, RIP, and FISH assays. The proliferation, apoptosis, migration and tube formation of HUVECs were evaluated by CCK-8, flow cytometry, wound healing, and tube formation assays, respectively. A murine diabetic cutaneous wound model was used to investigate therapeutic effects of exosomal KLF3-AS1 in vivo. Histological alterations in skin tissues were examined using HE, Masson staining, and immunostaining of CD31. RESULTS: BMSC-derived exosomal KLF3-AS1 sufficiently promoted proliferation, migration, and tube formation, while inhibited apoptosis of HUVECs challenged by high glucose. The protective effects of exosomal KLF3-AS1 were achieved at least partially by down-regulating miR-383, and boosting the expression of its target, VEGFA. In vivo, exosomes from KLF3-AS1-expressing BMSCs demonstrated the best effects in promoting cutaneous wound healing in diabetic mice, which were associated with minimal weight loss, increased blood vessel formation, reduced inflammation, decreased miR-383 expression, and up-regulated VEGFA. CONCLUSIONS: Exosomal lncRNA KLF3-AS1 derived from BMSCs induces angiogenesis to promote diabetic cutaneous wound healing.

[TCMATCOV--a bioinformatics platform to predict efficacy of TCM against COVID-19].

There is urgent need to discover effective traditional Chinese medicine(TCM) for treating coronavirus disease 2019(COVID-19). The development of a bioinformatic tool is beneficial to predict the efficacy of TCM against COVID-19. Here we deve-loped a prediction platform TCMATCOV to predict the efficacy of the anti-coronavirus pneumonia effect of TCM, based on the interaction network imitating the disease network of COVID-19. This COVID-19 network model was constructed by protein-protein interactions of differentially expressed genes in mouse pneumonia caused by SARS-CoV and cytokines specifically up-regulated by COVID-19. TCMATCOV adopted quantitative evaluation algorithm of disease network disturbance after multi-target drug attack to predict potential drug effects. Based on the TCMATCOV platform, 106 TCM were calculated and predicted. Among them, the TCM with a high disturbance score account for a high proportion of the classic anti-COVID-19 prescriptions used by clinicians, suggesting that TCMATCOV has a good prediction ability to discover the effective TCM. The five flavors of Chinese medicine with a disturbance score greater than 1 are mainly spicy and bitter. The main meridian of these TCM is lung, heart, spleen, liver, and stomach meridian. The TCM related with QI and warm TCM have higher disturbance score. As a prediction tool for anti-COVID-19 TCM prescription, TCMATCOV platform possesses the potential to discovery possible effective TCM against COVID-19.

Identification and characterization of a novel group of natural anti-sense transcripts from RNA1.2 gene locus of human cytomegalovirus.

BACKGROUND: Natural anti-sense transcripts (NATs), which are transcribed from the complementary DNA strand of annotated genes, exert regulatory function of gene expression. Increasing studies recognized anti-sense transcription widespread throughout human cytomegalovirus (HCMV) genome, whereas the anti-sense transcription of RNA1.2 gene locus has never been investigated. In this study, the transcription of the RNA1.2 anti-sense strand was investigated in clinically isolated HCMV strain. METHODS: Strand-specific high-through RNA-sequencing (RNA-seq) was performed to find possible anti-sense transcripts (ASTs). For analyzing and visualization of RNA-seq data sets, Integrative Genomics Viewer software was applied. To confirm these possibilities, Northern blotting and rapid amplification of cDNA ends (RACE) were used. RESULTS: Transcription of the opposite strand of RNA1.2 gene locus was detected by RNA-sequencing using RNAs extracted from human embryonic lung fibroblasts infected with HCMV clinical isolate HAN. At least three HCMV NATs, named RNA1.2 AST 1, RNA1.2 AST2, and RNA1.2 AST3, were characterized by Northern blotting and RACE analyses. These RNA1.2 ASTs orientated from the complementary strand of RNA1.2 locus during the late phase of HCMV infection. The 5'- and 3'-termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene. CONCLUSION: A cluster of novel NATs was transcribed from the opposite sequence of the HCMV RNA1.2 gene region.

Identification and BAC construction of Han, the first characterized HCMV clinical strain in China.

Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects, and may lead to severe or lethal diseases in immunocompromised individuals. Several HCMV strains have been identified and widely applied in research, but no isolate from China has been characterized. In the present study, we isolated, characterized and sequenced the first Chinese HCMV clinical strain Han, and constructed the novel and functional HCMV infectious clone Han-BAC-2311. HCMV Han was isolated from the urine sample of a Chinese infant with multiple developmental disorders. It expresses HCMV specific proteins and contains a representative HCMV genome with minor differences compared to other strains. By homologous recombination using mini-F derived BAC vector pUS-F6, the infectious clone Han-BAC-2311 was constructed containing representative viral genes across the HCMV genome. The insertion site and orientation of BAC sequence were confirmed by restriction enzyme digestion and Southern blotting. The reconstituted recombinant virus HanBAC-2311 expresses typical viral proteins with the same pattern as that of wild-type Han, and also displayed a similar growth kinetics to wild-type Han. The identification of the first clinical HCMV strain in China and the construction of its infectious clone will greatly facilitate the pathogenesis studies and vaccine development in China.

Expression pattern of neuregulin-1 type III during the development of the peripheral nervous system.

Neuregulin-1 type III is a key regulator in Schwann cell proliferation, committing to a myelinating fate and regulating myelin sheath thickness. However, the expression pattern of neuregulin-1 type III in the peripheral nervous system during developmental periods (such as the premyelinating stage, myelinating stage and postmyelinating stage) has rarely been studied. In this study, dorsal root ganglia were isolated from rats between postnatal day 1 and postnatal day 56. The expression pattern of neuregulin-1 type III in dorsal root ganglia neurons at various developmental stages were compared by quantitative real-time polymerase chain reaction, western blot assay and immunofluorescent staining. The expression of neuregulin-1 type III mRNA reached its peak at postnatal day 3 and then stabilized at a relative high expression level from postnatal day 3 to postnatal day 56. The expression of neuregulin-1 type III protein increased gradually from postnatal day 1, reached a peak at postnatal day 28, and then decreased at postnatal day 56. Immunofluorescent staining results showed a similar tendency to western blot assay results. Experimental findings indicate that the expression of neuregulin-1 type III in rat dorsal root ganglion was increased during the premyelinating (from postnatal day 2 to postnatal day 5) and myelinating stage (from postnatal day 5 to postnatal day 10), but remained at a high level in the postmyelinating stage (after postnatal day 10).

Antiinflammatory effects of matrine in LPS-induced acute lung injury in mice.

Matrine is one of the main active components of Chinese herb Sophora flavescens Ait (Kushen), which has been demonstrated to be effective in suppressing inflammation. The aim of the present study is to investigate the effect of matrine on LPS-induced lung injury. Lung injury was assessed by histological study and wet to dry weight ratios, as well as cell count and protein content in bronchoalveolar lavage fluid. We also detected MPO activity reflecting neutrophil infiltration and MDA activity examining oxidative stress in lung tissues. Cytokines and ROS production in cells were monitored by ELISA and flow cytometry, respectively. The results showed that high dose of matrine significantly reduced the mortality rate of mice with LPS administration. Treatment with matrine improved LPS-induced lung histopathologic changes, alleviated pulmonary edema and lung vascular leak, inhibited MPO and MDA activity,and reduced the production of inflammatory mediators including TNF-α, IL-6 and HMGB1. In vitro, matrine administration reduced the production of ROS and inflammatory factors, which was possibly associated with inhibition of NF-κB. In conclusion, the current study demonstrated that matrine exhibited a protective effect on LPS-induced acute lung injury by inhibiting of the inflammatory response, which may involve the suppression of ROS and tissue oxidative stress.

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study.

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 µL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 µm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.