Reshaped commensal wound microbiome via topical application of Calvatia gigantea extract contributes to faster diabetic wound healing.

Background: Calvatia gigantea (CG) is widely used as a traditional Chinese medicine for wound treatment. In this study, we aimed to determine the effects of CG extract (CGE) on diabetic wound healing and the commensal wound microbiome. Method: A wound model was established using leptin receptor-deficient db/db mice, with untreated mice as the control group and CGE-treated mice as the treatment group. The wound healing rate, inflammation and histology were analyzed. Additionally, wound microbiome was evaluated via 16S ribosomal RNA (rRNA) gene sequencing. Results: CGE significantly accelerated the healing of diabetic ulcer wounds, facilitated re-epithelialization, and downregulated the transcription levels of the inflammatory cytokines, interleukin-1ß and tumor necrosis factor-α. Furthermore, CGE treatment positively affected the wound microbiome, promoting diversity of the microbial community and enrichment of Escherichia-Shigella bacteria in the CGE-treated group. Conclusions: Overall, CGE enhanced diabetic wound healing by modulating the wound microbiome and facilitating macrophage polarization during inflammation. These findings suggest modulation of the commensal wound microbiome using medicinal plants as a potential therapeutic strategy for diabetic wounds.

Investigation of Tannins Transformation in Sanguisorbae Radix Over Carbonizing by Stir-Frying.

Carbonizing by stir-frying (CSF) is the most common technology in botanical folk medicines to enhance the convergence, hemostasis, and antidiarrheal effects. Sanguisorbae Radix (SR), a well-known herbal medicine in China, has extensive therapeutic functions, while charred SR is known as an additional product obtained from SR after CSF. In this study, mass spectrometry was used to investigate the effect of charring on tannins transformation of SR. The findings showed that the content level of tannins in SR decreased significantly after carbonizing process, while their three categories, gallotannins, ellagitannins, and procyanidins, had downward trends in general. Moreover, CSF also induced the polyphenol in SR to release relevant monomers from its origins. Significant amount of hydrolyzable tannins were detected by mass spectrometry, including gallotannins and ellagitannins, suggesting that hydrolysis during CSF yielded gallic and ellagic acid and their derivatives, in addition to sugar moieties. Subsequently, gallic and ellagic acid can further polymerize to form sanguisorbic acid dilactone. The amount of proanthocyanidins, the oligomers of catechin, including procyanidin, procyanidin C2, procyanidin B3, and 3-O-galloylprocyanidin B3, decreased to form catechin and its derivatives, which may further degrade to protocatechualdehyde. Quantitative analysis illustrated that the amount of gallic, pyrogallic, and ellagic acid and methyl gallate, the essential effectors in SR, significantly increased after CSF, with increased ratios of 1.36, 4.28, 10.33, and 4.79, respectively. In contrast, the contents of cathechin and epigallocatechin dropped remarkably with increased ratios of 0.04 and 0.02. Tannins exhibit moderate absorption, while their relevant monomers have a higher bioavailability. Therefore, CSF is proved here to be an effective technique to the release of active monomers from the original polyphenol precursor. This study explored the mechanism by which tannins are transformed upon CSF of SR.

miRNA-10a-5p inhibits cell metastasis in hepatocellular carcinoma via targeting SKA1.

A variety of microRNAs (miRNAs) are involved in the occurrence and development of hepatocellular carcinoma (HCC). However, the role of miR-10a-5p in the progression of HCC remains unclear. Therefore, the purpose of this study was to determine the role of miR-10a-5p in the development of HCC and the possible molecular mechanism. miR-10a-5p expression in HCC tissues and plasma from patients was detected by quantitative real-time polymerase chain reaction. Migratory changes in HCC cells were detected after the overexpression of miR-10a-5p. Epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. Finally, through luciferase assay and rescue experiments, the mechanism by which miR-10a-5p regulates its downstream gene, human spindle and kinetochore-associated complex subunit 1, SKA1 and the interaction between these molecules in the development of HCC were determined. The expression of miR-10a-5p was markedly downregulated in HCC tissues, cell lines, and plasma. The overexpression of miR-10a-5p significantly inhibited the migration, invasion, and EMT of HCC cells. Furthermore, SKA1 was shown to be a downstream gene of miR-10a-5p. SKA1 silencing had the same effect as miR-10a-5p overexpression in HCC. In particular, the overexpression of SKA1 reversed the inhibitory effects of miR-10a-5p in HCC. Taken together, low miR-10a-5p expression is associated with HCC progression. miR-10a-5p inhibits the malignant development of HCC by negatively regulating SKA1.

Circular RNA hsa_circ_0005556 Accelerates Gastric Cancer Progression by Sponging miR-4270 to Increase MMP19 Expression.

PURPOSE: Circular RNAs (circRNAs) are a new class of RNA molecules whose function is largely unknown. There is a growing evidence that circRNAs play an important regulatory role in the progression of a variety of human cancers. However, the exact roles and the mechanisms of circRNAs in gastric cancer are not clear. In this study, we aimed to elucidate the mechanism of hsa_circ_0005556. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction was used to detect the expression of hsa_circ_0005556, miR-4270, and matrix metalloproteinase-19 (MMP19) in gastric cancer tissues and cell lines. The expression of hsa_circ_0005556 in gastric cancer cells was silenced by lentivirus, and cell proliferation, invasion, migration, and tumorigenesis in nude mice were assessed to evaluate the function of hsa_circ_0005556 in gastric cancer. RESULTS: The expression of hsa_circ_0005556 in gastric cancer tissues and gastric cancer cell lines was higher compared to normal controls. In vitro, the downregulation of hsa_circ_0005556 significantly inhibited proliferation, migration, and invasion of gastric cancer cells. In vivo, the downregulation of hsa_circ_0005556 suppressed tumor growth in nude mice. CONCLUSIONS: Our study shows that the hsa_circ_0005556/miR-4270/MMP19 axis is involved in proliferation, migration, and invasion of gastric cancer cells through the competing endogenous RNA (ceRNA) mechanism.

Telomerase inhibition decreases esophageal squamous carcinoma cell migration and invasion.

Telomerase has been shown to be associated with a variety of cancer types. To elucidate the role of telomerase in esophageal squamous carcinoma (ESCC), tissue samples from 100 patients with ESCC, and paired paracancerous tissues from 75 of these patients, were collected for use in the present study. Using immunohistochemical analysis, the expression of telomerase reverse transcriptase (hTERT) in the cytoplasm of ESCC cells was revealed to be significantly higher compared with that in paracancerous tissues, and no significant difference was observed between hTERT expression in the nucleus of ESCC and paracancerous tissue cells. Combined analysis revealed that the cytoplasmic hTERT-positive rate of patients with ESCC was significantly associated with pathological grade, N stage and Tumor-Node-Metastasis (TNM) stage; these data support the association between hTERT expression and poor patient prognosis. In vitro experiments demonstrated that hTERT knockdown does not inhibit the proliferation of ESCC Kyse410 or Kyse520 cells, but inhibits their migration and invasion abilities. These findings indicate that hTERT expression is associated with ESCC metastasis. Interestingly, decreased colony-formation ability was observed in Kyse410 cells, but not in Kyse520 cells. Collectively, the results of the present study suggest that hTERT may serve as a potential therapeutic target for ESCC.

High Seroprevalence of Human Herpesvirus 8 Infection in HIV-positive Homosexual Men in Jiangsu Province, China.

BACKGROUND: HIV-infected homosexual men are more frequently diagnosed with Kaposi's sarcoma. With the increase of HIV-infected homosexual men in China, we urgently need to know the KS-related human herpesvirus (KSHV/HHV-8) seroprevalence in this population. To investigate HHV-8 prevalence among HIV-positive homosexual men, we recruited 183 patients naive of antiretroviral therapy (ART) whose blood samples presented with HIV-antibody positive as confirmed by western blot. METHODS: HIV viral load was tested using Cobas TaqMan HIV-1 test Version 2.0, and CD4 T cell counts were tested using a Flow cytometry instrument. All HIV-positive blood samples were collected and screened for KSHV. Immunofluorescence (IFA) test was conducted for HHV-8-Specific antibodies (anti-LANA) in the plasma. HHV-8 DNA in whole blood cells of IFA-positive subjects was quantified with Real-time polymerase chain reaction (RT-PCR). RESULTS: All samples showed HIV RNA positive. CD4+ T cell count was 23% cases (42/183) which showed ≤200 cells/µL, 51.3% cases (94/183) showed 201-500 cells/µL and 25.7% cases (47/183) showed ≥501 cells/µL. Immunofluorescence (IFA) test demonstrated an HHV-8 prevalence of 50.8% (93/183), among which 20.4% of the cases (19/93) were HHV-8 DNA positive. HHV-8 infection showed no difference among different age groups (p=0.96). Similarly, HHV-8 infection exhibited no significant difference among different HIV viral load groups (p=0.08). However, HHV-8 infection among different CD4+ T cell count showed significant difference (P=0.0004). CONCLUSION: This study showed a high seroprevalence of human herpesvirus 8 infection in HIVpositive homosexual men.

Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro.

A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks' adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His6-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation.

An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

Molecular structure, distribution, and immunology function of TNFSF13B (BAFF) in Nile tilapia (Oreochromis niloticus).

B cell-activating factor (BAFF)is a member of the tumor necrosis factor (TNF) family and plays roles in B cell survival and maturation. In this study, the full-length cDNA of Nile tilapia (Oreochromis niloticus) BAFF (tBAFF) was amplified from the spleen by reverse transcription PCR (RT-PCR). The open reading frame of this cDNA encodes a protein of 261 amino acids containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian, avian, and reptile BAFF. Real-time quantitative PCR (qPCR) analysis revealed that tBAFF is present in various tissues and is predominantly expressed in the spleen. The predicted three-dimensional (3D) structure of the Nile tilapia (Oreochromis niloticus) soluble BAFF (tsBAFF) monomer was determined by (3D) structure modeling monomeranalyzed by (3D) structure mouse counterpart. Both tsBAFF and EGFP/tsBAFF were efficiently expressed in Escherichia coli BL21 (DE3), as confirmed by SDS-PAGE and Western blot analysis. After purification, the EGFP/tsBAFF fusion protein showed a fluorescence spectrum similar to that of EGFP. Laser scanning confocal microscopy showed that EGFP/tsBAFF bound to its receptor. In vitro, tsBAFF promoted the proliferation of Nile tilapia and mouse splenic B cells together with/without a priming agent (Staphylococcus aureus Cowan 1, SAC) or anti-mouse IgM. Furthermore, tsBAFF showed a similar proliferation-stimulating effect on mouse B cells compared to msBAFF. These findings indicate that tsBAFF plays an important role in the proliferation of Nile tilapia B cells and has functional cross-reactivity among Nile tilapia and mammals. Therefore, BAFF may represent a useful factor for enhancing immunological efficacy in animals.

Molecular cloning, expression and functional characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene from mandarin fish (Siniperca chuatsi).

Interferon-γ-inducible lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. For this important function in the immune system, we cloned a GILT gene homologue from mandarin fish (designated mGILT), a kind of precious freshwater fish with high market value. Through reverse transcription PCR and rapid amplification of cDNA ends (RACE) strategies, we obtained the full-length cDNA of mGILT, which consists of 1008 bp with a 771 bp open reading frame, encoding a protein of 256 amino acids, with a putative molecular weight of 28.47 kDa. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 6 conserved cysteines. The result of real-time quantitative PCR showed that mGILT mRNA was expressed in a tissue-specific manner. In addition, the expression of mGILT mRNA was obviously up-regulated in splenocytes and kidney after induction with lipopolysaccharide (LPS). Recombinant mGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Further study revealed that mGILT exhibit thiol reductase activity on IgG substrate. These results suggest mGILT is highly likely to play a role in the immune responses in mandarin fish.

Molecular cloning and characterization of TNFSF14 (LIGHT) and its receptor TNFRSF14 (HVEM) in guinea pig (Cavia porcellus).

LIGHT (lymphotoxin-related inducible ligand that competes with herpes simplex virus (HSV) glycoprotein D for herpesvirus entry mediator on T cells) is a member of the tumor necrosis factor (TNF) ligand superfamily, which plays important roles in inflammatory and immune responses. In the present study, the cDNAs of guinea pig (Cavia porcellus) LIGHT (designated as gpLIGHT) and its receptor herpes virus entry mediator (designated as gpHVEM) were amplified from spleen by reverse transcription polymerase chain reaction (RT-PCR). The ORFs of gpLIGHT and gpHVEM cover 726 and 861 bp, encoding predicted proteins with 241 and 286 aas, respectively. The three-dimensional (3D) structure, phylogenetic relationships, and characterization of both genes were also analyzed. We also generated a 3D model to verify interaction between the two proteins. Real-time quantitative PCR (qPCR) analysis revealed that both LIGHT and HVEM are constitutively expressed in guinea pig various tissues. A fusion protein SUMO (Small Ubiquitin-like Modifier)-gpsLIGHT (the soluble mature part of gpLIGHT) was efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy (LSCM) showed that gpsLIGHT can bind its receptors on T cells. The LIGHT-HVEM signaling pathway plays an important role in the immune system, and our results might provide a platform for further research into the effects of LIGHT and HVEM.

Isolation and characterization of LIGHT (TNFSF14) gene homologue in zebrafish (Danio rerio).

The tumor necrosis factor superfamily (TNFSF) proteins are cytokines involved in many biological processes. In this study, the TNF superfamily member 14 (TNFSF14, LIGHT) has been isolated from zebrafish Danio rerio (designated zLIGHT). The full-length open reading frame (ORF) of zLIGHT cDNA consists of 708 bp encoding a protein of 235 amino acids. The zLIGHT open reading frame (ORF) genomic sequence consists of three introns and four exons, is about 9.9 kb in size. Real-time quantitative PCR (qPCR) analysis suggested that zLIGHT was predominantly expressed in zebrafish spleen. The soluble LIGHT (zsLIGHT) had been cloned into the pSUMO vector, SDS-PAGE and Western blotting analysis confirmed that the recombinant protein SUMO-zsLIGHT was efficiently expressed in Escherichia coli BL21 (DE3). Laser scanning confocal microscopy analysis showed that SUMO-zsLIGHT could bind to its receptors on T cells. LIGHT is involved in many important biological effects, including up-regulating proinflammatory chemokines, cytokines, inducing cell death, apoptosis, and enhancing T cell survival. Zebrafish may conduct as a model animal for further research on LIGHT.