Objective To investigate the microbiota composition and diversity between autogenous and anautogenous Culex pipiens pallens, so as to provide insights into unraveling the pathogenesis of autogeny in Cx. pipiens pallens. Methods Autogenous and anautogenous adult Cx. pipiens pallens samples were collected at 25 ℃, and the hypervariable regions of the microbial 16S ribosomal RNA (16S rRNA) gene was sequenced on the Illumina NovaSeq 6000 sequencing platform. The microbiota abundance and diversity were evaluated using the alpha diversity index, and the difference in the microbiota structure was examined using the beta diversity index. The microbiota with significant differences in the abundance between autogenous and anautogenous adult Cx. pipiens pallens samples was identified using the linear discriminant analysis effect size (LEfSe). Results The microbiota in autogenous and anautogenous Cx. pipiens pallens samples belonged to 18 phyla, 28 classes, 70 orders, 113 families, and 170 genera, and the dominant phyla included Proteobacteria, Bacteroidetes, and so on. At the genus level, Wolbachia was a common dominant genus, and the relative abundance was (77.6 ± 11.3)% in autogenous Cx. pipiens pallens samples and (47.5 ± 8.5)% in anautogenous mosquito samples, while Faecalibaculum (0.4% ± 0.1%), Dubosiella (0.5% ± 0.0%) and Massilia (0.5% ± 0.1%) were specific species in autogenous Cx. pipiens pallens samples. Alpha diversity analysis showed that higher Chao1 index and ACE index in autogenous Cx. pipiens pallens samples than in anautogenous samples (both P values > 0.05), and lower Shannon index (P > 0.05) and Simpson index (P < 0.05) in autogenous Cx. pipiens pallens samples than in anautogenous samples. LEfSe analysis showed a total of 48 significantly different taxa between autogenous and anautogenous Cx. pipiens pallens samples (all P values < 0.05). Conclusion There is a significant difference in the microbiota diversity between autogenous and anautogenous Cx. pipiens pallens.

Objective To investigate the MRI manifestations of acquired immunodeficiency syndrome complicated with subtentorial progressive multifocal leukoencephalopathy(PML).Methods Sixteen patients with acquired immunodeficiency syndrome complicated with subtentorial PML confirmed by surgery,pathology,or clinical diagnosis were selected.The MRI features of plain scan and enhanced scan were analyzed and summarized,retrospectively.Results Among the 16 acquired immunodeficiency syndrome patients complicated with subtentorial PML,in terms of lesion distribution,the left cerebellar hemisphere was involved in 10 cases(62.5％),and the right cerebellar hemisphere was involved in 10 cases(62.5％).The left pontine arm was involved in 11 cases(68.8％),the right pontine arm was involved in 10 cases(62.5％),the medulla oblongata was involved in 6 cases(37.5％),the pontine region was involved in 11 cases(68.8％),the midbrain was involved in 7 cases(43.8％),and the supratentorial lesions were involved in 5 cases(31.3％).Subtentorial lesions were found to be asymmetrically distributed in all patients.In terms of MRI imaging findings,acquired immunodeficiency syndrome patients complicated with subtentorial PML lesions exhibited low signal on T,WI and high signal on T2 WI/T2-fluid attenuated inversion recovery(FLAIR)images.The"shrimp sign"and"crescent cerebellar lesion"was respectively observed in 3 and 7 cases on T2 WI.There were no diffusion limitations on diffusion weighted imaging(DWI),no significant enhancement after contrast-enhanced scanning,and no mass effect in all the 16 cases.Conclusion The MRI imaging findings of acquired immunodeficiency syndrome complicated with subtentorial PML exhibit similar features to those observed in PML affecting the cerebral hemispheres.A characteristic"shrimp sign"is often observed on T2WI.The presence of typical MRI imaging findings,along with the patient's clinical history and the detection of JC virus deoxyribonucleic acid(DNA)in the cerebrospinal fluid(CSF),can aid in the accurate diagnosis of PML.

OBJECTIVE: To clarify the molecular signaling mechanism underlying the inhibitory effect of metformin on transforming growth factor-β1 (TGF-β1)-stimulated collagen I production in rat biliary fibroblasts. METHODS: Primary biliary fibroblasts were isolated under aseptic condition from 50 Sprague-Dawley rats (half male and half female), and microscopic observation identified no obvious difference in the morphology or viability of the cells from rats with different sexes or body weight. The cells were treated with TGF-β1 (10 ng/mL), Smad3 siRNA+TGF-β1, CTGF siRNA+TGF-β1, metformin (10 mmol/L)+ TGF-β1, or Compound C (10 μmol/L)+metformin+TGF-β1. The expressions of CTGF and collagen I in the treated cells were determined using ELISA kit or Western blotting; the phorsphorylated and total Smad3 and AMPK expressions were detected using immunoblotting. RESULTS: TGF-β1 time- and dose-dependently induced collagen I production in rat biliary fibroblasts. The activated AMPK by metformin dose-dependently inhibited TGF-β1-induced collagen I production. Pre-incubation of cells with the AMPK inhibitor Compound C restored the inhibitory effect of AMPK on TGF-β1-induced collagen I secretion ( < 0.01). Activation of AMPK by metformin significantly reduced TGF-β1-induced collagen I production by suppressing Smad3-driven CTGF expression ( < 0.01), and the application of Compound C reversed such changes in the fibroblasts ( < 0.01). CONCLUSIONS Metformin inhibits TGF-β1-stimulated collagen I production by activating AMPK and inhibiting Smad3- driven CTGF expression in rat biliary fibroblasts.
Animals ; Cells, Cultured ; Collagen ; Female ; Fibroblasts ; Male ; Metformin ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad3 Protein ; Transforming Growth Factor beta1

Objective To evaluate the effect of 8% emulsified isoflurane preconditioning on renal ischemia-reperfusion (I/R) injury in rats.Methods Thirty-two male Sprague-Dawley rats,aged 10-13 weeks,weighing 220-300 g,were randomly divided into 4 groups (n =8 each):sham operation group (group S); I/R group;emulsified isoflurane preconditioning group (group E) ; intralipid preconditioning group (group I).Renal ischemia was induced by occlusion of the left renal pedicle for 45 min with atraumatic microclips followed by 3 h reperfusion.8 % emulsified isoflurane and 30 % intralipid 4 ml· kg-1· h-1 were infused intravenously for 30 min followed by 15 min washout before renal UR in groups E and I,respectively.Arterial blood samples were taken at 3 h of reperfusion to determine the concentrations of serum creatinine (Cr),cystatin C (Cys C),TNF-α,IL-6 and IL-10.The animals were then sacrificed and left kidneys were removed and stained with hematoxylin-eosin for microscopic examination and assessment of necrosis of renal proximal convoluted tubules (0 =normal,4 =necrosis of whole segment of proximal convoluted tubules).Results Compared with group S,the serum Cr,Cys C,TNF-α,IL-6 and IL-10 concentrations and severity of necrosis of renal proximal convoluted tubules were significantly increased in groups I/R,E and I (P ＜ 0.05).The serum Cr,Cys C,TNF-α and IL-6 concentrations and severity of necrosis of renal proximal convoluted tubules were significantly lower,while the serum IL-10 concentration was higher in group E than in groups I/R and I (P ＜0.05).There was no significant difference in the indexes mentioned above between groups L and I/R (P ＞ 0.05).The damage to renal tissues was less serious in group E than in groups I/R and L.Conclusion Preconditioning with 8 % emulsified isoflurane can attenuate renal I/R injury by inhibiting inflammatory responses in rats.

Objective To investigate the local immune response in condyloma acuminatum treated with aminolevulinic acid-photodynamic therapy (ALA-PDT).Methods In vitro and in vivo studies were performed.A previously established keratinocyte cell line human papilloma virus (HPV) 16E7/HaCaT which stably expresses HPV16E7 protein was used in this study.Peripheral blood mononuclear cells (PBMCs) were separated from 10 healthy volunteers.After pretreatment with ALA-PDT,HPV16E7/HaCaT cells were cocultured with the PBMCs for 3 hours in a Transwell chamber followed by the observation of chemotactic migration of PBMCs.Tissue samples were obtained from the lesions of 10 patients with condyloma acuminatum before,and at 1,2,3 and 48 hours after the first session of ALA-PDT.Immunohistochemistry was conducted to determine the number of CD4+ T cells,CD8+ T cells and CD68+ macrophages as well as CD4/CD8 T-cell ratio in the tissue samples.Results After 3-hour coculture with HPV16E7/HaCaT cells pretreated by ALA-PDT,PBMCs showed apparent chemotactic migration.Immunohistochemistry revealed a statistical increase in the number of CD4+ T cells,CD8+ T cells and CD4/CD8 T-cell ratio at 48 hours (all P ＜ 0.05),as well as in the number of CD68+ macrophages at 3 hours and 48 hours (both P ＜ 0.05) after the first session of ALA-PDT.Conclusion ALA-PDT may induce local antiviral immune response in condyloma acuminatum.

Objective To investigate the efficacy and safety of lauromacrogol foam sclerosant in the treatment of peripheral venous malformations.Methods 21 patients with peripheral venous malformations were treated with foam sclerotherapy.The sclerosing foam was produced by Tessari's method using 1％ lauromacrogol.When necessary,the injections were repeated at an interval of four weeks,and this process contained 3 to 5 injections.Therapeutic outcomes and safety were established by evaluating size of lesion,symptom,duration of treatment and side-effects of treatment before and after treatment.Results 21 patients were followed up for 3 to 18 months.10 cases showed markedly shrinked or even disappeared of treated malformations,9 cases showed a reduction in size over 50 ％,and 2 cases showed a reduction in size less 50 ％.Local swelling occurred in almost per session,pyrexia in 5 patients,which resolved spontaneously within several days to 1 week.No major complication occurred.Conclusions Foam sclerotherapy using lauromacrogol seems to be a safe and effective therapeutic method for peripheral venous malformations.

Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting (HRM) and study its preliminary clinical application.Methods Two common mutations [ IVS-2-654 ( C ＞ T ) and -28 ( A ＞ G ) ]of β-thalassemia in Wenzhou city population were selected.The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls.A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity,sensitivity and repeatability were methodologically evaluated.One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children's Hospital of Wenzhou Medical College were enrolled into this study.The genomic DNA was extracted from whole blood cells and detected by HRM method.The results were compared with the direct sequencing data.Results HRM method could detect the mutations [ IVS-2-654( C ＞ T) and -28 ( A ＞ G ) ]of β-thalassemia and the results did not show any non-specific amplified fragments.All within-run and between-run coefficients of variation for different DNA types' Tm were smaller than 0.1％.And minimum 103 copies of DNA of each assay and 10％ mutation could be determined by this method.One hundred and seventeen patients with clinically suspected β-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing.There were 45 IVS-2-654 ( C ＞ T)heterozygous mutation and 9-28 ( A ＞ G)heterozygous mutation and none homozygous mutation.Conclusion The method of rapid identification of β-thalassemia gene mutations based on HRM analysis is successfully established,which is a convenient,rapid,specific,sensitive and accurate technique for screening gene mutations in β-thalassemia as well as a general technical platform to identify other gene mutations.

Penicillium decumbens T. is an important filamentous fungus for the production of cellulases to effectively degrade lignocellulose for second generation biofuel production. In order to enhance the capability of Penicillium decumbens to produce cellulases, we constructed a creB (a deubiquitinating enzyme encoding gene) deletion cassette, and generated a creB knockout strain with homologous double crossover recombination. This mutation resulted in a detectable decrease of carbon catabolite repression (CCR) effect. The filter paper activity, endoglucanase activity, xylanase activity and exoglucanase activity of the deltacreB strain increased by 1.8, 1.71, 2.06 and 2.04 fold, respectively, when comparing with the parent strain Ku-39. A 2.68 fold increase of extracellular protein concentration was also observed. These results suggest that the deletion of creB results in CCR derepression. These data also suggest that CREB influences cellulase production of Penicillium decumbens. In generation, this study provides information that can be helpful for constructing cellulase hyper-producing strain.
Cellulase ; biosynthesis ; Endopeptidases ; genetics ; metabolism ; Gene Knockout Techniques ; Lignin ; metabolism ; Mutant Proteins ; metabolism ; Penicillium ; enzymology ; genetics ; Recombination, Genetic ; Ubiquitinated Proteins ; genetics ; Ubiquitination

Objective To establish a model for cutaneous squamous cell carcinoma by irradiation of SKH-1 hairless mice with solar-simulated ultraviolet (solar UV), and to explore the biological characteristics of the model. Methods A total of 91 SKH-1 hairless mice were randomly divided into seven experimental groups (n = 10) and seven control groups (n = 3). The mice in experimental groups were irradiated with minimal erythema dose of solar UV 4 times per week for various durations (4, 8, 12, 16, 20, 24, 28 weeks), while the control mice received no irradiation. The general status and skin appearance of mice were observed during the treatment process. Mice were killed immediately after the last irradiation at different time points and pathological examination was carried out to observe the histological changes of skin lesions. Results Papules measuring equal to or more than 1 mm in diameter began to develop in some mice in experimental group 10 weeks after the first irradiation; tumors began to appear in 39.3% (11/28) of the remaining mice in experimental group on week 20, and in 100% (10/10) of the remaining mice on week 28. The cumulative dose approximated to 26.99 J/cm2 for UVB and 242.91 J/cm2 for UVA after 28-week irradiation. No tumor was observed in the control mice. Pathological examination revealed characteristic changes of squamous cell carcinoma in 30% of the mice on week 12, 33.3% on week 16, 60% on week 20, 87% on week 24, and 100% on week 28. Conclusions Ultraviolet could induce the hyperplasia of skin in SKH-1 hairless mice, and even cause the development of cutaneous squamous cell carcinoma after prolonged irradiation.

This study is to investigate the antitumor activity of CIP-36 on multidrug resistant human oral squamous carcinoma cell line (KBV200 cells) in vitro and the possible anticancer mechanisms. MTT assay, Hoechst fluorescein stain, RT-PCR and immunohistochemistry were carried out on KBV200 and KB cells. The growth of many tumor cells was obviously inhibited by CIP-36, especially the multidrug resistant cells KBV200. Obvious apoptosis could be observed in the Hoechst 33342 staining experiments. The results of RT-PCR showed that the levels of p53, p21, caspase-3 and bax mRNA increased, and meanwhile the expression of mdr-1 and bcl-2 mRNA decreased in a dose-dependent manner. The data were significantly different from that of vehicle. The expression of P-gp significantly decreased with the increasing dosage of CIP-36 examined by immunohistochemistry. It can be concluded that CIP-36 could change resistance-related genes and proteins to overcome multidrug resistance in the KBV200 cell line.