BAG6 negatively regulates the RLR signaling pathway by targeting VISA/MAVS.

The virus-induced signaling adaptor protein VISA (also known as MAVS, ISP-1, Cardif) is a critical adaptor protein in the innate immune response to RNA virus infection. Upon viral infection, VISA self-aggregates to form a sizeable prion-like complex and recruits downstream signal components for signal transduction. Here, we discover that BAG6 (BCL2-associated athanogene 6, formerly BAT3 or Scythe) is an essential negative regulator in the RIG-I-like receptor signaling pathway. BAG6 inhibits the aggregation of VISA by promoting the K48-linked ubiquitination and specifically attenuates the recruitment of TRAF2 by VISA to inhibit RLR signaling. The aggregation of VISA and the interaction of VISA and TRAF2 are enhanced in BAG6-deficient cell lines after viral infection, resulting in the enhanced transcription level of downstream antiviral genes. Our research shows that BAG6 is a critical regulating factor in RIG-I/VISA-mediated innate immune response by targeting VISA.

[Effect of Wei's triple nine needling on eye regulation in patients with presbyopia complicated with visual fatigue of liver depression and spleen deficiency].

OBJECTIVE: To compare the clinical efficacy between Wei's triple nine needling combined with esculin and digitalis glycosides eye drops and esculin and digitalis glycosides eye drops alone for presbyopia complicated with visual fatigue of liver depression and spleen deficiency. METHODS: Forty-six cases (92 eyes) with presbyopia complicated with visual fatigue of liver depression and spleen deficiency were randomly divided into an observation group (23 cases) and a control group (23 cases, 2 cases dropped off). The cases in the observation group were treated with Wei's triple nine needling and esculin and digitalis glycosides eye drops. The acupoints included Shangming (Extra), Chengqi (ST 1), Cuanzhu (BL 2) to Jingming (BL 1), Sizhukong (TE 23) to Taiyang (EX-HN 5), etc; the needling was given once every other day, three times a week, and the eye drops were given one drop each time, three times a day. The cases in the control group were only treated with the eye drops. Both groups were treated for 7 days as one course of treatment, and 2 courses of treatment were given. The visual fatigue core symptoms score, adjustment amplitude, adjustment lag and best average corrected visual acuity were observed in the two groups before treatment, 1 week and 2 weeks into treatment, respectively. RESULTS: Compared before treatment, the visual fatigue core symptoms scores in the two groups were decreased after 1-week and 2-week treatment (P<0.05); in the observation group, the adjustment amplitude was increased after 2-week treatment (P<0.05), while in the control group, the adjustment amplitude was increased after 1-week and 2-week treatment (P<0.05); in the observation group, the adjustment lag was decreased after 1-week and 2-week treatment (P<0.05). After 2-week treatment, the visual fatigue core symptoms score in the observation group was lower than that in the control group, and the adjustment amplitude was higher than that in the control group (P<0.05). There were no significant differences in adjustment lag and best average corrected visual acuity between the two groups after 1-week and 2-week treatment (P>0.05). CONCLUSION: Wei's triple nine needling combined with esculin and digitalis glycosides eye drops could improve the visual fatigue and eye regulation ability in patients with presbyopia complicated with visual fatigue of liver depression and spleen deficiency, and the effect is better than esculin and digitalis glycosides eye drops alone.

New insights into the phylogeny of Sinocarum (Apiaceae, Apioideae) based on morphological and molecular data.

Sinocarum is a Sino-Himalayan endemic genus of Apiaceae and distributed in high-elevations from Nepal to SW China. In this study, morphological characteristics were combined with nuclear internal transcribed spacer (ITS) and two chloroplast DNA (cpDNA) intron sequences (rpl16 and rps16) to determine the phylogenetic placement of Sinocarum and the infrageneric relationships between five Sinocarum species. The results confirmed that Sinocarum was a polyphyletic group separated into two clades, Acronema and East Asia clades. S. coloratum, the generic type of Sinocarum, S. cruciatum, S. vaginatum and S. filicinum are in the Acronema clade. Among them, the first three species are clustered into a subclade and are closely related to the genus Acronema. While S. filicinum has a close affinity with Meeboldia. S. schizopetalum did not ally with its congeners we collected and is allied closely with members of the distantly related East Asia clade. In addition, the fruit of the Acronema clade Sinocarum species is usually oblong-ovoid or ovoid, and the pollen is super-rectangular, while the Sinocarum species in the East Asia clade have broad-ovoid fruit and sub-rhomboidal pollen. This study has furnished cumulative evidence to reduce phylogenetic uncertainty and provide a more comprehensive description of the plant morphology, fruit morphology and anatomy, and pollen morphology of these five Chinese Sinocarum species.

Astaxanthin inhibiting oxidative stress damage of placental trophoblast cells in vitro.

Oxidative stress from the trophoblasts is one of the possible pathological mechanisms of Preeclampsia (PE). This study aimed at exploring the potential effects of astaxanthin (ATX) on oxidative stress damaged placental trophoblast cell line HTR-8/SVneo. Oxidative stress-induced damaged through H2O2 treatment was checked by MTS CellTiter 96® cell viability, 2',7'-dichlorofluorescein diacetate (DCFH-DA) induced fluorescence, the level of the intracellular malondialdehyde (MDA), and the detection of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT). Different concentrations of ATX were applied, and then the proliferation rate, apoptotic percentage, cell cycle distribution, invasion test and relative biological function of the rescued cells were followed. We provide evidence that ATX had an anti-oxidative effect against oxidative stress induced by H2O2 on the trophoblast cell line and had beneficial role in promoting cell proliferation, inhibiting cell apoptosis, and inducing cell invasion.Abbreviations: UV: ultraviolet; DCFH-DA: 2',7'-dichlorofluorescein diacetate; EVT: extravillous trophoblast; MMPs: matrix metalloproteinases; IUGR: intrauterine growth restriction.

Tongoloa arguta (Apiaceae), a new species from southwest China.

A new species Tongoloa arguta (Apiaceae) is described and illustrated in this article. The new species grows in alpine bushes and meadows in south-western China. It resembles T. silaifolia, but differs from the latter by the length of the stem, ultimate segments of leaf and rays of the umbel. Phylogenetic analysis, based on nuclear ribosomal DNA internal transcribed spacer (ITS) sequences, is provided, as well as comparative morphology between related species.

Characterization of Pneumocystis jirovecii pneumonia at three tertiary comprehensive hospitals in southern China.

Due to the increasing use of immunosuppressant therapy, Pneumocystis jirovecii pneumonia (PJP) has become an emerging concern in human immunodeficiency virus (HIV)-negative patients. In this study, we conducted a retrospective study of 96 hospitalized patients with PJP from January 2015 to June 2019 at three tertiary comprehensive hospitals in Southern China. Information was collected regarding patient demographics, clinical manifestations, risk factors, laboratory analyses, radiological images, and treatment outcomes. PJP infection was most commonly found in middle-aged men. Kidney diseases (35.5%) and connective tissue diseases (38.7%) were the predominant risk factors for PJP. About half of the patients (48.4%) received glucocorticoid, immunosuppressant, and/or chemotherapy in a low dose or in a short-term (< 3 months). None of the patients had previously received trimethoprim-sulfamethoxazole (TMP-SMX) for PJP prophylaxis. All patients had two or more clinical manifestations (cough, dyspnea, fever, and chest pain). Biochemical investigations of CRP, ESR, PaO2, LDH, and KL-6 showed that over 90% of the patients exceeded the reference range of indicators. Our analyses revealed the dominant risk factors (HIV, kidney diseases, and connective tissue diseases) and the most consistent biochemical indicators (LDH, BG, and KL-6) for PJP. Moreover, early prophylaxis, diagnosis, and treatment should contribute to improve the survival of these PJP patients.

Characterization of the complete chloroplast genome of Taibaisanqi (Tongoloa silaifolia).

Tongoloa silaifolia, known as a traditional Chinese medicine Taibaisanqi, is a perennial herb of Apiaceae. In this study, the complete chloroplast genome of T. silaifolia was determined through Illumina sequencing method. The chloroplast genome of T. silaifolia was 161,122 bp in length and contained a pair of IR regions (30,824 bp) separated by a small single-copy region (17,553 bp) and a large single-copy region (81,921 bp). This chloroplast genome is encoded with 137 genes including 92 CDS, 37 tRNA genes, and 8 rRNA genes. The overall GC content of T. silaifolia cp genome is 37.7%. By phylogenetic analysis using maximum likelihood method, T. silaifolia showed the closest relationship with Chuanminshen violaceum and Hansenia species.

Effects of inoculation with arbuscular mycorrhizal fungi on maize grown in multi-metal contaminated soils.

Pot culture experiments were established to determine the effects of colonization by arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. sp) on maize (Zea mays L.) grown in Pb, Zn, and Cd complex contaminated soils. AMF and non-AMF inoculated maize were grown in sterilized substrates and subjected to different soil heavy metal (Pb, Zn, Cd) concentrations. The root and shoot biomasses of inoculated maize were significantly higher than those of non-inoculated maize. Pb, Zn, and Cd concentrations in roots were significantly higher than those in shoots in both the inoculated and non-inoculated maize, indicating the heavy metals mostly accumulated in the roots of maize. The translocation rates of Pb, Zn, and Cd from roots to shoots were not significantly difference between inoculated and non-inoculated maize. However, at high soil heavy metal concentrations, Pb, Zn, and Cd in the shoots and Pb in the roots of inoculated maize were significantly reduced by about 50% compared to the non-inoculated maize. These results indicated that AMF could promote maize growth and decrease the uptake of these heavy metals at higher soil concentrations, thus protecting their hosts from the toxicity of heavy metals in Pb, Zn, and Cd complex contaminated soils.

[Expression of vascular smooth muscle cell markers during early stage of embryonic stem cell-derived embryoid bodies differentiation].

AIM: To observe expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC during early embryonic vascular development, and to initially investigate the differentiation effect of platelet derived growth factor-BB (PDGF-BB) on vascular smooth muscle cells (VSMCs) during that period. METHODS: Murine embryonic stem cell line expressing the enhanced green fluorescent protein (GFP) under the transcriptional control of the smooth-muscle-specific SM22alpha promoter was used to make embryoid bodies,and to analyze the expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC by immunofluorescence stainings, RT-PCR and Western blot. Then AG1296 (PDGF receptor inhibitor) 0 micro-mol/L(control group), 10 micromol/L and 50 micromol/L were used to treat EBs respectively in order to analyze the differences of SMa-actin, SM22alpha, myocardin and SMMHC at gene and protein levels among the three groups. RESULTS: SMalpha-actin, myocardin, SM22alpha and SMMHC expression in EBs were found to begin at day 0 (ESCs), 8, 11, 13 respectively during early embryonic vascular development. There were no clear differences in SMa-actin, SM22alpha, myocardin and SMMHC protein expression and SM22alpha, myocardin and SMMHC mRNA level among the three groups of different concentrations of AG1296. CONCLUSION: A spontaneous VSMCs differentiation occurs during EBs development, SMalpha-actin is the first to be detected,the following are myocardin, SM22a and SMMHC. PDGF-BB may not be indispensable for the regulation of expression of VSMCs markers during early EBs differentiation.

Transgenic mice can express mutant human coagulation factor IX with higher level of clotting activity.

To improve the available values of transgenic animals, we produced a mutant human coagulation factor IX minigene (including cDNA and intron I) with arginine at 338 changed to alanine (R338A-hFIX) by using a direct mutation technique. The R338A-hFIX minigene was then cloned into a plasmid carrying the goat beta-casein promoter to get a mammary gland-specific expression vector. The clotting activity in the supernatant of the transfected HC-11 cells increased to approximately three times more than that of wild-type hFIX. Nine transgenic mice (three females and six males) were produced, and the copy number of the foreign gene was very different, ranging from 1 to 43 in different lines. ELISA, Western blot, and clotting assay experiments showed that the transgenic mice could express R338A-hFIX, showing higher average levels of clotting activity than wild-type hFIX in the milk (103.76% vs. 49.95%). The highest concentration and clotting activity of hFIX reached 26 mug/mL and 1287% in one founder (F(0)-7), which was over 10 times higher than that in human plasma. Furthermore, RT-PCR, APTT assay, and histological analysis indicated that hFIX was expressed specifically in the mammary gland without affecting the intrinsic coagulation pathway and physiologic performance of the local tissue.

Construction of an allogenic chimeric mouse model for the study of the behaviors of donor stem cells in vivo.

BACKGROUND: It is essential to establish an animal model for the elucidation of the biological behaviors of stem cells in vivo. We constructed a chimeric animal model by in utero transplantation for investigation of stem cell transplantation. METHODS: This chimerism was achieved by injecting the stem cells derived from the bone marrow of green fluorescence protein (GFP)-transgenic mice into fetal mice at 13.5 days of gestation. Several methods such as polymerase chain reaction (PCR), real-time PCR, fluorescence-assisted cell sorting (FACS) and fluorescence in situ hybridization (FISH) were used for the observation of donor cells. RESULTS: Under a fluorescence microscope, we observed the GFP cells of donor-origin in a recipient. PCR, FACS analysis and FISH indicated chimerism at various intervals. Real-time PCR indicated that some donor cells existed in chimera for more than 6 months. CONCLUSIONS: Allogenic stem cells may exist in recipients for a long time and this allogenic animal model provides a useful tool for studying the behavior of hematopoietic stem cells and also offers an effective model system for the study of stem cells.

[Prevalence of major external birth defects in high and low risk areas in China, 2003].

OBJECTIVE: To study the prevalence rates of birth defects in high and low risk areas in China. METHODS: A population-based surveillance system on birth defects was used to obtain the prevalence rates of 24 kinds of major external birth defects from > or = 20 weeks of gestation to 7 days of life in selected areas in Shanxi and Jiangsu provinces. RESULTS: The birth prevalence of birth defects (232.4 per 10,000 births) and neural tube defects (NTDs) (138.7 per 10,000 births) in four counties of Shanxi province were significantly higher than that in Taiyuan city (75.3 and 28.2 per 10,000 births, respectively). There was no significant difference for all selected birth defects between Wuxi city and Xishan counties in low risk areas. There was a 6.1-fold of higher prevalence for NTDs in Taiyuan city compared with that in Wuxi areas (4.6 per 10,000 births). In four counties of Shanxi province, the prevalence rates of anencephaly, spina bifida, hydrocephaly, cleft palate alone and polydactyly were significantly higher than in Wuxi areas. The NTDs prevalence rate in four counties of Shanxi was 30.2 times higher than in Wuxi areas. When compared with previous surveillance data, the NTDs prevalence rate did not present obvious declining trend in high risk areas. The birth prevalence rate had a 31.8% decrease when births were calculated after 28 gestational weeks and compared with those from 20 gestational weeks. CONCLUSION: NTDs remained to be the most common birth defect seen in Shanxi province. The birth prevalence rate of NTDs in some areas of Shanxi province was among the highest that ever reported in the world in comparison with data from other countries and regions. The current prevalence rate in high risk areas in Shanxi province did not clearly show a declining trend. Programs on surveillance and prenatal diagnosis were proved to have made big impact on the rates of major external birth defects.

In utero transplantation of human hematopoetic stem cells into fetal goats under B-type ultrasonographic scan: an experimental model for the study of potential prenatal therapy.

OBJECTIVE: Using fetal goats as animal models, to establish the methodology of in utero transplantation of human hematopoeitic stem cell (HSC) under B-scan ultrasonographic guidance for prenatal therapy. STUDY DESIGN: Human HSC were directly injected into the peritoneal cavities of the recipient fetal goats at 45-55 days of gestation (term: 145 days) under the guidance of B-type ultrasound scan. After birth, the peripheral blood was collected for fluorescence assisted cell sorting (FACS), quantitative real-time PCR and fluorescence in situ hybridization (FISH) to detect and analyze the presence of human cells in the recipients. RESULTS: The 32 recipients were born alive except one miscarriage. To test for the presence of human-goat chimeras, cells from 13 randomly selected transplanted goats were collected. FACS analyses showed the presence of human cells in all the transplanted goats tested. The average proportion of CD34+ cells and GPA+(glycophorin A) cells in the peripheral blood were 1.34 +/- 1.10% and 2.80 +/- 2.10%, respectively. No CD34+ or GPA+ cells were found in the non-transplanted goats tested. The results of the quantitative real-time PCR in three engraftment goats were 1.2 x 10(4), 2.9 x 10(4), and 3.2 x 10(4) copies of human GPA DNA per mug of genomic DNA. FISH experiments showed that cells containing human specific alpha-satellite DNA sequence were present in the peripheral blood of the transplanted goats. CONCLUSIONS: The method described herein is safe and reliable, with low miscarriage risk and high chimerism rate. This approach may provide a promising animal model for potential prenatal treatment.

[A parental case control study on the association between reduced folate carrier gene polymorphism and neural tube defects].

OBJECTIVE: To study the association between reduced folate carrier gene (RFC1 A80G) polymorphism and the risk for child with neural tube defects (NTDs), and to provide epidemiological evidence for the existence of NTDs genetic marker. METHODS: RFC1 (A80G) genotypes were detected using RFLP-PCR for blood DNA of 104 families with NTDs-affected children and 100 control families with no history of child-affected birth defects. Case-control study and transmission/disequilibrium test(TDT) for the RFC1 genotype of NTDs pedigree were carried out. RESULTS: The G allele frequency of children with NTDs was higher than that of controls when compared to A allele( OR = 1. 64, 95% CI :1.08-2.49). The offspring of the GG genotype were associated with a 2.56-fold increased risk of NTDs when compared to the AA genotype (OR = 2.56, 95% CI: 1.04-6.36) in our study population. There was evidence of association between G allele and the risk of parent having a child with NTDs (OR = 1.56, 95% CI: 1.07-2.28) in the TDT analysis. CONCLUSION: Our findings indicated that there was potential association between offspring RFC1 GG genotype and the risk of NTDs, and the G allele was a possible susceptible gene marker for an increased NTDs risk in the Chinese population.

Effects of human locus control region elements HS2 and HS3 on human beta-globin gene expression in transgenic mouse.

The locus control region (LCR) is the most important cis-element in the regulation of beta-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of beta-LCR. To examine the effect of HS2, HS3, and HS2-HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human beta-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by beta-globin promoter and under the control of HS2, HS3, and HS2-HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of beta-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2-HS3), the two cis-elements showed a marked synergy in regulating beta-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer beta-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same beta-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of beta-globin gene in the developmental stages and the switch of gamma-->beta-globin genes. Our results indicate that the mechanism of gamma-->beta switch could be best explained by the "divided model."

[Analysis of human cells in transplanted goats using fluorescence in situ hybridization].

OBJECTIVE: To analyze the existence and the dynamic cell frequencies of human cells in goats transplanted in utero with human hematopoietic stem cell (hHSC) by using fluorescence in situ hybridization (FISH) technique. METHODS: Interphase FISH (IFISH) with human-specific 17-chromosome satellite DNA and/or human-specific Y-chromosome satellite DNA as probes was performed to analyze the presence and proportions of human cells in 13 transplanted goats. Samples were peripheral blood cells, bone marrow smears and liver touch imprint preparations. RESULTS: Of the 13 transplanted goats, eleven were identified to present human cells. Among them, two goats transplanted with human male HSC were found to have human male cells. The results demonstrated that these transplanted goats were human/goat HSC xenogeneic chimeras. Human cell frequencies decreased with the goat age (months), but the longest survival reached 21 months. During the detected life periods of goats, human cell frequencies in peripheral blood, bone marrow and liver tissues were less than 1@1000, but local human cell frequencies of 207.92@1000 and 392.41@1000 were detected in the liver tissues of 2 transplanted goats. CONCLUSIONS: The existence and long-term survival of human cells in transplanted goats detected by FISH indicated that goats were appropriate recipients for hHSC in utero transplantation. The lower human cell frequencies in blood and bone marrow, and the higher local human cell frequencies in liver tissues suggested that the microenvironment of goat liver tissues might favor the survival, proliferation and differentiation of human cells.

A chimeric mouse model established by allogenic in utero transplantation.

In utero stem cells transplantation is a promising approach to the prenatal treatment of diseases. In order to investigate the fate of the stem cells after in utero transplantation, we have established a chimeric mouse model with the method of in utero transplantation. Mononuclear cells (including stem cells/progenitor cells) derived from male mouse bone marrow were injected into fetal mouse peritoneal cavity during the pre-immune period. The donor cells in the circulatory blood of female recipients were identified by fluorescence in situ hybridization (FISH), and the Y-chromosome specific DNA was detected by PCR as well as real-time quantitative PCR after the recipient mice were born. The results showed that a total of 4 female recipient mice were chimeric in their peripheral blood. Significantly, the donor cells in three chimeric mice persisted up to six months.

[High expression of human FIX(hFIX) in transgenic mice directed by goat beta-casein gene promoter].

To probe the feasibility of efficient production of human clotting factor IX(hFIX) with the approach of mammary gland bioreactor of transgenic animals, we constructed hFIX mammary gland expression vector containing promoter, exon 1, intron 1 and exon 2 of the goat beta-casein gene about 6.7 kb fragment as well as full-length of hFIX cDNA and its modified intron 1 sequence. By using transgenic products and 12 transgenic founders (9 female, 3 male) were produced, and the integration rate thus was 11.2%. ELISA assay and Western blot showed that the milk of 8 female transgenic mice had hFIX expression with high clotting activities. The highest hFIX expression in the milk of one transgenic mouse reached 52.9 mg/L, and the highest clotting activity of the transgene milk was 279.2%. FISH experiments indicated that hFIX DNA was integrated in different chromosomes in different mice. This result indicated that the hFIX mammary gland expression vector based on the goat beta-casein promoter can efficiently direct high expression of hFIX gene in the milk of transgenic mice, which maintained high clotting activity.

[Detection of the integration of human FIX (hFIX)on chromosomes of transgenic mice by fluorescence in situ hybridization].

Fluorescence in situ hybridization (FISH) was used to detect the integration of hFIX on chromosomes of transgenic mice from F1 to F4 generation in two strains. For transgenic mice, 98%-100% of metaphases and 85%-94% of interphases showed hybridization signal. For negative control mice,100% of metaphases and 95%-96% of interphases showed no hybridization signal. The results demonstrated that FISH developed to detect the integration sites of hFIX was high efficient and specific. The integration sites of the transgenic mice analyzed were both single but different between the two strains. The integration chromosomes can be found in the transgenic mice from F1 to F4 generation and the integration sites were the same as each of the strains,which indicated that the transgene was stably integrated and transmitted to offspring.