[The effect of ubiquitin ligase Cullin3 on the proliferation, migration and invasion of triple-negative breast cancer cells and exploration of its mechanism].

Objective: To investigate the effects of ubiquitin ligase Cullin3 (CUL3) on the proliferation, migration and invasion ability of triple-negative breast cancer (TNBC) cells and its mechanism of action. Methods: Bioinformatics-based methods were used to obtain CUL3 gene and protein expression data in TNBC tissues, and to assess the expression of CUL3 in tumour tissues of TNBC patients (n=160) and in normal breast tissues (n=572), and its relationship with clinical prognosis. The effects of overexpression of CUL3 on the proliferation, migration and invasion ability of TNBC cells in vitro were detected by CCK8 cell proliferation assay, scratch assay and transwell assay; proteins that might interact with CUL3 were screened by immunoprecipitation combined with mass spectrometry analysis, and the substrate protein regulated by CUL3 was identified as Glutathione S-Transferase Pi 1 (GSTP1); the effects of overexpression of GSTP1 on the migration and invasion ability of TNBC cells were detected by scratch assay and Transwell assay, and it was explored whether overexpression of CUL3 could reverse the effects of GSTP1 on the migration and invasion ability of cells; and the effects of overexpression of GSTP1 on the migration and invasion ability of cells were detected by Western blot and IP (Immunoprecipitation) to detect the effect of CUL3 on the ubiquitination modification of GSTP1 protein, and to verify the molecular mechanism by which CUL3 regulates the expression of GSTP1 to affect TNBC migration and invasion. Results: CUL3 expression was significantly higher in TNBC (P<0.000 1), and high CUL3 expression was closely associated with poor prognosis of TNBC patients (OS, P=0.018; RFS, P=0.008); overexpression of CUL3 significantly increased the proliferation of TNBC cells (F=11.97, P=0.002 for the 231-cell group, F=51.92, P<0.001 for the 468-cell group), migration [74.7±4.0 and 128.0±6.1 perforating cells in the overexpression groups of 231 and 468 cell lines, compared with 21.0±2.7 and 70.0±6.6 in the blank control (NC) group, and the t-values of 231 and 468 cell groups were-19.24 and-11.23, with P-values<0.001] and invasive ability (48 h cell proliferation rates were 56.6%±4.4% and 51.6%±3.7% in the 231 and 468 cell line overexpression groups, compared with 40.5%±2.9% and 32.9%±4.8% in the NC group, respectively, t=-5.26, P=0.006 3 in the 231 cell group; t=-5.38 in the 468 cell group, P=0.005 8); GSTP1 expression was reduced in TNBC, and up-regulation of GSTP1 inhibited TNBC cell migration (the number of membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines were 16.3±6.5 and 33.0±6.2, respectively, compared with 34.3±2.5 and 77.3±5.0 in the NC group, and t=5.44 in the 231 cell group, P=0.006; 468 cell group t=7.20, P=0.002) and invasion (48 h cell proliferation rates of 49.6%±1.7% and 36.2%±1.4% in the 231 and 468 cell line overexpression groups, compared to 59.4%±4.7% and 53.0%±1.7% in the NC group, t=3.42, P=0.027 in the 231 cell group; 468 cell group t=13.18, P<0.001), whereas up-regulation of CUL3 reversed the effects of GSTP1 on cell migration (37.0±1.0 and 67.0±5.3 membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines, respectively, 231 cell group t=-3.97, P=0.017; 468 cell group t=-6.12, P=0.004), and invasion (48 h cell proliferation rates of 71.9%±3.6% and 59.4%±2.1% in the 231 and 468 cell line overexpression groups, respectively, with t-values of -9.61 and -16.01 in the 231 and 468 cell groups, respectively, P-values<0.001) inhibitory effects; and CUL3, by increasing GSTP1 ubiquitylation modification, promotes ubiquitin-proteasome system to degrade GSTP1 protein, thereby reducing the stability of GSTP1 protein. Conclusion: Overexpression of CUL3 promotes TNBC development by promoting GSTP1 ubiquitination degradation inducing cell migration and invasion.

[Clinicopathological features of adult Wilms tumor with BRAF V600E mutation].

Objective: To identify BRAF V600E mutations in adult Wilms tumor (WT) with overlapping histologic features of metanephric adenoma (MA) and to investigate the clinicopathological features of adult WT. Methods: The clinical features of adult WT diagnosed at the Fudan University Shanghai Cancer Center, Shanghai, China from 2012 to 2021 were reviewed. HE-stained slides of all cases were reviewed by 2 expert pathologists. Representative tissues were selected for BRAF V600E immunohistochemical (IHC) staining and gene sequencing. Results: In adult WT with MA-like areas (cohort Ⅰ, n=6), 5 of the 6 cases were composed of epithelial-predominant and were positive for WT-1 and CD56, respectively, and all were positive for CD57. All 6 cases revealed highly variable Ki-67 indices, ranging from 1% in some areas to 60% in others. 5 of the 6 cases harbored a BRAF V600E mutation. All cases in cohort I were followed up for 23 to 71 months, and all survived. In classical adult WT without MA-like areas cohort (cohort Ⅱ, n=13), all 7 cases with available material were negative for BRAF by IHC and none of them had any BRAF mutation. Conclusions: BRAF V600E mutations are frequently present in adult WT with overlapping morphologically features of MA, but not in those without. More importantly, adult WTs with overlapping histologic features of MA may be an intermediate entity between typical MA and WT that may have a favorable prognosis and possible therapeutic targets.

[Analysis on impact of four major non-communicable diseases on life expectancy of local population in Ji'nan, 2015-2020].

Objective: To analyze the impact of malignant neoplasms, cardiovascular diseases, respiratory diseases and diabetes (four major chronic diseases) on the increase of life expectancy of local population in Ji'nan during 2015-2020. Methods: Based on 2015-2020 cause-of-death surveillance information and population data of Ji'nan, we used abbreviated life expectancy table and Arriaga decomposition method to analyze the overall and disease specific contributions of four major chronic diseases to the increase of life expectancy, and the gender and age specific contributions of mortality rates of four major chronic diseases to the increase of life expectancy of local population from 2015 to 2020. Results: Life expectancy of the local population in Ji'nan increased by 1.59 years from 2015 to 2020. The decline in mortality rates of the four major chronic diseases contributed 1.25 years, with a contribution of 78.62%. The life expectancy in men increased by 1.66 years, with a contribution of 1.18 years from the decline in mortality rate of four major chronic diseases, and the life expectancy in women increased by 1.52 years, with a contribution of 1.35 years from the decline in mortality of four major chronic diseases. The declines in the mortality rates of malignant neoplasms, cardiovascular diseases, respiratory diseases and diabetes contributed 0.42, 0.62, 0.20, and 0.01 years to life expectancy, respectively. The decline mortality rate of gastric cancer contributed more to the increase of life expectancy compared with lung cancer. The increase in the mortality rate of hypertensive heart disease resulted in a negative contribution. Asthma and diabetes contributed very little to the increase of life expectancy. Conclusions: The increase in the life expectancy of local population in Ji'nan from 2015 to 2020 was mainly attributed to the decline in mortality of four major chronic diseases. It is necessary to pay close attention to the diseases which contributed very little or even had negative contribution to the increase of life expectancy, such as lung cancer, diabetes and hypertensive heart disease.

[Evaluation of bladder function and sleep patterns in children with primary mono-symptomatic nocturnal enuresis by polysomnography combined with ambulatory urodynamic monitoring].

Objective: To evaluate the bladder function and sleep pattern in the children with primary mono-symptomatic nocturnal enuresis (PMNE) by the polysomnography (PSG) and ambulatory urodynamic monitoring (AUM). Methods: From October 2019 to October 2021, forty-three patients with PMNE were selected as PMNE group from the First Affiliated Hospital of Zhengzhou University and further subdivided into the severe PMNE group (enuresis>4 times/week) and the non-severe PMNE group (enuresis times 4 times/week) according to the severity. The conventional urodynamics (CUD), AUM, and PSG examinations and bladder diary were completed in the PMNE group. The control group consisted of 23 children with normal PSG findings and without the lower urinary tract symptoms. Results: The severe PMNE group included 9 males and 14 females, aged(12.1±3.2)years, and nocturnal enuresis number per week is 6.7±1.7. The non-severe PMNE group included 9 males and 11 females, aged(12.0±3.4)years, and nocturnal enuresis number per week is 2.3±1.0. The incidences of nocturnal polyuria and the reduction in maximum bladder capacity in the PMNE group was 34.9% and 11.6%, respectively. The incidence and frequency of detrusor overactivity (DO) in the severe PMNE group were significantly higher than those in the non-severe PMNE group [78.3% vs 45.0%, (5.5±1.8) times/h vs (3.4±1.0) times/h, respectively, all P<0.05]. It was found by the PSG that the severe PMNE group had significantly higher cortical arousal index, apnea hypopnea index (AHI), and percentage of N1+N2 phase in total sleep time, compared with the control group[(58.6±9.8)% vs (49.3±9.5)%, (9.4±4.4) times/h vs (3.1±1.5) times/h, (2.7±0.9) times/h vs (0.9±0.7) times/h] (all P<0.05). While the sleep efficiency of the severe PMNE group was substantially lower than that of the non-severe PMNE group [(86.4±4.3)% vs (91.0±3.9)%], the cortical arousal index and AHI were significantly greater than those of the non-severe PMNE group[(9.4±4.4) times/h vs (5.7±3.2) times/h, (2.7±0.9) times/h vs (1.9±0.7) times/h] (all P<0.05). In the PMNE group, there were positive correlations between cortical arousal index and nocturnal DO frequency or AHI (r=0.705, 0.765, P=0.001). Conclusions: Children with PMNE have nocturnal bladder dysfunction and abnormal sleep pattern, and there is a certain correlation between them. PSG and AUM are necessary for the evaluation and treatment of children with PMNE.

A practical study regarding the effect of adaptive roller-skating on emotion regulation ability of autistic children.

OBJECTIVE: Our aim is to analyze the effect of adaptive roller-skating on emotional regulation of autistic children. MATERIALS AND METHODS: Adaptive roller-skating course was designed for autistic children based on adaptive sports and A-B-A experiments. RESULTS: The adaptive roller-skating intervention focuses on improving emotional regulation ability, and directs the children to reasonably vent, recognize and stabilize their emotions. Adaptive roller-skating has a significant effect in intervening sadness, anger, anxiety and fear in autistic children; the intervention content setting and difficulty setting of the course have a certain impact on the intervention effect. Highly difficult and risky content can stimulate children. CONCLUSIONS: Adaptive roller-skating intervention course should obey the concept of adaptive movement in view of the differences between autistic children and the fun of roller-skating and guide the autistic children in emotion regulation with positive emotions.

[Outcomes at discharge of preterm infants born <34 weeks' gestation].

Objective: To investigate the incidence and trend of short-term outcomes among preterm infants born <34 weeks' gestation. Methods: A secondary analysis of data from the standardized database established by a multicenter cluster-randomized controlled study "reduction of infection in neonatal intensive care units (NICU) using the evidence-based practice for improving quality (REIN-EPIQ) study". This study was conducted in 25 tertiary NICU. A total of 27 192 infants with gestational age <34 weeks at birth and admitted to NICU within the first 7 days of life from May 2015 to April 2018 were enrolled. Infants with severe congenital malformation were excluded. Descriptive analyses were used to describe the mortality and major morbidities of preterm infants by gestational age groups and different admission year groups. Cochran-Armitage test and Jonckheere-Terpstra test were used to analyze the trend of incidences of mortality and morbidities in 3 study-years. Multiple Logistic regression model was constructed to analyze the differences of outcomes in 3 study-years adjusting for confounders. Results: A total of 27 192 preterm infants were enrolled with gestational age of (31.3±2.0) weeks at birth and weight of (1 617±415) g at birth. Overall, 9.5% (2 594/27 192) of infants were discharged against medical advice, and the overall mortality rate was 10.7% (2 907/27 192). Mortality for infants who received complete care was 4.7% (1 147/24 598), and mortality or any major morbidity was 26.2% (6 452/24 598). The incidences of moderate to severe bronchopulmonary dysplasia, sepsis, severe intraventricular hemorrhage or periventricular leukomalacia, proven necrotizing enterocolitis, and severe retinopathy of prematurity were 16.0% (4 342/27 192), 11.9% (3 225/27 192), 6.8% (1 641/24 206), 3.6% (939/25 762) and 1.5% (214/13 868), respectively. There was a decreasing of the overall mortality (P<0.001) during the 3 years. Also, the incidences for sepsis and severe retinopathy of prematurity both decreased (both P<0.001). However, there were no significant differences in the major morbidity in preterm infants who received complete care during the 3-year study period (P=0.230). After adjusting for confounders, infants admitted during the third study year showed significantly lower risk of overall mortality (adjust OR=0.62, 95%CI 0.55-0.69, P<0.001), mortality or major morbidity, moderate to severe bronchopulmonary dysplasia, sepsis and severe retinopathy of prematurity, compared to those admitted in the first study year (all P<0.05). Conclusions: From 2015 to 2018, the mortality and major morbidities among preterm infants in Chinese NICU decreased, but there is still space for further efforts. Further targeted quality improvement is needed to improve the overall outcome of preterm infants.

[Pathological features of primary extragonadal germ cell tumor of the prostate with small round blue cell morphology].

Objective: To investigate the clinicopathological features and clinical management of primary extragonadal germ cell tumor of the prostate. Methods: Two cases of primary extragonadal germ cell tumor in the prostate were collected at Fudan University Shanghai Cancer Center, in January and September 2016, respectively. Their pathological features, clinical treatments and follow-up were retrospectively analyzed. Results: The two patients were 41 and 32 years old, respectively, and both presented with obstructive symptoms of the lower urinary tract. Histologically, both cases showed small round blue cells and an invasive growth pattern. The immunohistochemistry (IHC) stains of SALL4, OCT3/4, CD117 and PLAP were all positive, while those of PSA, AR and syn were negative. Moreover, case 1 demonstrated perinuclear dot-like staining for CKpan, which might be a diagnostic pitfall. There was no evidence of disease in other areas via physical examination or radiographic studies. Based on these IHC findings and the morphology, the two cases were diagnosed as primary seminoma of the prostate, which were consequently treated with six cycles of bleomycin, etoposide and cisplatin-based chemotherapy. A complete response was achieved in case 1. Case 2 was followed up and showed tumor recurrence, and progression with elevated tumor marker AFP. The subsequent radical removed specimens of case 2 were finally diagnosed as mixed germ cell tumor of the prostate. Conclusion: As a rare neoplastic entity, primary germ cell tumor of the prostate can show small blue round cell morphology. Pathologically, the morphology of small round blue cells combined with a perinuclear dot-like pattern of CKpan IHC staining may be a diagnostic pitfall. The clinical treatment strategy should be evaluated with consideration of the pathological diagnosis and comprehensive evaluation of the tumor markers.

[Study on the influence of sleeve height and implant length on accuracy of static computer-assisted implant surgery].

Objective: To evaluate the influence of the sleeve lengths and implant lengths on accuracy of static computer-assisted implant surgery (sCAIS). Methods: Twenty-eight models of bilateral mandibular single tooth loss were included. Fifty-five implants were placed under the guidance of sCAIS (Straumann Bone Level 4.1 mm×10 mm). According to the height of metal sleeve of static guide plate, 55 implants were divided into 11 groups (free hand group, 1 mm group, 2 mm group, 3 mm group, 4 mm group, 5 mm group, 6 mm group, 7 mm group, 8 mm group, 9 mm group, 10 mm group), with 5 implants in each group. Eight research models were included. Group with 5 mm sleeve guides were used to place implants of different length, (Straumann Bone Level width 4.1 mm, height was 8 mm, 10 mm and 14 mm), 5 implants in each group. Eighteen patients with mandibular single tooth loss were included in the Department of Oral Implantology, Tianjin Stomatological Hospital from October 2018 to June 2019. There were 10 males and 8 females, 18-46(33.7±7.9) years old. A total of 18 implants were implanted and divided into 3 groups (free hand group, 3 mm group and 5 mm group) with 6 implants in each group. Digital software was used to compare the implant positions before and after implantation. Non-parametric Kruskal-Wallis test or one-way ANOVA were used to analyze the results. Results: There was no significant difference in implant vertical deviation between different sleeve height groups (1-10 mm) and free hand group, but the neck deviation in free hand group[(1.04±0.13) mm] was significantly higher than that in different sleeve height groups (1-10 mm) (P<0.05). The tip deviations of free hand group, 1 mm group and 2 mm group [(1.32±0.43), (0.83±0.10) and (0.78±0.11) mm, respectively] was significantly higher than that of 10 mm group [(0.31±0.14) mm](P<0.05). The angle deviation of free hand group and 1 mm group (3.99°±0.85° and 2.59°±0.69°), respectively] was significantly higher than that of 10 mm group (0.61°±0.03°) (P<0.05). The tip deviations of implants in the 14 mm group [(0.83±0.22) mm] was significantly higher than that in the 8 mm and 10 mm groups [(0.44±0.07) and (0.49±0.06) mm, respectively]. Clinical studies showed that there was no significant difference in neck deviation, tip deviation and angle deviation between 3 mm group and 5 mm group (P>0.05), but deviations were significantly lower than those in free hand group (P<0.05). Conclusions: The length of the sleeves has significant influence on the accuracy of the surgical guide. There was no significant difference in accuracy of the implant guide with 3 mm or 5 mm metal sleeves. The vitro study has some limitations and needs further systematic research.

[Strategy of schistosomiasis elimination and its effects in Jinhu County, Jiangsu Province].

OBJECTIVE: To understand the strategy of schistosomiasis elimination and its effects in Jinhu County, Jiangsu Province. METHODS: The data of schistosomiasis control in Jinhu County at different stages from 1970 to 2017 were collected and analyzed. RESULTS: From 1970 to 2017, there were three stages of schistosomiasis control, including transmission control, transmission interruption, and monitoring and elimination stages in Jinhu County. The main measures included Oncomelania hupensis snail control, infectious source control, and health education. A total of area of 290 691.78 hm2 was detected in Jinhu County, and the area with snails was 3 420.98 hm2. There were 8 729.37 hm2 area with snails was controlled. Since 2014, no O. hupensis snails were found. A total of 525 377 person-times were examined for schistosomiasis, with 2 815 schistosomiasis patients identified, and 2 844 person-times were treated by chemotherapy. In addition, 977 cases received the expand chemotherapy. Since 1990, no local schistosome-infected persons were found. In 2017, the awareness rate of schistosomiasis control knowledge and the correct rate of health behavior were increased by 54.59% and 14.23% respectively compared with those in 1992. CONCLUSIONS: The comprehensive schistosomiasis control measures implemented in Jinhu County at different periods have achieved remarkable outputs and accelerated the schistosomiasis elimination process. However, the precise control measures should be implemented in the future to consolidate the prevention and control achievements.

Effect of treating renal failure with decorin gene therapy.

OBJECTIVE: To explore a new approach for treating renal insufficiency with gene therapy by implanting decorin (DCN)-expressing fibroblasts within the renal tissue of rats with renal failure to neutralize TGF-ß1 activity. MATERIALS AND METHODS: The 5/6 kidney of the selected male SD rats were removed under aseptic conditions. The rats were grouped randomly after the establishment of the model. There were 10 rats in the sham-operated group (Group A), 10 in the operation control group (without treatment, Group B), 10 in the blank control group [treated with empty vector-transfected fibroblasts (FB (LXSN) cells), Group C], and 10 in the treatment group [treated with FB (LDCNSN) cells, Group D]. The pathological changes of rats including body weight, blood lipids, renal function, and renal histology, were observed. The expression of TGF-ß1 and DCN in renal tissue was detected by immunohistochemistry. RESULTS: There were no significant differences in body weight and blood lipids between the groups at 4 weeks after treatment. The levels of blood urea nitrogen and serum creatinine in rats in Group D were significantly decreased compared with those in Group C (p < 0.05). Although the differences were not statistically significant, the levels of those pathological indicators are higher than baseline values. The expression of DCN in renal tissue increased significantly after 4 weeks in rats of Group D and the differences were significant compared with the other groups. There were no significant differences in TGF-ß1 expression between any two groups of Group D, B, and C. Furthermore, pathological damage to the renal interstitium of rats in Group D was significantly decreased compared with that of Group B and C. CONCLUSIONS: DCN can alleviate fibrosis and delay the progression of renal failure.

SETD2-mediated crosstalk between H3K36me3 and H3K79me2 in MLL-rearranged leukemia.

Previously, we identified SETD2 loss-of-function mutations in 22% of MLL-rearranged (MLLr) acute leukemia patients, implicating a mechanism for cooperativity between SETD2 mutations and MLL fusions. However, the detailed mechanism of how SETD2-H3K36me3 downregulation accelerates MLLr leukemia remains unclear. Here, we show that in MLLr leukemia, both H3K79me2 and H3K36me3 are aberrantly elevated and co-enriched in a group of genes. SETD2 inactivation leads to a global reduction of H3K36me3 and a further elevation of H3K79me2, but does not change the expression of known MLL fusion target genes. Instead, this pattern of histone changes is associated with transcriptional deregulation of a novel set of genes; downregulating tumor suppressors (for example, ASXL1) and upregulating oncogenes (for example, ERG). Taken together, our findings reveal a global crosstalk between the oncogenic DOT1L-H3K79me2 axis and the tumor suppressive SETD2-H3K36me3 axis in gene regulation, provide molecular insights into how SETD2 mutations accelerate MLLr leukemogenesis through differential regulation of additional tumor suppressors and oncogenes.

Pollinators shift to nectar robbers when florivory occurs, with effects on reproductive success in Iris bulleyana (Iridaceae).

Studies have indicated that florivory and nectar robbing may reduce reproductive success of host plants. However, whether and how these effects might interact when plants are simultaneously attacked by both florivores and nectar robbers still needs further investigation. We used Iris bulleyana to detect the interactions among florivory, nectar robbing and pollination, and moreover, their effects on plant reproductive success. Field investigations and hand-pollination treatments were conducted on two experimental plots from a natural population, in which Experimental plot was protected from florivores and Control plot was not manipulated. The flower calyx was bitten by sawflies to consume the nectary, and three bumblebee species were pollinators. In addition, the short-tongued pollinator, Bombus friseanus, was the only robber when there was a hole made by a sawfly. The bumblebee had significantly shortened flower handling time when robbing, as compared to legitimate visits. Pollinator visitation and seed production decreased significantly in damaged flowers. However, seed production per flower after supplementary hand-pollination did not differ significantly between damaged and undamaged flowers. Compared to the Experimental plot, bumblebees visited fewer flowers per plant in a foraging bout in the Control plot. The flowers damaged by florivory allowed B. friseanus to shift to a nectar robber. Florivory and nectar robbing collectively decreased plant reproductive success by consuming nectar resources, which may reduce attractiveness to pollinators of the damaged flowers. However, the changes in pollinator behaviour might be beneficial to the plant by reducing the risk of geitonogamous mating.

Osteoblast differentiation of rabbit adipose-derived stem cells by polyethylenimine-mediated BMP-2 gene transfection in vitro.

The aim of this study was to examine the efficiency of polyethylenimine-mediated transfection of the human bone morphogenetic protein-2 (BMP-2) gene into rabbit adipose-derived stem cells (ADSCs), and its effect on osteoblast differentiation. Adipose tissue was isolated from the necks of adult Japanese white rabbits and cultured in vitro to obtain ADSCs. Gene delivery of BMP-2 was mediated by polyethylenimine and stable transformants were selected by G-418. The expression of BMP-2 mRNA was confirmed by reverse transcription-polymerase chain reaction, and of the BMP-2 protein by ELISA. Osteocalcin and collagen type I were detected by western blot and by an alkaline phosphatase kit. Alizarin red S stain was also utilized to examine osteogenesis. The non-transfected group was considered as a control. In this study, we successfully derived ADSCs from rabbit adipose tissue. Through passages 3-6, the expression of CD29 and CD44 gradually increased, whereas the expression of CD34 and CD45 gradually decreased. Both mRNA and protein expression of BMP-2 were confirmed following polyethylenimine-mediated BMP-2 gene delivery. In addition, the expression of alkaline phosphatase, osteocalcin, and collagen type I was found to be upregulated and alizarin red S staining was positive in transfected ADSCs, indicating BMP-2-induced osteogenesis. Therefore, this study determined that polyethylenimine was able to mediate BMP-2 gene delivery and induce osteogenic differentiation of ADSCs.

Development of EST-SSR markers in the relict tree Davidia involucrata (Davidiaceae) using transcriptome sequencing.

Davidia involucrata, reputed to be a "living fossil" in the plant kingdom, is a relict tree endemic to China. Extant natural populations are diminishing due to anthropogenic disturbance. In order to understand its ability to survive in a range of climatic conditions and to design conservation strategies for this endangered species, we developed genic simple sequence repeats (SSRs) from mRNA transcripts. In total, 142,950 contigs were assembled. Of these, 30,411 genic SSR loci were discovered and 12,208 primer pairs were designed. Dinucleotides were the most common (77.31%) followed by trinucleotides (16.44%). Thirteen randomly selected primers were synthesized and validated using 24 individuals of D. involucrata. The markers displayed high polymorphism with the number of alleles per locus ranging from 3 to 12 and the observed and expected heterozygosities ranging from 0.083 to 1.0 and 0.102 to 0.69, respectively. This large expressed sequence tag dataset and the novel SSR markers will be key tools in comparative studies that may reveal the adaptive evolution, population structure, and resolve the genetic diversity in this endangered species.

Development of expressed sequence tag-based microsatellite markers for the critically endangered Isoëtes sinensis (Isoetaceae) based on transcriptome analysis.

Isoëtes sinensis is a critically endangered quillwort. To facilitate studies on the conservation genetics of this species, we developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of 50,063 unigenes were predicted by transcriptome sequencing, 5294 (10.6%) of which significantly matched 3011 Gene Ontology annotations and 2363 were assigned to Kyoto Encyclopedia of Genes and Genomes metabolic pathways. Most of these (2297) were involved in metabolism. A total of 1982 SSR motifs were identified, with trinucleotides being the dominant repeat motif, and 1438 (72.6%) SSR primers were designed. Eighteen randomly selected primer pairs were used to genotype 24 I. sinensis accessions, which confirmed the suitability of these novel markers for molecular studies of I. sinensis. The heterozygosity index value ranged between 0.0799 and 0.9106, while the Shannon-Wiener diversity index value ranged between 0.1732 and 2.5589. The EST-SSRs reported in this study are linked to genic sequences, and are therefore ideal for investigating the evolutionary history of I. sinensis. These markers, together with the large EST dataset generated in this study, will greatly facilitate conservation genetic studies of I. sinensis.

Development of microsatellite markers in Hagenia abyssinica (Bruce) J.F. Gmel, an endangered tropical tree of eastern Africa, using next-generation sequencing.

Hagenia abyssinica (Bruce) J.F. Gmel is an endangered tree species endemic to the high mountains of tropical Africa. We used Illumina paired-end technology to sequence its nuclear genome, aiming at creating the first genomic data library and developing the first set of genomic microsatellites. Seventeen microsatellite markers were validated in 24 individuals. The average number of alleles per locus was 7.6, while the observed and expected heterozygosities ranged from 0.000 to 0.958 and from 0.354 to 0.883, respectively. These polymorphic markers will be used as tools for further molecular studies to facilitate formulation of appropriate conservation strategies for this species.

Low vaspin levels are related to endothelial dysfunction in patients with ankylosing spondylitis.

Vaspin is a novel adipocytokine associated with glucose tolerance and chronic inflammation. Some studies reveal that vaspin may be involved in cardiovascular diseases. Our objective was to investigate the relationship between serum vaspin levels and endothelial function in patients with ankylosing spondylitis. One hundred and twenty patients with newly diagnosed ankylosing spondylitis and 100 healthy subjects were studied. Serum vaspin levels were measured with enzyme-linked immunosorbent assay. High resolution ultrasound was used to measure brachial artery diameter at rest, after reactive hyperemia (flow-mediated dilation, FMD) and after sublingual glyceryltrinitrate. Serum vaspin level in patients was 1.92±1.03 ng/mL, which was significantly lower than that in healthy subjects (2.88±0.81 ng/mL). By dividing the distribution of serum vaspin levels into quartiles, FMD levels increased gradually with the increase of serum vaspin levels in patients (P<0.01). Univariate analysis showed a correlation between vaspin and FMD (r=0.73, P=0.003), low-density lipoprotein cholesterol (r=-0.45, P=0.033), high-density lipoprotein cholesterol (r=0.63, P=0.025), fasting blood glucose (r=-0.79, P=0.006), triglycerides (TG) (r=-0.68, P=0.036), systolic blood pressure (r=-0.35, P=0.021), C-reactive protein (r=-0.67, P=0.011), homeostatic model assessment of insulin resistance (HOMA-IR) (r=-0.77, P=0.023) and erythrocyte sedimentation rate (r=-0.88, P=0.039) in patients. Multivariate analysis indicated that serum vaspin levels were independently associated with FMD, HOMA-IR and TG in patients. Our study found that serum vaspin levels were decreased in patients with ankylosing spondylitis and were associated with FMD levels. Vaspin may serve as an independent marker for detecting early stage atherosclerosis in patients with ankylosing spondylitis.

Association of CD59 and CFH polymorphisms with acute anterior uveitis in Chinese population.

PurposeCD59 complement regulator and complement factor H (CFH) have important roles in complement activation pathways, which are known to affect the development of uveitis. The present study was performed to investigate whether an association exists between CD59 and CFH genetic polymorphisms and acute anterior uveitis (AAU).MethodsA total of 600 individuals (300 patients diagnosed with AAU and 300 healthy controls) were recruited for this case-control study. Five single-nucleotide polymorphisms (SNPs) in CD59 (rs831626, rs12272807, rs831625, rs11585, and rs12576440) and CFH-rs1065489 were genotyped using Sequenom MassARRAY technology. Allele and genotype frequencies were statistically compared between patients and controls using χ2 test. Analyses were stratified for gender, human leukocyte antigen (HLA)-B27, and ankylosing spondylitis (AS) status.ResultsNo significant association was found between any of the six polymorphisms and AAU. In HLA-B27-negative AAU patients, the frequencies of the G allele and GG homozygosity were lower in CD59-rs831626 when compared with controls (P=0.032). There were also significant decreases in the frequencies of T allele and TT homozygosity in CFH-rs1065489 in AAU patients with AS compared with controls (P=0.002). Furthermore, the frequencies of the T allele and TT homozygosity in CFH-rs1065489 were lower in the AAU male patients with AS compared with controls (P=0.015).ConclusionOur results revealed that SNPs CD59-rs831626 and CFH-rs1065489 were associated with the susceptibility of AAU. The influence on AAU could be gender specific and dependent on the HLA-B27 and AS status. No positive results were found in the overall group.