[Prevalence of CYP2C19 gene mutations in patients with coronary heart disease and its biological activation effect in clopidogrel antiplatelet response].

Objective: The purpose of this study was to investigate the effects of CYP2C19 gene mutations on clopidogrel antiplatelet activity in the patients with coronary heart disease treated by percutaneous coronary intervention. Methods: Patients with coronary heart disease, who hospitalized in the Second Affiliated Hospital of Nanchang University from March 2011 to June 2019, and healthy individuals with matching genetic background, gender, and age as controls were included in this study. Basic clinical data were analyzed and blood samples of all research subjects were obtained for extraction of DNA, and Sanger first-generation sequencing method was used to detect CYP2C19 gene mutation from full exon and exon and intron junction. CYP2C19 gene variations in patients with coronary heart disease were compared with the 1000 Genomes Browse database and the sequencing results of healthy controls to determine whether the gene variation was a genetic mutation or a genetic polymorphism. After that, PolyPhen-2 prediction software was used to analyze the harmfulness of gene mutations to predict the effect of mutations on protein function. The same dose of CYP2C19 wild-type plasmid and the CYP2C19 gene mutant plasmids were transfected into human normal liver cells HL-7702. After transfection of 24 h, the expression of CYP2C19 protease in each group was detected. The liver S9 protein was incubated with clopidogrel, acted on platelets to detect the platelet aggregation rate and the activity of human vasodilator-activated phosphoprotein (VASP). Results: A total of 1 493 patients with coronary heart disease (59.36%) were enrolled, the average age was (64.5±10.4) years old, of which 1 129 were male (75.62%). Meanwhile, 1 022 healthy physical examination volunteers (40.64%) were enrolled, and the average age was (64.1±11.0) years old, of which 778 were male (76.13%). A total of 5 gene mutations of CYP2C19 gene were identified in 12 patients (0.80%), namely, 4 known mutations T130K (1 case), M136K (6 cases), N277K (3 cases), V472I (1 case) and one new mutation G27V (1 case), no corresponding gene mutation was found in healthy controls. It was found that T130K and M136K were probably damaging, G27V was possibly damaging, and N277K and V472I were benign mutations. In vitro, we demonstrated that the platelet aggregation rate of the M136K gene mutation group was 24.83% lower than that of the wild type (59.58% vs. 34.75%; P<0.05), and the phosphorylated VASP level was 23.0% higher than that of the wild type (1.0 vs. 1.23; P<0.05). However, the platelet aggregation rate and phosphorylated VASP level were similar between of G27V, T130K, N277K, V472I gene mutation groups and wild type group (P>0.05). Conclusions: In this study, 5 gene mutations are defined in patients with coronary heart disease, namely G27V, T130K, M136K, N277K, V472I. In vitro functional studies show that CYP2C19 gene mutation M136K, as a gain-of-function gene mutation, can enhance the activation of CYP2C19 enzyme on clopidogrel, thereby inhibiting the platelet aggregation rate.

A novel multiplex PCR assay for rapid detection of virulent Aeromonas in cultured eels.

AIMS: Under intensive and stressful aquaculture conditions, cultured eels are highly susceptible to virulent Aeromonas sp. infections. To rapidly and simultaneously confirm Aeromonas isolate and its virulence, a two-tube multiplex PCR (mPCR) assay incorporating gyrB gene for genus-specific recognition and seven major virulence genes for virulence assessment was developed. METHODS AND RESULTS: Eight pairs of primers were designed and divided into two groups-gyrB, ahpA, epr and aerA in tube 1 and alt, act, ast and hlyA in tube 2. The optimized mPCR conditions were the same except for their final concentrations. The specificity of the mPCR was validated by the extracted DNA of 10 Aeromonas and 8 non-Aeromonas species, or mixed DNA templates. Detection limits were determined to be 200 copies per µl in tube 1 and 20 copies per µl in tube 2. The mPCR reproducibility was tested by both artificial challenge and clinical samples. CONCLUSIONS: The results showed this two-tube mPCR assay was rapid, specific, sensitive and reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report to distinguish virulent Aeromonas isolates from nonvirulent ones by seven popular and major virulence genes at the genus-specific level. And it will be useful for large-scale screening of virulent Aeromonas sp. in cultured eels.

[Effect of different parts of skull thickness on stereotactic electroencephalogram in children].

Objective: To investigate the thickness of cranial bone in different parts of children skull during stereotactic electroencephalogram (SEEG) and its effect on electrode fixation. Methods: From October 2016 to March 2017, 13 children with SEEG by robot of surgery assistant (ROSA) were selected. The basic case information and electrode design scheme were collected. The skull thickness of each electrode channel was measured on post-operation CT, and the loosening of the fixed screws were recorded. The thickness of skull in frontal bone, temporal bone, parietal bone and occipital bone was statistically processed by SPSS statistical software. Results: There were total 113 electrodes in 13 children with epilepsy. There were 45 electrodes at frontal bone, of which the thickness was (5.7±2.8)mm. There were 34 electrodes at temporal bone, of which the thickness was (3.5±1.3)mm.There were 16 electrodes at parietal bone, of which the thickness was (6.0±2.5)mm.There were 18 electrodes at occipital bone, of which the thickness was (6.9±0.5)mm. Statistics showed that there was significant difference between differnt bone (F=15.340, P<0.01). There were 4 electrodes loosening, 1 at frontal bone and 3 at temporal bone, when the screws were removed. There was no adverse event related to the implantation of electrodes. Conclusions: The children's skull thickness is thinner than adults. The screw loosening is exist in some cases, but it has no effect on SEEG recording. No SEEG related adverse events are found in this group. Therefore, ROSA guided SEEG is safe and reliable in children with epilepsy.

[Role of nuclear factor of activated T-cells cytoplasmic 1 on vascular calcification in rats with chronic renal failure].

Objective: To explore the role of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) on vascular calcification in chronic renal failure rats. Methods: Nineteen male Sprague-Dawley (SD) rats were randomly divided into three groups: sham-operated group (n=6), 5/6 Nephrectomy (Nx) group (n=6), 5/6 Nx+ calcitriol group (n=7). Vascular calcification was determined by von Kossa staining and orthocresolphthalein complexone (OCPC) method. Protein expressions of NFATc1 and runt-related transcription factor 2 (Runx2) in aortas were measured by immunohistochemistry.In vitro, vascular smooth muscle cells (VSMCs) were primarily cultured and calcification was induced by ß-glycerophosphate (ß-GP). These cells were then randomly divided into control group, calcification group (10 mmol/L ß-GP) and cyclosporin A (CsA) intervention group (10 mmol/L ß-GP+ 1 µg/ml CsA). Calcium deposition was measured by Alizarin red staining and OCPC method; alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. RT-PCR and Western blotting were used to observe the mRNA and protein expression of VSMCs NFATc1 and Runx2 respectively. Results: Compared to that in sham-operated and 5/6 Nx group, the expression of NFATc1 was obviously up-regulated in 5/6 Nx+ calcitriol group (7.20±0.46 vs 1.52±0.77, 2.04±1.31, P<0.05). In vitro, VSMCs calcification was successfully induced by high phosphorus environment, and RT-PCR and Western blotting showed that the expressions of NFATc1 and Runx2 were up-regulated (P<0.05). The calcification level in CsA intervention group was lower than that in calcification group [(60.86±7.95) vs (107.20±11.07) mg/g, P<0.05], and expression of Runx2 (mRNA and protein level) and ALP activity [(48.63±3.02) vs (98.75±3.46) U/g, P<0.05] decreased as well. Conclusion: NFATc1 contributes to accelerating vascular calcification in rat with chronic renal failure, the possible mechanism of which is that NFATc1 promotes VSMCs transformation to osteogenic phenotype.

[Effect and mechanism of intermittent alkaline stimulation on high phosphorus induced calcification in vascular smooth muscle cells of rats].

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L ß-glycerophosphate and alkalized by 7.4% NaHCO(3) to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel ß(3) subunit(LTCC ß(3)) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC ß(3) were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.5 group (P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC ß(3) were higher in high phosphorus+ pH7.7 group (all P<0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P<0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P<0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P<0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P<0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P<0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P<0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P<0.05). Conclusion: Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC ß(3) subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

[Polymorphism at the miR-502 binding site in the 3' untranslated region of SET8 gene is associated with the risk of clear cell renal cell carcinoma].

OBJECTIVE: To investigate the relationship between single nucleotide polymorphism of SET8 gene and the risk of clear cell renal cell carcinoma (CCRCC). METHODS: We selected 140 CCRCC patients and 130 healthy controls in this case-control study.Genotype of single nucleotide polymorphism (rs16917496) at the miR-502 binding site in the 3'UTR of SET8 mRNA in the CCRCC patients and healthy controls was tested and the association between genotype and risk of cancer was assessed. The expression of SET8 was determined by immunohistochemistry and the relationship between expression of SET8 and genotype of rs16917496 was analyzed. RESULTS: In the control group, CC, CT and TT genotypes were found in 30, 32 and 68 persons, respectively, while in the CCRCC patients, CC, CT and TT genotypes were found in 14 , 47 and 79 cases, respectively.The frequencies of rs16917496 CT and TT genotypes in the CCRCC group were significantly higher than those in the control group (P<0.05). Compared with the CC genotype, patients with CT and TT genotypes were more susceptible to develop CCRCC (P<0.05). CT and TT genotypes of rs16917496 at the miR-502 binding site of the SET8 gene were associated with expression of SET8. CONCLUSIONS: Genotype of the SNP rs16917496 at the miR-502 binding site in the 3' untranslated region of the SET8 gene is associated with the expression of SET8 protein. Analysis of genetic polymorphisms in miRNA binding sites may help to identify the subgroups of population susceptible to CCRCC.

[Effects of intermediate conductance calcium-activated potassium channel blocker TARAM-34 on ß-glycerophosphate induced vascular smooth muscle cells calcification].

OBJECTIVE: To observe the role of TRAM-34 (1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole), the blocker of intermediate conductance calcium-activated potassium channel (KCa3.1), on ß-glycerophosphate induced vascular calcification in vitro. METHODS: Vascular smooth muscle cells(VSMCs) were obtained from rat thoracic aorta, and VSMCs after the fourth passage and aortic rings were divided into control group (cultured in DMEM with 10% fetal bovine serum), high phosphorus group (cultured in DMEM with 10% fetal bovine serum and 10% ß-glycerophosphate) and TRAM-34 group(20 nmol/L TRAM-34 was added into high phosphorus DMEM). Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method.Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM.The expression of runt-related transcription factor 2(Runx2)was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings.ALP activity was measured by alkaline phosphatase activity detection kit. RESULTS: (1) Compared with control group, calcification was significantly increased in high phosphorus group ((121.67±6.17) mg/g vs. (84.38±8.17) mg/g, P<0.05) and this effect could be attenuated by TRAM-34 ((93.31±11.36) mg/g, P<0.05 vs. high phosphorus group) after 12 days culture. Similar results were found in aortic rings cultured for 12 days-high phosphorus group: (7.17±0.57) mg/g vs. CONTROL: (1.18±0.13) mg/g (P<0.05) and TRAM-34: (4.71±0.42) mg/g, P<0.05 vs. high phosphorus group.(2) Compared with control group, the calcium influx was higher in high phosphorus group (349.22±40.47 vs. 151.67±16.94, P<0.05) and reduced in TRAM-34 group (194.67±22.21, P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days. (3) Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group (0.630±0.033 vs.0.340±0.058 and 0.865±0.031 vs.0.414±0.011, both P<0.05) and lower in TRAM-34 group (0.399±0.023 and 0.575±0.014, both P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days.Besides, compared with high phosphorus group, the expression of Runx2 was decreased in control group(0.113±0.010 vs.0.067±0.008, P<0.05) and TRAM-34 group (0.069±0.006, P<0.05) after aortic rings were cultured for 4 days. (4) Compared with control group, the activity of ALP was significantly increased in high phosphorus group (96.56±9.84 vs.46.92±4.60, P<0.05) and decreased in TRAM-34 group(70.20±8.41, P<0.05 vs. high phosphorus group) in VSMCs simulated for 12 days. CONCLUSION: KCa3.1 blocker TRAM-34 can inhibit ß-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx, downregulating Runx2 expression and attenuating osteogenic differentiation.

Epidemiology of Mycoplasma acquisition in male HIV-1 infected patients: a multistage cross-sectional survey in Jiangsu, China.

Mycoplasma infections are most frequently associated with disease in the urogenital or respiratory tracts and, in most cases, mycoplasmas infect the host persistently. In HIV-infected individuals the prevalence and role of genital mycoplasmas has not been well studied. To investigate the six species of Mycoplasma and the risk factors for infection in Jiangsu province, first-void urine and venous blood samples were collected and epidemiological questionnaires were administered after informed consent. A total of 1541 HIV/AIDS patients were recruited in this study. The overall infection rates of six Mycoplasma species were: Ureaplasma urealyticum (26·7%), Mycoplasma hominis (25·3%), M. fermentans (5·1%), M. genitalium (20·1%), M. penetrans (1·6%) and M. pirum (15·4%). The Mycoplasma infection rate in the unmarried group was lower than that of the married, divorced and widowed groups [adjusted odds ratio (aOR) 1·432, 95% confidence interval (CI) 1·077-1·904, P < 0·05]. The patients who refused highly active antiretroviral therapy (HAART) had a much higher risk of Mucoplasma infection (aOR 1·357, 95% CI 1·097-1·679, P < 0·05). Otherwise, a high CD4+ T cell count was a protective factor against Mycoplasma infection (aOR 0·576, 95% CI 0·460-0·719, P < 0·05). Further research will be required to confirm a causal relationship and to identify risk factors for Mycoplasma infection in HIV/AIDS populations.

Lack of estrogen down-regulates CXCR4 expression on Treg cells and reduces Treg cell population in bone marrow in OVX mice.

Postmenopausal osteoporosis (PMO) is the most common metabolic bone disease in women after menopausal. Recent works focused on cross—talk between immune regulation and bone metabolism pathways and suggested Treg cells suppressed bone resorption and osteoclasts (OC) differentiation in bone marrow via cell—cell contact interaction and/or secreting of IL—10 and TGF—beta. In this study, we investigated the impact of estrogen on regulatory T cells (Treg cells) trafficking and staying in bone marrow and we found that a significant reduction of Treg cell population in bone marrow in estrogen deficiency ovariectomied (OVX) mice. We then studied the expressions of chemokines CXCL12/CXCR4 axes, which were critical to Treg cells migration and our data show the expression of CXCR4 on Treg cells was relative with oestrogen in vivo, however, the expression of CXCL12 was not. Furthermore, the loss of trafficking ability of Treg cells in OVX mice was recoverable in our system. These findings may mechanistically explain why Treg cells lose their suppressive functions on the regulation of OC cells and demonstrate a previously unappreciated role for estrogen, which may be critical to the novel therapy in clinical practice of PMO patients.

Alarming incidence of genital mycoplasmas among HIV-1-infected MSM in Jiangsu, China.

Males who have sex with men (MSM) are considered at high risk of blood-borne and sexually transmitted infections (STIs), mainly due to the practice of unsafe sex, often combined with drug use and needle-sharing. A cross-sectional study was designed for the detection of genital mycoplasmas during the period from March 2009 to May 2010 in Jiangsu province. This work was approved by the Research ethics Committee of Jiangsu Centers for Diseases Prevention and Control (CDC), and written consent was obtained from all participants. In total, 243 human immunodeficiency virus-1 (HIV-1)-infected MSM were screened in this study. Over half of them reported a history of sexual activity with females (65.0 %), and 26.3 % reported a history of sexually transmitted diseases (STDs) other than HIV. 44.0 % of patients were in the first 2 years of their HIV infection, and 72.4 % were still in HIV progression. Of the 243 analyzed samples, all were positive for at least one kind of mycoplasma. The infection rates of Mycoplasma genitalium, M. fermentans, M. penetrans, and M. pirum were 25.5, 9.9, 2.5, and 18.5 %, respectively. The M. genitalium infection was associated with a history of sexual activity with females, and those who had sex with females showed higher infection rates. Six M. penetrans-positive patients were still in HIV infection progression and did not receive highly active antiretroviral therapy (HAART). Men who perform this particular behavior are at higher risk of Mycoplasma infections. Further molecular and epidemiological cohort studies with larger populations are needed in order to identify the role of Mycoplasma infections in HIV-1-infected MSM.

First Report of Fusarium Maize Ear Rot Caused by Fusarium meridionale in China.

Fusarium is an important genus of fungal pathogens that are responsible for devastating diseases, such as Fusarium ear rot on maize, which may result in yield losses and/or mycotoxin contamination. In September 2013, a survey to determine population composition of Fusarium species on maize was conducted at 22 fields in 18 counties in Gansu Province. Maize ears with clear symptoms (with a white to pink- or salmon-colored mold at the ear tip) were collected. Symptomatic seeds were surface-sterilized with 70% ethanol and 10% sodium hypochlorite and rinsed three times with sterile water to eliminate hypochlorite residues. After drying on sterile filter paper, the seeds were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 3 days. Mycelium that was characteristic of Fusarium spp. (2) was purified by transferring single spores to fresh PDA. Fusarium species were identified by morphological characteristics (2), multilocus genotyping assay (MLGT) (3), and sequence analysis of the translation elongation factor-1α (TEF) gene. Several Fusarium species were identified and Fusarium verticillioides and F. proliferatum were the predominant species. Based on MLGT, two strains from Chenghong County were identified as F. meridionale with NIV chemotype, a species in F. graminearum species complex (FGSC). Morphological characteristics were also identical to FGSC. Colonies grew rapidly on PDA and produce relatively large amounts of dense mycelia and red pigments. Slender, thick-walled, and moderately curved or straight macroconidia were observed with 5- to 6-septate. Furthermore, conidia on SNA also showed typical characteristics of F. meridionale, as the dorsal and ventral lines were often parallel and gradually curved. Sequences comparison of the partial translation elongation factor (TEF-1α, 644 bp) gene (1) was used to validate these observations. BLASTn analysis with the FUSARIUM-ID database revealed 100% sequence identity to F. meridionale (GenBank Accession No. KJ137017). Thus, both morphological and molecular criteria supported identification of the strains as F. meridionale. A pathogenicity test was performed on Zhengdan958, the maize variety with the largest planted acreage in China. Four days after silk emergence, 2 ml conidial suspension (105 macroconidia/ml) of each isolate were injected into each of 10 maize ears through silk channel. Control plants were inoculated with sterile distilled water. Typical FER symptoms (reddish-white mold) was observed on inoculated ears and no symptoms were observed on water controls. Koch's postulates were fulfilled by re-isolating the same fungus from the infected seeds. F. meridionale was one of the pathogens causing Fusarium head blight on wheat and barley in China and produced nivalenol (4,5) and it also has been isolated from maize in Korea and Nepal. To our knowledge, this is the first report of F. meridionale causing Fusarium ear rot on maize in China. Further studies on biological characteristics such as temperature sensibility and fungicide resistance are needed to gain a better understanding of this new pathogen. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) T. J. Ward et al. Fungal Genet. Biol. 45:473, 2008. (4) L. Yang et al. Phytopathology 98:719, 2008. (5) H. Zhang et al. Plos one 7:e31722, 2012.

First Report of Fusarium temperatum Causing Fusarium Ear Rot on Maize in Northern China.

In China, several diseases of maize (Zea mays L.) including ear rot are caused by Fusarium spp., leading to significant yield losses and potential risk of mycotoxin contamination (2,3). In 2013, a survey was conducted to determine the population composition of Fusarium species on maize ears in Jilin Province. Symptomatic maize ears with pink or white mold were collected and surface disinfested with 70% ethanol and 10% sodium hypochlorite, followed by three rinses with sterile distilled water and placed onto potato dextrose agar (PDA). After 3 days of incubation at 25°C in the dark, newly grown-out mycelia were transferred onto fresh medium and purified by the single-spore isolation method (4). Fusarium spp. were identified by morphological characteristics (2) and sequence analysis of translation elongation factor-1α (TEF) gene (1). A large number of Fusarium spp. were found including F. graminearum species complex and F. verticillioides. In addition, a new species, F. temperatum, recently described in Belgium (2), was also identified. F. temperatum was originally described as F. subglutinans, but a robust polyphasic approach proved it to be a new biological species closely related to F. subglutinans (2). Previous studies had reported ~15% of Fusarium maize ear rot in Jilin was F. subglutinans. In this study, we found both F. subglutinans s. str. and F. temperatum in the proportion of 16.3% and 9.2%, respectively. Similar to previous studies (2), colonies of our strains on PDA were initially white cottony mycelium that become pinkish white. Conidiophores formed abundantly on SNA that were erect, branched, and terminated in 1 to 3 phialides. Microconidia were abundant, hyaline, 0 to 2 septa, obovoid to oval, and not produced in chains. Chlamydospores were absent. Typically macroconidia were falcate, 3 to 5 septate (mostly 4 septate), hyaline with a curved and blunt apical cell and a distinct foot-shaped basal cell. In order to validate this result, partial translation elongation factor (TEF-1α, 629 bp) gene sequences of isolates were generated (GenBank Accession No. KJ137018) (1). BLASTn analysis revealed 100% sequence identity to F. temperatum (HM067690). A pathogenicity test was performed on maize cv. Zhengdan958. Four days after silk emergence, 2 ml conidial suspension (105 macroconidia/ml) of each isolate was injected into each of 10 maize ears through silk channel. Control plants were inoculated with sterile distilled water. Twenty days after inoculation, typical Fusarium ear rot symptoms (reddish-white mold) was observed on inoculated ears and no symptoms were observed on water controls. Koch's postulates were fulfilled by re-isolating the same fungus from the infected seeds. Although F. temperatum was reported to attack maize kernels in southern China where the annual average temperatures are moderately high (3), to our knowledge, this is the first report of F. temperatum causing Fusarium ear rot in northern China, where the winter is long and very cold, the annual average temperature is 4 to 5°C, and the lowest temperature is lower than -35°C. This indicated that F. temperatum was widely distributed in different ecological regions in China. Furthermore, the northeast spring corn region that includes Jinlin is the most important corn belt, with corn production of this region accounting for 42% of the total corn production in China. Therefore, we should pay more attention to the new species in this region and consider them in the development of maize cultivars with broad-based resistance to the pathogens. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) J. Scauflaire et al. Mycologia 103:586, 2011. (3) J. H. Wang et al. J. Phytopathol. 162:147, 2014. (4) L. Yang et al. Phytopathology 98:719, 2008.

First Report of Fusarium Ear Rot of Maize Caused by Fusarium andiyazi in China.

Maize (Zea mays L.) is an important food crop worldwide. Some Fusarium species cause maize ear rot leading to significant yield losses and, for some Fusarium species, potential risk of mycotoxin contamination. In 2013, a survey was conducted to determine the population composition of Fusarium species on maize in Dongyang, Zhejiang Province, China, where about 5% of maize ears in each field were found with reddish-white mold. Symptomatic maize ears were collected from several cultivars including forage corn Zhedan724 and Zhengdan958, sweet corn Chaotian4 and Chaotian135, and waxy corn Heinuo181 and Zhenuoyu6; no association between the disease and maize cultivars was observed. Maize kernels showing a pink or white mold were surface-disinfested with 70% ethanol and 10% sodium hypochlorite, followed by three rinses with sterile distilled water and placed onto potato dextrose agar (PDA). After 3 days of incubation at 25°C in the dark, mycelia were transferred to fresh PDA and purified by the single-spore isolation method (4). Species were identified based on morphological characteristics (2), and sequence analysis of the translation elongation factor-1α (TEF) gene. The results indicated that Fusarium verticillioides Sacc. (84.6%) is the main causal agent of maize ear rot in this region. However, morphological characteristics of two strains (7.7%) from the same field were found to be identical to F. andiyazi Marasas, Rheeder, Lampr., K.A. Zeller & J.F. Leslie. Colonies on PDA showed floccose to powdery mycelium and pale-purple pigmentation. Hyaline and straight or slightly curved macroconidia were observed with 3- to 6-septate and a slightly curved apical cell. Chlamydospores were absent. In order to validate this result, partial translation elongation factor (TEF-1α, 646 bp) gene sequences of isolates were generated (GenBank Accession No. KJ137019) (1). BLASTn analysis of TEF-1α with the GenBank database revealed 99.7% sequence identity to F. andiyazi (JN408195 and JN408196), and much lower (94 to 98%) identity with other Fusarium spp. Thus, both morphological and molecular criteria supported identification of the strains as F. andiyazi. A pathogenicity test was performed on maize cv. Zhengdan958 in a greenhouse. Four days post-silk emergence, a 2-ml conidial suspension (105 macroconidia/ml) of each isolate was injected into each of 10 maize ears through the silk channel. An equal amount of sterile distilled water was injected into 10 ears as a control. Typical Fusarium ear rot symptoms (reddish-white mold), which were observed in the ears inoculated with these strains 20 days after inoculation, were similar to the original symptoms in the sampling sites, and no symptoms were observed on the water control ears. The same fungus was re-isolated from the infected kernels using the method described above. F. andiyazi are the major pathogens of sorghum (2) and also proved to attack maize kernels recently (3). To our knowledge, this is the first report of F. andiyazi causing Fusarium ear rot on maize in China. Further investigation is needed to gain a better understanding of the spatial and temporal dynamics of this new pathogen. Also, the new species must be considered in the development of maize cultivars with broad-based resistance to the pathogens. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) A. Madania et al. J. Phytopathol. 161:452, 2013. (4) H. Zhang et al. PLoS ONE 7:e31722, 2012.

Helical distraction is superior to linear and circular distraction in mandibular distraction osteogenesis: an in silico study.

Helical mandibular distraction is theoretically better than linear or circular distraction. However, it is not known whether this more complex treatment will result in unquestionably better outcomes. Therefore, the best attainable outcomes of mandibular distraction osteogenesis were evaluated in silico, given the constraints of linear, circular, and helical motion. This cross-sectional kinematic study included 30 patients with mandibular hypoplasia who had been treated with distraction, or to whom this treatment had been recommended. Demographic information and the computed tomography (CT) scans showing the baseline deformity were collected. The CT scans of each patient were segmented and three-dimensional models of the face created. Then, the ideal distraction outcomes were simulated. Next, the most favorable helical, circular, and linear distraction movements were calculated. Finally, errors were measured: misalignment of key mandibular landmarks, misalignment of the occlusion, and changes in intercondylar distance. Helical distraction produced trivial errors. In contrast, circular and linear distractions resulted in errors that were statistically and clinically significant. Helical distraction also preserved the planned intercondylar distance, while circular and linear distractions led to unwanted changes in the intercondylar distance. It is now evident that helical distraction offers a new strategy to improve the outcomes of mandibular distraction osteogenesis.

Therapeutic effect of micropump intravenous infusion of ambroxol hydrochloride on respiratory distress syndrome in premature infants.

OBJECTIVE: This work aimed to explore the therapeutic effect of micropump intravenous infusion of ambroxol hydrochloride (AH) on respiratory distress syndrome (RDS) in premature infants. PATIENTS AND METHODS: 56 premature infants from 28 to 34 weeks were recruited for analysis in this work. According to the treatment methods, they were randomly divided into two groups, with 28 patients in each group. Patients in the experimental group were given intravenous AH by micropump, while those in the control group inhaled atomized AH. The therapeutic effects were evaluated by comparing the data after treatment. RESULTS: The results showed that the serum 8-iso-PGP2α level in the experimental group was 166.32 ± 49.52, which was substantially inferior to that in the control group (183.32 ± 52.54), p < 0.05. In the experimental group, PaO2, SaO2, and PaO2/FiO2 were 95.88 ± 12.82 mmHg, 95.86 ± 2.27%, and 346.81 ± 51.93 mmHg, respectively, after 7 days of treatment. Compared with the control group (88.21 ± 12.82 mmHg, 93.18 ± 3.13%, and 266.83 ± 48.09 mmHg), the difference was statistically significant, p < 0.05. The oxygen duration, respiratory distress relief time, and length of stay were 95.12 ± 12.53 h, 4.4 ± 0.6 d, and 19.84 ± 2.8 d, respectively, in the experimental group, while they were 145.92 ± 13.85 h, 6.9 ± 0.9 d, and 28.42 ± 3.7 d, respectively, in the control group, showing great differences (p < 0.05). CONCLUSIONS: Micropump infusion of AH in the treatment of premature RDS patients was more conducive to efficacy. It can alleviate the clinical symptoms of children with RDS, improve their blood gas indicators, relieve and repair the damage to alveolar epithelial cell lipids in children with RDS, and ultimately improve the therapeutic effect, which can be used for the clinical treatment of premature RDS.

[One Health: Re-thinking of zoonoses control].

Under the dual pressure of emerging zoonoses and the difficulty in eliminating conventional zoonoses, many uncertainties in global control of infectious diseases are challenging the achievement of sustainable development goals set by the United Nations General Assembly. One Health, developed on the basis of understanding the relationship between human diseases and animal diseases, is conducive to the prevention and control of zoonoses. The connotation of "One Health" is mainly explained by three aspects, namely the systems thinking mode of "unity of environment and man", the practice guidance of "multi-sectoral concert" and the economic evaluation strategy of "cost-effectiveness analysis". One Health approach has been successfully applied in the control of major infectious diseases in China, such as schistosomiasis, leading to remarkable achievements; however, there are still multiple challenges. This review proposes that much attention should be paid to top-level design, the difference between emerging zoonoses and conventional zoonoses, and the dynamic process of One Health governance during the development and application of One Health.

Alteration of the faecal microbiota composition in patients with constipation: evidence of American Gut Project.

There is limited information is known about the composition difference of the gut microbiota in patients with constipation and healthy controls. Here, the faecal 16S rRNA fastq sequence data of microbiota from the publicly available American Gut Project (AGP) were analysed. The tendency score matching (PSM) method was used to match in a 1:1 manner to control for confounding factors age, gender, body mass index (BMI), and country. A total of 524 participants including 262 patients with constipation and 262 healthy controls were included in this analysis. The richness and evenness of the gut microbiota in the constipation group were significantly lower than those in the control group. The dominant genera in the constipation group include Escherichia_Shigella, Pseudomonas, and Citrobacter. The dominant genera in the control group include Faecalibacterium, Prevotella, Roseburia, Clostridium_XlVa, and Blautia. The abundance of three butyrate production-related pathways were significantly higher in the constipation group than in the control groups. There was no significant difference in the diversity and gut microbiota composition in patients with constipation at different ages. In conclusion, patients with constipation showed gut microbiota and butyrate metabolism dysbiosis. This dysbiosis might provide a reference for the diagnosis and clinical therapy of diseases.

Association of Loss of Occlusal Pairs of Teeth with Sarcopenia in a Chinese Population of Community-Dwelling Elderly.

OBJECTIVES: Tooth loss, which usually leads to malnutrition, is common in the elderly. However, limited information is available regarding its association with sarcopenia. This study aimed to investigate the relationship between loss of occlusal pairs of tooth and sarcopenia. DESIGN: A cross-sectional retrospective study was performed. SETTING: The elderly who participated in the National Basic Public Health Project in the Maigaoqiao Community Medical Center in Nanjing, Jiangsu Province, China. PARTICIPANTS: A total of 2850 individuals aged ≥60 years were enrolled. MEASUREMENTS: Sarcopenia was defined according to the criteria proposed by the Asian Working Group for Sarcopenia. A trained dentist assessed oral health status and counted the number of present teeth. Logistic regression analyses were performed to evaluate the association between the loss of occlusal pairs and sarcopenia. RESULTS: The prevalence of sarcopenia was 7.1% (201/2850). Univariate logistic regression analysis showed that loss of occlusal pairs was associated with sarcopenia [anterior occlusal pairs (AOPs): odd ratio (OR) = 1.292, 95% confidence interval (CI) = 1.158-1.442; posterior occlusal pairs (POPs): OR = 1.147, 95% CI = 1.018-1.221]. Multivariate logistic regression analysis indicated that loss of POPs was still an independent risk for sarcopenia (OR = 1.108, 95% CI = 1.007-1.220) after adjustment for traditional confounders. Subgroup analysis showed that loss of POPs was more significantly linked to sarcopenia in those with advanced age (≥80years) (OR = 1.307, 95% CI = 1.116-1.532) and in females (OR = 1.165, 95%CI = 1.038-1.308). Compared to individuals with ≥5 occluding pairs of POPs, those with <5 occluding pairs of POPs had a higher incidence of sarcopenia. CONCLUSIONS: Loss of POPs is associated with an increased risk of sarcopenia in the elderly in a Chinese population. Further research on the mechanism of the observed causal relationship is needed.

First Report of Fusarium cerealis Causing Fusarium Head Blight on Barley in China.

The fungus Fusarium cerealis (Cooke) Sacc. (1886) (synonym F. crookwellense L.W. Burgess, P.E. Nelson et Toussoun (1982)) is one of the causal agents of Fusarium head blight (FHB), a significant fungal disease of small grains and maize in many countries (3). Miller et al. identified trichothecene chemotypes from three F. cerealis (F. crookwellense) strains collected from Chinese wheat in 1991 (2). However, there have been no other reports of this Fusarium species in China since then. The disease has been an important threat to the production of wheat and barley in China, and has caused yield losses of approximately 212,000 t (data from China National Agro-Technical Extension and Service Center) in 2008, one of the most severe FHB epidemic years in Chinese wheat in the past 10 years. Scabby heads of barley and wheat were collected from Yuxi and Zhaotong in Yunnan Province, respectively, in 2008. Each of 15 strains was isolated from a different spike of barley and eight isolates were derived from different wheat spikes. All strains were single spored. On the basis of a multilocus genotyping assay (4), all 15 isolates from barley and six of the eight strains from wheat were identified as F. cerealis with the nivalenol (NIV) genotype. To validate this result, partial translation elongation factor (TEF-1α, ~700 bp) gene sequences of isolates were generated (GenBank Accession Nos. HQ824369 and HQ824370) and then compared with the FUSARIUM-ID database (1). The TEF-1α sequences of all 21 isolates were highly conserved and showed 100% identity with the sequences of F. cerealis in the database. A pathogenicity test was performed on a winter wheat cultivar, Yangmai158, using a three-replicate randomized complete block design in Langfang Experimental Farm. Langfang is not an FHB-endemic area and is very dry, with almost no infection source in normal years. At anthesis, the central floret of each of five spikes was injected with 20 µl of a conidial suspension (106 macroconidia/ml) of each isolate in each block, such that 105 spikes were injected per block. An equal amount of water was injected into five heads in each block as a control. The inoculated spikes were enclosed in sandwich bags misted with water for 3 days to ensure infection by the inoculated pathogen and prevent infections from other sources. The plots were misted twice daily after inoculation. The average night and day temperatures were 19.0 and 25.2°C, respectively. Typical FHB symptoms (light tan or bleached spikelets, bases of infected spikelets, and portions of the rachis were dark brown), which were observed in the heads inoculated with F. cerealis 7 days after inoculation, were similar to the original symptoms in the sampling sites. The control plants remained asymptomatic. F. cerealis was reisolated from the infected heads, indicating it was the pathogen causing this disease. To our knowledge, this is the first report of F. cerealis causing FHB on barley in China, and also the first time that a high proportion of F. cerealis strains were isolated from one sampling site (15 of 15 in Yuxi and 6 of 8 in Zhaotong). More isolates must be collected in Yunnan Province and further studies conducted to gain a better understanding of the spatial and temporal dynamics of this pathogen. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2010. (2) J. D. Miller et al. Mycologia 83:121, 1991. (3) D. W. Parry et al. Plant Pathol. 44:207, 1995. (4) T. J. Ward et al. Fungal Genet. Biol. 45:473, 2008.