A guide to human microbiome research: study design, sample collection, and bioinformatics analysis / 中华医学杂志(英文版)

The purpose of this review is to provide medical researchers, especially those without a bioinformatics background, with an easy-to-understand summary of the concepts and technologies used in microbiome research. First, we define primary concepts such as microbiota, microbiome, and metagenome. Then, we discuss study design schemes, the methods of sample size calculation, and the methods for improving the reliability of research. We emphasize the importance of negative and positive controls in this section. Next, we discuss statistical analysis methods used in microbiome research, focusing on problems with multiple comparisons and ways to compare β-diversity between groups. Finally, we provide step-by-step pipelines for bioinformatics analysis. In summary, the meticulous study design is a key step to obtaining meaningful results, and appropriate statistical methods are important for accurate interpretation of microbiome data. The step-by-step pipelines provide researchers with insights into newly developed bioinformatics analysis methods.

Clinical and laboratory features of macrophage activation syndrome / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical and laboratory features of macrophage activation syndrome (MAS) at the early stage of diagnosis, and to explore a method for early identification of MAS.</p><p><b>METHODS</b>A retrospective analysis was performed for the demographic data, clinical and laboratory features, and treatment outcomes of 21 MAS patients.</p><p><b>RESULTS</b>Of the 21 MAS patients, 14 had systemic juvenile idiopathic arthritis, 5 had Kawasaki disease (KD), and 2 had connective tissue disease (CTD) as primary diseases. The median time of MAS onset was 19 days. The KD patients had the shortest time of MAS onset, while the CTD patients had the longest onset time (P=0.009). The top 10 clinical symptoms were fever (95%), rash (86%), lymph node enlargement (67%), hemophagocytic phenomenon in bone marrow (63%), pulmonary disease (62%), serous effusion (62%), hepatomegaly (52%), cerebrospinal fluid abnormalities (50%), central nervous system damage (43%), and splenomegaly (38%). The median of hemoglobin level was lower than the normal value. The medians of C-reactive protein level and erythrocyte sedimentation rate were higher than the normal values. There were significant increases in serum ferritin, glutamic-pyruvic transaminase, aspartate aminotransferase, lactate dehydrogenase, and triglyceride. The median of fibrinogen level was lower than the normal value. There were significant increases in D-dimer, interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon-γ (IFN-γ). Of the 21 patients, 20 were improved and discharged.</p><p><b>CONCLUSIONS</b>If patients with rheumatic disease have persistent fever, hepatic dysfunction, coagulation disorders, multiple organ impairment, significantly increased IL-10 and IFN-γ, and a persistent increase in serum ferritin, the development of MAS should be considered.</p>

Association of vitamin D concentrations with juvenile idiopathic arthritis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>25-Hydroxyvitamin D3 [25(OH)D3] is the main product of vitamin D and can reflect the absolute concentration of active vitamin D in the body. This study examined serum 25(OH)D3 levels in children with juvenile idiopathic arthritis (JIA) in order to explore the association of vitamin D concentrations with the pathogenesis and disease activity of JIA.</p><p><b>METHODS</b>Serum samples were collected from 53 children confirmed as having JIA between January 2013 and March 2014, as well as 106 healthy children (control group) who underwent physical examination in the same period. Serum concentrations of 25(OH)D3 were measured using ELISA and compared between the cases and healthy controls. The association of serum 25(OH)D3 levels with JIA subtypes, ACR Pediatric 30 Score, peripheral blood C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) were analyzed in children with JIA.</p><p><b>RESULTS</b>Compared with the control group, the JIA group had significantly reduced serum 25(OH)D3 levels (median: 42.6 nmol/L vs 49.9 nmol/L; P<0.01). The percentage of subjects with severe deficiency of vitamin D in the JIA group was significantly higher than that in the control group (17.0% vs 6.6%; P<0.05). Serum 25(OH)D3 showed no significant correlations with JIA subtypes, ACR Pediatric 30 Score, CRP, and ESR in children with JIA.</p><p><b>CONCLUSIONS</b>Vitamin D concentrations are significantly decreased in children with JIA. Decreased vitamin D concentrations may be associated with the pathogenesis of JIA. However, vitamin D concentrations may have no correlations with JIA subtypes, disease severity, and disease activity.</p>

Serum cytokine levels in children with newly diagnosed active systemic juvenile idiopathic arthritis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the changes in serum cytokines levels in children with newly diagnosed active systemic juvenile idiopathic arthritis (SJIA) and to explore the role of cytokines in the development and progression of SJIA.</p><p><b>METHODS</b>Seventy-four pediatric patients with active SJIA between January 2010 and December 2013 were included in the study. Serum levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukine-10 (IL-10), tumor necrosis factor (TNF), and interferon gamma (IFN-γ) were measured by flow cytometry in these patients. The levels of cytokines were also determined in 202 healthy children as the control group. Routine laboratory parameters including white blood cell (WBC) count, percentage of neutrophils, hemoglobin level, platelet count, hypersensitive C-reactive protein (hs-CRP), and erythrocyte sedimentation rate (ESR) were monitored in the patient group.</p><p><b>RESULTS</b>The WBC count, percentage of neutrophils, hs-CRP, and ESR in 74 cases of SJIA were significantly above the normal range, their platelet counts were within the normal range, whereas hemoglobin levels were below the normal range. Compared with the control group, the patient group showed a significantly increased level of IL-6 (P<0.01) and significantly reduced levels of IL-4, IL-10, and TNF (P<0.01). However, there were no significant changes in serum levels of IL-2 and IFN-γ in the patient group (P>0.05). In SJIA children, IL-6 level, which was significantly elevated, was negatively correlated with hemoglobin level, which was significantly reduced (r=-0.244, P<0.05).</p><p><b>CONCLUSIONS</b>Serum level of IL-6 is significantly increased in children with SJIA, and it has a negative correlation with anemia.</p>

Study on mechanism of trimethyltin chloride-induced apoptosis in PC12 cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of trimethyltin chloride (TMT) on proliferation, apoptosis, oxidative damage, and NF-κB expression in PC12 cells in vitro.</p><p><b>METHODS</b>PC12 cells were treated with 0, 0.3125, 0.6250, 1.2500, 2.5000, 5.0000, 10.0000, and 20.0000 µmol/L TMT for 24 and 48 h, and MTT assay was used to evaluate cell viability. PC12 cells were treated with 1.25, 2.50, 5.00, and 10.00 µmol/L TMT for 12 and 24 h, and flow cytometry was used to measure the apoptotic rates of cells. PC12 cells were treated with 1.25, 2.50, 5.00, and 10.00 µmol/L TMT for 6 h, and the reactive oxygen species (ROS) and glutathione (GSH) levels were measured. PC12 cells were treated with 1.25, 2.50, 5.00, and 10.00 µmol/L TMT for 12 h, and Western blot was used to measure NF-κB levels.</p><p><b>RESULTS</b>Compared with solvent controls, the PC12 cells treated with 2.5000, 5.0000, 10.0000, and 20.0000 µmol/L TMT for 24 h showed significantly decreased cell viability (P < 0.05); the PC12 cells treated with 1.2500, 2.5000, 5.0000, 10.0000, and 20.0000 µmol/L TMT for 48 h showed significantly decreased cell viability (P < 0.05). The PC12 cells treated with 1.2500, 2.5000, 5.0000, and 10.0000 µmol/L TMT for 12 h had apoptotic rates of 15.30% ± 0.75%, 18.90% ± 0.61%, 22.00% ± 0.60%, and 36.50% ± 0.66%, respectively, and the PC12 cells treated with 1.25, 2.50, 5.00, and 10.00 µmol/L TMT for 24 h had apoptotic rates of 28.6% ± 0.40%, 43.54% ± 2.00%, 65.73% ± 0.71%, and 74.67% ± 0.40%, respectively, all significantly higher than those of the control group (12 h: 12.80% ± 1.00%, 24h: 16.83% ± 0.25%) (P < 0.05). The ROS fluorescence intensities of the PC12 cells treated with 1.25, 2.50, 5.00, and 10.00 µmol/L TMT were 1.42, 1.71, 1.78, and 1.89 times that of the control group (P < 0.05); the PC12 cells treated with 2.50, 5.00, and 10.00 µmol/L TMT had GSH levels of 0.17 ± 0.0, 0.20 ± 0.04, and 0.07 ± 0.03 µmol/µg protein, significantly lower than that of the control group (0.30 ± 0.01 µmol/L protein) (P < 0.05). The PC12 cells treated with 2.50, 5.00, and 10.00 µmol/L TMT had significantly higher expression of NF-κB p65 than the control group (P < 0.05).</p><p><b>CONCLUSION</b>Under our laboratory conditions, TMT can significantly inhibit proliferation and induce apoptosis in PC12 cells, which may be related to oxidative stress and NF-κB signaling pathway activation.</p>

Effects of melatonin on nerve cell apoptosis and expression of Bcl-2 & cytochrome C genes in rat cerebrum with deltamethrin induction / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the effects of melatonin (MT) on nerve cell apoptosis and the expression of bcl-2 and cytochrome C genes in rat cerebrum with deltamethrin induction.</p><p><b>METHODS</b>35 male Wistar rats were randomly divided into five groups (eight rats per group): olive oil control, deltamethrin-treated (12.5 mg/kg), deltamethrin plus melatonin (25.0 mg/kg, 50 mg/kg and 100 mg/kg respectively) group. Animal models were established by intraperitoneal injection deltamethrin in rats. Nerve cell apoptosis and the protein expression of bcl-2 and cytochrome C genes were detected by flow cytometry with PI staining and immunohistochemistry respectively.</p><p><b>RESULTS</b>Compared with DM group (20.73 +/- 3.34), the positive expression gradation of the bcl-2 protein in nerve cell was increased significantly in MT groups (DM + MT(25) was 45.26 +/- 3.84, DM + MT(50) 39.4 +/- 4.04 and DM + MT(100) 34.4 +/- 4.52) (P < 0.05) but significantly lower than the control group (59.33 +/- 4.03). Compared with DM group (34.86 +/- 4.15), the cytochrome C protein in nerve cell was decreased significantly in MT groups (20.53 +/- 3.17, 28.73 +/- 2.61 and 28.66 +/- 4.82 respectively) (P < 0.05). Compared with DM group (23.06 +/- 3.63), the apoptotic rate in nerve cell was decreased significantly in MT groups [(15.0 +/- 1.77)%, (14.88 +/- 1.84)% and (11.75 +/- 1.93)% respectively] (P < 0.05).</p><p><b>CONCLUSION</b>MT can protect nerve cell against deltamethrin induced brain injury by inhibiting nerve cell apoptosis, downregulate the protein expression of cytochrome C gene and upregulate the protein expression of bcl-2 gene.</p>

Clinical evaluation for sublingual immunotherapy of allergic asthma and atopic rhinitis with Dermatophagoides Farinae Drops / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the safety and efficacy of sublingual immunotherapy with 'Dermatophagoides Farinae Drops' in D. farinae allergic asthma and/or rhinitis patients.</p><p><b>METHODS</b>A 25-week double-blind, placebo-controlled, multi-centered trail was conducted in 278 children (aged 4 - 18 yr) with mite-induced asthma and/or rhinitis. Patients were randomly assigned to receive sublingual immunotherapy (SLIT) with 'Dermatophagoides Farinae Drops' (n = 139) or placebo (n = 139) for 25 weeks and the dosage and administration strictly followed the manufacturer's instructions. At the beginning of the 2nd, 3rd, 4th, 6th, 10th, 14th, 18th, 22nd week of the treatment, the patients were asked to accept follow-up visit, during the clinical trial all patients and parents were asked to keep a daily record of their asthma symptom scores, rescue medicine use, rhinitis symptom scores, morning and evening peak expiratory flow. Asthma symptom scores, reduction in use of rescue medicine, rhinitis symptom scores, lung function tests, skin sensitivity to mite, mite-specific immunoglobulin (Ig) E and IgG4, and quality of life and adverse effect were assessed during the study.</p><p><b>RESULT</b>(1) Of the 278 children, 27 dropped out before the study completion. (2) After 25 weeks of treatment, the median variability of PEFR was -1.38 for SLIT group and -0.90 for the placebo (P < 0.05). (3) Besides, the mean variability of medicine score of asthma was -0.08 for SLIT group and 0.52 for the plcebo (P < 0.05). (4) The median variability of rhinitis symptom score was -1.96 for SLIT group and -1.03 for the placebo (P < 0.01). (5) The rescue medicine usage of SLIT reduced but did not show significant differences between SLIT and placebo. (6) After 25 weeks treatment, the increase of D. farinae specific IgE antibody of two groups were similar, while specific IgG4 increased significantly in SLIT compared to the patients in control one (P < 0.01); (7) No severe adverse events happened in the trial and the most-likely adverse events were mild asthma and local rash.</p><p><b>CONCLUSION</b>Dermatophagoides Farinae Drops is safe and effective in treating allergic asthma and atopic rhinitis.</p>

Effect of allergen on function of peripheral blood CD4+T cells in patients with asthma / 上海第二医科大学学报

Objective To investigate the relationship among the proliferation of CD4+T cells, the intracellular levels of interleukin-10 (IL-10) and transforming growth factor-?1(TGF-?1), and allergic bronchial asthma. Methods Dermatophagoides farinae antigen were prepared as allergen. Twenty-five patients with asthma and 15 healthy individuals were enrolled and divided into blank control group, allergen group and self control group, respectively, after venous blood sample collection. The proliferation of CD4+T cells and the distributions of CD4+/IL-10+, CD4+/TGF-?+1 and CD4+/IL-10+/TGF-?+1 were measuredby flow cytometry (FCM). Results The distributions of CD4+/IL-10+, CD4+/TGF-?+1 and CD4+/IL-10+/TGF-?+1 could hardly be detected in the peripheral blood samples of the blank controls of the patients with asthma and healthy ones. In the allergen group of the healthy individuals, the peripheral blood CD4+T cells were significantly proliferated, and the proportions of CD4+T cells andCD4+/IL-10+ cells were much higher than the self control group, while there was no significantly increase in the proportions of CD4+/TGF-?+1 and CD4+/IL-10+/TGF-?+1 subgroups. In the allergen group of those with asthma, the proportions of peripheral blood CD4+, CD4+/IL-10+, CD4+/TGF-?+1 and CD4+/IL-10+/TGF-?+1 cells were not found significantly increased compared with those self controls. After being activated by allergen, the proportion of peripheral blood CD4+/IL-10+ cells was significantly lower in the patients with asthma than the healthy individuals(P

The study on pathogenesis of latex allergy / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>In order to study the pathogenesis of latex allergy and the significance in the prevention and cure of occupational diseases.</p><p><b>METHODS</b>651 cases in the out-patient department were tested with skin prick test (SPT), and the specific IgE (sIgE) to latex were detected by means of disk ELISA and Western-blot.</p><p><b>RESULTS</b>It was found that the positive rate of latex SPT (37.5%) and sIgE (31.25%) were rather higher in patients in comparison with those of the normal. The positive rate of latex sIgE was much higher in the high-risk group than that of the low-risk group and the normal. The serum of the patients can react with multi-bands in the latex glove extracts.</p><p><b>CONCLUSION</b>The prevalence of latex allergy is rather high, this disease is mediated by IgE. The people in high-risk should be tested by latex allergy in order to take proper occupational and daily protection.</p>

Study on a Pre-enrichment Medium for the Simultaneous Recovery of Salmonella spp., Escherichia coli and Staphylococcus aureus / 微生物学通报

This text involved with a pre-enrichment medium for the simultaneous recovery of Salmonella spp.,Escherichia coli and Staphylococcus aureus.This medium is named buffered saline broth(BSB),which contains peptone 10g,beef exact 3g,disodium hydrogen phosphate 9g,potassium dihydrogen phosphate 1.5g,additive 50g,deionized water 1 000mL,pH7.2.1cfu per one milliliter of Salmonella spp.,Escherichia coli and Staphylococcus aureus in saline were stimutaneously added to 97 mL of BSB,buffered peptone water,lactose broth,nutrient broth,Escherichia coli broth,rappaport-vassiliadis enrichment broth,7.5% sodium chloride enrichment medium,respectively,and incubated for 18h at 37℃.The results showed BSB was the best enrichment medium,in which Salmonella spp.,Escherichia coli and Staphylococcus aureus multiplied at nearly same speed,and reached at 10~6 、10~6 、10~7 cfu/mL,respectively.Multiplex PCR produced specific amplicons of expected sizes,284bp for Salmonella spp.invA gene,622bp for Escherichia coli phoA gene,484bp for Staphylococcus aureus nuc gene.In contrast,the three bacteria couldn't multiply harmoniously in the other six media.So BSB might be considered as the medium,which could enrich above mentioned three bacteria.

Detection of Salmonella spp.,Escherichia coli and Staphylococcus aureus by Multiplex PCR / 微生物学通报

According to DNA sequences of the invA gene of Salmonella spp.,the phoA gene of Escherichia coli and the nuc gene of Staphylococcus aureus,three pairs of oligonucleotide primers were designed and synthesized to amplify the special DNA sequences by multiplex PCR. Moreover,the reaction conditions of multiplex PCR were optimized. The results showed the multiplex PCR using the three pairs of primers produced specific amplicons of expected sizes,284bp for Salmonella spp.,622bp for Escherichia coli,484bp for Staphylococcus aureus. The optimized reaction conditions followed as the concentration of primer 40nmol/L for Salmonella spp.,40nmol/L for Escherichia coli,80nmol/L for Staphylococcus aureus,2.4mmol/L Mg 2+ ,200?mol/L dNTP,1.5U Taq DNA polymerase,anneal temperature from 55.0℃ to 57.4℃. Under the condition,the detection limits for DNA template were 10.2pg,10.2pg and 102.0pg for Salmonella spp.,Escherichia coli and Staphylococcus aureus,respectively. The whole process could be completed within 4h. The multiplex PCR assay was a specific,sensitive,rapid and reliable method for detecting Salmonella spp.,Escherichia coli and Staphylococcus aureus,which establish important foundation for simultaneous detection for these three bacteria in food.

Screening Citrinin Mutants from the Transformants Library of Monascus ruber M-7 by Agrobacterium-mediated DNA transfer / 微生物学通报

200 citrinin mutants were screened with the inhibition zone method from the transformants library of Monascus ruber M-7 by Agrobacterium-mediate DNA transfer, which contains more than 5,000 transformants.Then 53 mutants, whose citrinin contents ranged from 0.04?g/g to 154.57?g/g in the red fermented rice (RFR), were achieved by high performance liquid chromatography (HPLC).Color values of RFR prepared by these mutants were also detected.The results showed that there was a positive correlation between the citrinin content and the color value among the mutants.These results provide materials and research bases for ferrther studying the relationship between the production of citrinin and pigment of Monascus ruber at molecular level.