[Value of serum gamma-glutamyl transpeptidase combined with direct bilirubin in the diagnosis of biliary atresia in infants].

OBJECTIVE: To study the value of serum gamma-glutamyl transpeptidase (GGT) combined with direct bilirubin (DB) in the diagnosis of biliary atresia. METHODS: A total of 667 infants with cholestasis who were hospitalized and treated from July 2010 to December 2018 were enrolled as subjects. According to the results of intraoperative cholangiography and follow-up, they were divided into biliary atresia group with 234 infants and cholestasis group with 433 infants. The two groups were compared in terms of age of onset, sex, and serum levels of total bilirubin (TB), DB, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), and GGT. A receiver operating characteristic (ROC) curve analysis was performed for indices with statistical significance, and the area under the ROC curve (AUC) and the optimal cut-off value for diagnosis were calculated. RESULTS: The biliary atresia group had a significantly younger age of onset than the cholestasis group (P<0.001). There were no significant differences in sex, ALT, and AST between the two groups (P>0.05), while the biliary atresia group had significantly higher serum levels of TB, DB, TBA, and GGT than the cholestasis group (P<0.05). GGT combined with DB had the highest AUC of 0.892 (95% confidence interval: 0.868-0.916) in the diagnosis of biliary atresia. At the optimal cut-off values of 324.0 U/L for GGT and 115.1 µmmol/L for DB, GGT combined with DB had a sensitivity of 79.8% and a specificity of 83.2% in the diagnosis of biliary atresia. CONCLUSIONS: GGT combined with DB has high sensitivity and specificity in the diagnosis of biliary atresia and can be used as an effective indicator for diagnosis of biliary atresia in infants.

The role and clinical significance of programmed cell death- ligand 1 expressed on CD19+B-cells and subsets in systemic lupus erythematosus.

BACKGROUND: Programmed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1)-targeted therapies have enhanced T-cell response and demonstrated efficacy in the treatment of multiple cancers. However, the role and clinical significance of PD-L1 expression on CD19+ B-cells and their subsets, with particular reference to systemic lupus erythematosus (SLE), have not yet been studied in detail. OBJECTIVE: The present study aimed to investigate PD-L1 expression on CD19+ B-cells and their subsets, in addition to exploring its possible role in Tfh-cell activation and B-cell differentiation in SLE. METHODS: Frequencies of CD19+ B-cells, their subsets, PD-L1 and Tfh cells in the peripheral blood of SLE patients and healthy controls (HCs) were determined using cytometry. The clinical data of SLE patients were recorded in detail, and the correlation between their laboratory parameters, clinical parameters and disease activity indices was statistically analyzed. CD19+PD-L1+B-cells and CD19+PD-L1- B-cells were sorted and cultured with a stimulant, following which the supernatants were collected for immunoglobulin G and anti-double stranded DNA detection via enzyme-linked immunosorbent assay. RESULTS: In SLE patients, CD19+B-cells and partial subgroups were enriched in peripheral blood. Also, the observed increase in the frequency of CD19+PD-L1+B-cells was significantly associated with a higher disease activity index. An in vitro culture test demonstrated that the amounts of anti-dsDNA and immunoglobulin G secreted by the CD19+PD-L1+B-cells of SLE patients and HCs were vastly different. In addition, a strong correlation existed between the frequencies of CD19+PD-L1+B-cells and defined Tfh cells of SLE patients. CONCLUSION: This study demonstrated that the expression of CD19+PD-L1+B-cells in the peripheral blood of SLE patients was abnormal, and that disease-related laboratory parameters and clinical indicators were correlated. CD19+PD-L1+B-cells were enriched and played a critical role in activating the pathogenic T-cell and B-cell responses in patients with SLE.

[Protective effects of glycyrrhizic acid against lupus nephritis in MRL/lpr mice].

OBJECTIVE: To investigate protective effect of glycyrrhizic acid (GA) against lupus nephritis in MRL/lpr mice and explore its underlying mechanisms. METHODS: Forty MRL/lpr mice were randomized equally into blank control group, dexamethasone (1.5 mg/kg) group, GA (20 mg/kg) group, and GA (40 mg/kg) group with corresponding treatments for 7 days, with 10 wild-type mice as the control group. Serum levels of uric acid and creatinine and inflammatory cytokines in the serum and kidney were tested after the treatments using enzyme-linked immunosorbent assays (ELISA). The pathological changes in the kidneys were detected using HE staining, and the protein expressions of NLRP3, ASC, caspase-1, IL-1ß, p-NF-κB, NF-κB, p-IκBα, and IκBα were detected with Western blotting. RESULTS: GA obviously decreased serum levels of uric acid and creatinine, decreased inflammatory cytokines in the serum and kidney, ameliorated renal pathologies and inhibited the expressions of NLRP3, ASC, caspase-1, IL-1ß, p-NF-κB, and p-IκBα proteins in MRL/lpr mice. CONCLUSION: GA has protective effects against lupus nephritis in MRL/lpr mice.

[The roles and mechanisms of p-STAT3 signaling pathway in acute pancreatitis].

OBJECTIVE: To detect the expression ofsignal transducer and activator of transcription 3 (STAT3) in pancreatic tissue of the mouse model of pancreatitis, and to explore its role in the evolution of acute pancreatitis. METHODS: Forty-eight healthy male balb/c mice were randomly divided into 3 groups (n=16):control group (Con) 0.09% NaCl, intraperitoneal injection; mild acute pancreatitis group (MAP) caerulein, intraperitoneal injection; severe acute pancreatitis group (SAP) caerulein plus lipopolysaccharide(LPS), intraperitoneal injection. The mice were sacrificed after 2 h and 6 h after intraperitoneall injection. Serum was isolated for amylase activity. Pancreatic was isolated and weighedto calculate the pancreatic wet weight ratio. Myeloperoxidase (MPO) activity was measured to assess the degree of inflammatory cell infiltration in lung tissue. Using HE staining, the pathological changes of pancreatic and lung were observed under the light microscope. The expression of phosphorylated STAT3 (p-STAT3) was detected by Western blot. RESULTS: Compared with control group, serum amylase activity, pancreatic wet weight ratio and lung MPO activity were significantlyincreased (P<0.05) in MAP and SAP group at each time point, especially SAP group showed higher levels of MPO activity than that in MAP group (P<0.01). The pathological changes of pancreas and lung were observed after modeling in 2 h. Western blot showed the expression of p-STAT3 could be detected in SAP group, the level increased most significantly after modeling 2 h, and decreased slightly after 6 h. The level of p-STAT3 was low in MAP group and negative in Con group at each time point. CONCLUSIONS: The expression of p-STAT3 in MAP and SAP groups are significantly different from that in control group, which indicates that STAT3 isclosely related in acute pancreatitis. Inhibition of STAT3 activity is a potential target to alleviate acute pancreatitis progression.

Sequential loss of genetic variation in flea beetle Agasicles hygrophila (Coleoptera: Chrysomelidae) following introduction into China.

The flea beetle (Agasicles hygrophila) was imported to Florida, USA and then introduced from Florida into China in 1987 as a biological control agent for the invasive plant alligator weed (Alternanthera philoxeroides). The initial beetle population was subsequently used for sequential introductions in other areas of China, but little is known about the genetic consequences of the introductions. In this study, the genetic diversity and population structure of five beetle populations, the source Florida population, three intentionally introduced China populations and one accidentally dispersed China population, were examined using amplified fragment length polymorphisms. The results showed a clear pattern of decreasing genetic diversity with the sequential introductions. The diversity was highest in the Florida population followed by the first introduction to Chongqing and then in Kunming and Fuzhou. The lowest diversity was found in the accidentally dispersed Guangzhou population that was first recorded in 1996. Both loci parameters and Nei's genetic diversity showed a high variation among these populations. Genetic differentiation among populations was further verified by the GST statistic (0.136-0.432). Beetles in Kunming had the highest gene flow with those in Guangzhou, and therefore lowest differentiation and closest genetic distance. These data show that sequential introduction influenced the genetic diversity of populations in China. Genetic diversity should be considered in planning introduction and long-term maintenance of populations.

Expression of yeast acyl-CoA-∆9 desaturase leads to accumulation of unusual monounsaturated fatty acids in soybean seeds.

An acyl-CoA-Δ9 desaturase from Saccharomyces cerevisiae was expressed by subcellular-targeting in soybean (Glycine max) seeds with the goal of increasing palmitoleic acid (16:1Δ9), a high-valued fatty acid (FA), and simultaneously decreasing saturated FA in oil. The expression resulted in the conversion of palmitic acid (16:0) to 16:1Δ9 in soybean seeds. 16:1Δ9 and its elongation product cis-vaccenic acid (18:1Δ11) were increased to 17 % of the total fatty acids by plastid-targeted expression of the enzyme. Other lipid changes include the decrease of polyunsaturated FA and saturated FA, suggesting that a mechanism exists downstream in oil biosynthesis to compensate the FA alternation. This is the first time a cytosolic acyl-CoA-∆9 desaturase is functionally expressed in plastid and stronger activity was achieved than its cytosolic expression. The present study provides a new strategy for converting 16:0 to 16:1Δ9 by engineering acyl-CoA-Δ9 desaturase in commercialized oilseeds.

[A clinical study of Leber hereditary optic neuropathy].

OBJECTIVE: To investigate the clinical characteristics of Leber hereditary optic neurology (LHON) patients with different primary site mutation. METHODS: Four hundred and fourteen patients with optic neuropathy were divided into three groups: clinically diagnosed LHON group (group A), probable LHON group (group B), optic neuropathy of unknown reason group (group C). Visual acuity (VA), colour vision, Intraocular pressure (IOP), virtual field and visual evoked potential (VEP) were tested for all the patients. Some (64 cases) had optical coherence tomography (OCT) measurement. Mutations of mtDNA were detected for all the groups, and clinical analysis were carried out emphatically in the patients with the 11778 mutation confirmed by gene assessment. T paired test was used to evaluate two group patients of different Mitochondrial DNA mutation. RESULTS: Gene mutations were found in 215 of the 414 patients (52%). Approximately 93% (199/255) of the patients were caused by the common primary mutations (11778, 14484, 3460 mutation), in which 100% mutation (106/106) in group A, 65% (91/139) in group B, and 11% (18/169) in group C. No cases were diagnosed with confirmed LHON in the patients with unilateral optic neuropathy. Fundus examination in 334 eyes of 167 cases showed pseudo papilledema (54 eyes), normal (67 eyes), pale disc or pale on the temporal side of the optic disc (213 eyes). On the basis data of OCT from 64 patients and 84 normal person, RNFL was found thickening at the early stage and thinning gradually at the later stage in the LHON patients. But, the RNFL thickness of patients with 1-2 years history was not significantly different from the patients with over 2 years history(P = 0.051), and there was no difference among the patients with different mitochondrial DNA mutations. The initial mean VA of patients with the 14484 mutation and 11778 mutation were 3.6 ± 0.65, 3.75 ± 0.54 (t = 0.536, P > 0.05), but the follow-up VA were 4.29 ± 0.55 (t = 4.034, P < 0.001) and 3.93 ± 0.49 respectively (t = 1.857, P > 0.05). CONCLUSIONS: The symptoms and fundus manifestation were similar in the LHOH patients with different primary site mutation. Gene mutation analysis is helpful to assess the prognosis of visual acuity.

Breviscapine attenuates acute pancreatitis by inhibiting expression of PKCα and NF-κB in pancreas.

AIM: To study the effect of breviscapine (Bre) on activity of protein kinase Cα (PKCα) and nuclear factor (NF)-κB in pancreas, and the mechanism of Bre attenuating acute pancreatitis (AP). METHODS: One hundred and eight rats were randomly divided into acute necrotizing pancreatitis (ANP) group, Bre group (ANP + Bre group) and sham operation (SO) group, 36 rats in each group. ANP model was induced by a retrograde injection of 4% sodium deoxycholate into the bilio-pancreatic duct. Fifteen minutes after the ANP model was induced, the rats in Bre group were intraperitoneally injected with Bre (0.4 mg/100 g body weight or 0.1 mL/100 g body weight). Survival time and mortality of rats were calculated. Serum amylase and malondialdehyde levels were measured, volume of ascites was recorded and morphology of pancreas and lung was evaluated at 1, 5 and 10 h, after the ANP model was induced, respectively. Expressions of PKCα and subunit p65 of NF-κB in pancreas were detected by immunohistochemistry and Western blotting. RESULTS: The life span of rats was longer and the mortality was lower in Bre group than in ANP group 13.51 ± 5.46 vs. 25.36 ± 8.11 (P < 0.05). The amylase and MDA levels as well as the volume of ascites were lower and the pathological changes in pancreas and lung were less in Bre group than ANP group (P < 0.05), indicating that the pancreatitis is less severe in Bre group than ANP group. The activation of PKCα and NF-κB p65 in pancreas was induced rapidly and reached their peak at 1 h or 5 h after ANP, but their activity in Bre group was significantly inhibited. CONCLUSION: Bre exerts its therapeutic effect on AP by inhibiting the activation of PKCα and NF-κB p65 in pancreas.

Calreticulin: conserved protein and diverse functions in plants.

Calreticulin (CRT) is a key Ca2+-binding protein mainly resident in the endoplasmic reticulum (ER), which is highly conserved and extensively expressed in all eukaryotic organisms investigated. The protein plays important roles in a variety of cellular processes including Ca2+ signaling and protein folding. Although calreticulin has been well characterized in mammalian systems, increased investigations have demonstrated that plant CRTs have a number of specific properties different from their animal counterparts. Recent developments on plant CRTs have highlighted the significance of CRTs in plants growth and development as well as biotic and abiotic stress responses. There are at least two distinct groups of calreticulin isoforms in higher plants. Glycosylation of CRT was uniquely observed in plants. In this article, we will describe our current understanding of plant calreticulin gene family, protein structure, cellular localization, and diverse functions in plants. We also discuss the prospects of using this information for genetic improvements of crop plants.

Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses.

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca(2+)-binding protein in multicellular eukaryotes. CRT plays a crucial role in many cellular processes including Ca(2+) storage and release, protein synthesis, and molecular chaperone activity. To elucidate the function of CRTs in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cDNA encoding calreticulin protein namely TaCRT was isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence of TaCRT shares high homology with other plant CRTs. Phylogenetic analysis indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. Southern analysis suggests that the wheat genome contains three copies of TaCRT. Subcellular locations of TaCRT were the cytoplasm and nucleus, evidenced by transient expression of GFP fused with TaCRT in onion epidermal cells. Enhanced accumulation of TaCRT transcript was observed in wheat seedlings in response to PEG-induced drought stress. To investigate further whether TaCRT is involved in the drought-stress response, transgenic plants were constructed. Compared to the wild-type and GFP-expressing plants, TaCRT-overexpressing tobacco (Nicotiana benthamiana) plants grew better and exhibited less wilt under the drought stress. Moreover, TaCRT-overexpressing plants exhibited enhanced drought resistance to water deficit, as shown by their capacity to maintain higher WUE (water use efficiency), WRA (water retention ability), RWC (relative water content), and lower MDR (membrane damaging ratio) (P < or = 0.01) under water-stress conditions. In conclusion, a cDNA clone encoding wheat CRT was successfully isolated and the results suggest that TaCRT is involved in the plant response to drought stress, indicating a potential in the transgenic improvements of plant water-stress.

[Influence of annealing and sputtering ambience on the photoluminescence of silicon nitride thin films].

The radio frequency (r. f.) magnetron sputtering was used for preparing silicon-rich silicon nitride films deposited on polished Si substrates at 80 degrees C substrate temperature. The high-purity Ar was used as a sputtering gas and the high-purity N2 as a reactive gas. The silicon nitride films with different Si-rich degrees were obtained by changing the flow ratio of Ar/N2, and subsequently the samples were annealed at a high temperature in pure N2 ambience. The influence of annealing on the properties of films was investigated by X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and photoluminescence (PL). The appearance of Si-N bonds can be confirmed by the XPS, from which the ratio of Si/N can be rough estimated. Therefore, the XPS reveals that the sample before annealing has a high content of Si which is the premise to come into being nanometer Si. However, the PL peak of the films before annealing in the visible light region was not observed obviously. The XRD results indicate that the presence of Si clusters buried in the films after annealing was confirmed by two novel diffraction peaks, which are related to nanometer Si. As the flow ratio of Ar/N2 decreased, the emission intensity of PL peak in the visible light region was enhanced, accompanied with a blue-shift of emission peak. According to the quantum confinement effect, the blue-shift of PL peak should be attributed to the enlarged band gap of Si clusters in the sample, and the increased intensity of the PL peak turns out to be due to the size of nanometer Si. The two important factors of annealing treatment and flow ratio of Ar/N2 were studied, which have an intimate connection with emitting mechanism in PL. The blue-shift of PL peak caused by nanometer Si embodied in the silicon nitride thin films depends on the sputtering condition, such as flow ratio, deposition temperature and sputtering pressure.

[Clinical features and the mutation of Leber's hereditary optic neuropathy in Chinese patients].

OBJECTIVE: To analyze the mutation of Leber's hereditary optic neuropathy (LHON) and the clinical features in Chinese patients. METHODS: The primary mtDNA mutations (3460A, 11778A and 14484C) of 156 patients (110 probands and 46 maternal relatives with LHON) were detected by mutation-specific priming polymerase chain reaction, heteroduplex-single strand conformation polymorphism polymerase chain reaction, restriction fragment length polymorphisms and measurement of DNA sequence. The clinical features were analyzed by retrospective study. RESULTS: The 11778A mutation was found in 100 probands (90.9%), the 3460A mutation was found in 2 (1.8%), and the 14484C was found in 8 (7.3%) of the 110 probands. The visual acuity at onset of the disease was 0.01 or worse in 44 (17.6%) of 250 eyes with the 11778A mutation, but in none of 79 eyes with the 14484C mutation. The visual acuity was 0.1 or better in 76 (29.6%) of 250 eyes with the 11778A mutation, but in 49 (87.3%) of 56 eyes with the 14484C mutation. And 6.8% of 250 eyes with the 11778A mutation recovered a mean final visual acuity of 0.03, whereas 50% of 56 eyes with the 14484C mutation recovered a mean final visual acuity of 0.8. CONCLUSION: In Chinese LHON patients the 11778A, 14484C primary mutations are common. The clinical features are associated with the site of primary mutation. The visual acuity at onset of the disease and the visual recovery of the eyes with 14484C mutation were better than the eyes with the 11778A mutation.

[Variations of the zinc finger protein 161 gene in Chinese with or without high myopia].

To investigate the association between variations of ZFP161 gene and high myopia, A total of 204 probands with simple high myopia(< or = -6.0 dipoters) were collected while 116 normal persons from different families without high myopia or related disease were used as controls. Genomic DNA was prepared from the peripheral leucocytes. The coding sequences of ZFP161 gene in 320 subjects were analyzed by using exon-by-exon PCR-heteroduplex-SSCP analysis. Identification of the Variations by cloning and sequencing, combinated with controls and family analysis, was used to disclose the correlation between ZFP161 gene and high myopia. A mutation of ZFP161 gene was identified as an insertion of AT before the 58th nucleotide of intron 1 (IVS1 58-59)(1/204) and a variation of ZFP161 gene was identified as a heterozygous C to A of the 168th nucleotide in exon 2 (Codon56, GCC-->GCA, Ala56Ala). Ala56Ala is a non-sense mutation identified in 5 of the 204 patients and 3 of 116 controls. No evidence shows that these variations are responsible for high myopia.

[The SNPs analysis of encoding sequence of interacting factor gene in Chinese population].

OBJECTIVE: To screen the variations of TG interacting factor(TGIF) gene in encoding sequence in Chinese high myopia patients and normal controls and to analyze the SNPs of TGIF gene encoding sequence in Chinese population. METHODS: Genomic DNA was collected from 204 probands with high myopia and 112 unrelated persons without high myopia. The coding sequences of TGIF gene in 316 subjects were analyzed by using exon-by-exon PCR heteroduplex-SSCP analysis and sequencing. RESULTS: There were 3 types of SNP and one single nucleotide mutation in the coding sequence of TGIF gene: IVS-2 nt350 G --> T(36/204), codon140 CCA --> CCG; Pro140Pro codon163 CCG --> CTG;Pro163Leu and codon126 GTG --> GCG; Val126Ala(1/204). The SNPs of codon140 CCA --> CCG and codon163 CCG --> CTG were composed of 3 alleles and 5 genotypes in Chinese population which abide by Hardy-Weinberg law. CONCLUSION: There was no evidence to prove that mutations in the TGIF gene are responsible for the high myopia in Chinese. Three SNPs of coding sequence TGIF gene in Chinese population abide by Hardy-Weinberg law.

[Variation of the peripherin gene in Chinese with or without high myopia].

To analyze the relationship of the peripherin gene(PRPH, OMIM17071) mutations with high myopia,genomic DNA was collected from 180 probands with high myopia (TTT(Phe21Phe,4/180), nt2138C-->G(IVS3,1/180), codon277 GCC-->ACC(Ala277Thr,8/180), codon237 CCA-->TCA (Arg237stop,1/180), codon292CCG-->CCA (Ala292Ala,1/180),codon361CUG-->CUC(Leu361Leu,12/180), codon369 AAA-->AAG(Lys369Lys,12/180),nt3331G-->C(IVS7,3/180)were detected in a number of probands as indicated in the blanket. Of the 8 variations one( codon 277,G-->A,Ala277Thr) is a missense mutation identified in 8 of the 180patients and one of 60 controls; The mutation of codon361 and codon 369 were synonymous one and linkage each other; Another one(codon237,CCA-->TCA,Arg237stop) is a heterozygous nonsense mutation identified in one patient with autosomal recessive inheritance mode population but not in the 60 normal controls. The others were synonymous mutations. Eight nucleotide variations were found in the PRPH gene. We found no evidence that mutations in the PRPH gene are responsible for the high myopia in Chinese.