种植体黏膜下微生物在健康种植体和种植体周炎中的构成与差异：一项横断面研究

目的：探索种植体黏膜下微生物在健康种植体和种植体周炎中的构成与差异,并分析与临床指标存在相关性的菌种,为种植体周炎的病因学研究提供参考。方法：采用横断面研究,共纳入49例患者,20例为健康种植体,29例为种植体周炎,共采集49份黏膜下微生物样本进行16S核糖体RNA(16S ribosomal RNA, 16S rRNA)基因高通量测序。对两组样本的多样性、菌群构成和差异物种进行分析和比较,采用Spearman相关性分析评价菌种与探诊深度(probing pocket depth, PPD)之间的相关性。结果：健康组的α多样性显著低于种植体周炎组[Chao1指数：236.85±66.13 vs. 150.54±57.43,P<0.001; Shannon指数：3.42±0.48 vs. 3.02±0.65,P=0.032]。主成分分析显示,两组样本的群落结构差异有统计学意义[相似性分析(analysis of similarities, ANOSIM),R²=0.243,P=0.001]。与健康种植体相比,种植体周炎黏膜下菌斑中的牙周致病菌显著增加,包括红色...     

Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity

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Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR?<?0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR?<?0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community.

• HIGHLIGHTS

• Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds.

• Differences were found between infected and healthy animals in gut microbiota.

• Candidatus Arthromitus was closely associated with infected birds.

• M. ornithogaster can affect intestinal microbiota composition of canaries.

     

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

TP53 mutations in prostatic cancer. Analysis of pre- and post-treatment archival formalin-fixed tumour tissue

The TP53 gene mutation pattern in prostatic cancer was examined in relation to progression and survival, using archival formalin-fixed pre-and post-treatment tumour specimens from 84 prostatic cancer patients. Thirty-four had hormone-sensitive tumours and 50 were hormone-resistant. Six of the 34 (18 per cent) therapy-responding tumours and 19 of the 50 (38 per cent) hormone-resistant tumours showed p53 protein accumulation in the post-treatment specimen. Both pre- and post-treatment specimens from these 25 patients were analysed for mutation of the conserved regions of the TP53 gene (exons 5–8), using constant denaturant gel electrophoresis (CDGE) followed by DNA sequencing. In the post-treatment samples, mutations were detected in three of the six patients with hormone-responsive tumours and in 11 of the 19 patients with hormone-resistant tumours. The three (100 per cent) patients with therapy-responsive tumours with mutations and nine of the 11 (82 per cent) patients with therapy-resistant tumours with mutations died of the disease. Thirteen of the 14 mutations in the post-treatment specimens were transitions, 11 occurring at CpG dinucleotides in which codon 273 was involved in ten. A significantly higher proportion of tumours with mutations were poorly differentiated compared with tumours without mutation (P<0·04). Our findings indicate that TP53 mutation is a late event in tumour development of the prostate gland and that codon 273 might be a ‘hotspot’ for mutation in the progression of the disease.     

Simple,rapid, and accurate determination of deletion mutations by automated dna sequencing of heteroduplex fragments of the adenomatous polyposis coli (APC) gene generated by PCR amplification

Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were “bulge” heteroduplexes, while the other two were homoduplexes made up of two wild-type strands and two deletion-mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion-carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases. © 1993 Wiley-Liss, Inc.     

新疆出血热病毒核蛋白基因的高效表达及其在诊断中的初步应用

目的　克隆并测定了克里米亚 刚果出血热病毒 (CCHFV)中国分离株 (新疆出血热病毒 ,XHFV)BA8816 6株核蛋白 (NP)基因的序列并实现其在细菌中的高效表达与临床诊断的应用。方法　病毒RNA经RT PCR扩增出完整的NP基因。将扩增产物进行序列分析并克隆至融合表达载体pET32a ,使重组质粒在大肠杆菌BL 2 1中高效表达。将融合蛋白经初步纯化后包被ELISA板用于抗体检测。结果　XHFVBA8816 6株NP基因序列以及推导的氨基酸序列与其它XHFV的NP基因和蛋白序列同源性较高 ,在进化树上形成独立的分支。BA8816 6株NP基因编码 4 82个氨基酸的核蛋白 ,推测的相对分子质量 (Mr)约为 5 4× 10 3。在细菌中表达的融合蛋白经印迹试验证明具有良好的抗原性。以所建立的ELISA方法检测疫区人和动物血清的结果与IFA一致 ,并与临床诊断有很好的符合率。结论　BA8816 6株与其它XHFVBA6 6 0 19、BA84 0 2的NP基因在进化上关系密切 ,综合M基因的序列分析结果 ,人源分离株BA8816 6可能是来自蜱的BA84 0 2变异株。表达于细菌中的核蛋白可作为安全的诊断性抗原用于临床检测及流行病学调查 ,所建立的方法准确、特异、简便、快速     

Female‐restricted syndromic intellectual disability in a patient from Thailand

Female‐restricted syndromic intellectual disability (ID) is a neurodevelopmental disorder with developmental delay (DD)/ID, facial dysmorphism, and diverse congenital anomalies comprising heart defects, anal anomalies, choanal atresia, postaxial polydactyly, scoliosis, and brain abnormalities. Loss‐of‐function mutations in the USP9X gene inherited as X‐linked dominance were identified as its etiology in females of different ethnic groups. Here, we report a 15‐year‐old Thai girl harboring a novel de novo heterozygous one‐base pair deletion (c.3508delG, p.Val1170TrpfsX9) in exon 23 of USP9X. Her profound DD, dysmorphic face including attached earlobes, short stature, and congenital malformations including s‐shaped thoracolumbar scoliosis, hip dislocation, and generalized brain atrophy shared common characteristics of X‐linked syndromic ID. We have observed severely malformed oro‐dental organs and a choledochal cyst, which have never been reported. Our study presents the first patient from Thailand expanding the phenotypic and mutational spectra of the syndrome.     

Phylogenetic Analysis of the DNA Polymerase Gene of a Novel Alphaherpesvirus Isolated from an Indian <Emphasis Type="Italic">Gyps</Emphasis> Vulture

The DNA polymerase gene of a novel herpesvirus, vulture herpesvirus (VHV), isolated from an Indian Gyps vulture was completely sequenced using primer walking and transposon insertion strategies. DNA sequencing analysis revealed a single open reading frame (ORF) of 3660 nucleotides (53% G-C content) able to encode 1219 amino acids. Identification was based on a nucleotide sequence identity of approximately 50% to other herpesvirus sequences found in Genbank. Nine motifs were identified that are conserved amongst all known herpesviruses and are found within the 3–5 exonuclease and DNA binding functional domains of the DNA polymerase enzyme. Phylogenetic analysis using Clustal W with neighbour-joining revealed VHV to group within the subfamily Alphaherpesvirinae, more closely related to the avian herpesviruses than to those of other species. Partial sequence data also revealed VHV to contain other genes fundamental to the structure and replication of all herpesvirus genomes. A Real Time PCR Taqman assay specific for the VHV DNA polymerase gene was designed to detect the presence of VHV genomic material in post mortem tissue samples from diseased birds. Positive tissues included the spleen, rectum, thymus, kidney and brain. A herpesvirus specific to vultures may pose a threat to the management of captive breeding programs being established to assist the survival of wild populations of Gyps vultures.