16S rRNA gene sequencing analysis of gut microbiome in a mini-pig diabetes model

Background : Currently, increasing attention is being paid to the important role of in-testinal microbiome in diabetes. However, few studies have evaluated the characteris-tics of gut microbiome in diabetic miniature pigs, despite it being a good model animal for assessing diabetes. Methods : In this study, a mini- pig diabetes model (DM) was established by 9- month high- fat diet (HFD) combined with low- dose streptozotocin, while the animals fed standard chow diet constituted the control group. 16S ribosomal RNA (rRNA) gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini- pigs. Results : The results showed that microbial structure in diabetic mini- pigs was altered, reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1 , which were positively correlated with diabetes, and decreases in levels of the bac-teria Rikenellaceae , Clostridiales_vadinBB60_group , and Bacteroidales_RF16_group , which were inversely correlated with blood glucose and insulin resistance. Moreover, PICRUSt- predicted pathways related to the glycolysis and Entner- Doudoroff super-pathway, enterobactin biosynthesis, and the L - tryptophan biosynthesis were signifi-cantly elevated in the DM group. Conclusion : These results reveal the composition and predictive functions of the in-testinal microbiome in the mini- pig diabetes model, further verifying the relationship between HFD, gut microbiome, and diabetes, and providing novel insights into the application of the mini- pig diabetes model in gut microbiome research.     

The microbiome of a striped dolphin (Stenella coeruleoalba) stranded in Portugal

Infectious diseases with epizootic consequences have not been fully studied in marine mammals. Presently, the unprecedented depth of sequencing, made available by high-throughput approaches, allows detailed comparisons of the microbiome in health and disease. This is the first report of the striped dolphin microbiome in different body sites. Samples from one striped female edematous dolphin were acquired from a variety of body niches, including the blowhole, oral cavity, oral mucosa, tongue, stomach, intestines and genital mucosa. Detailed 16S rRNA analysis of over half a million sequences identified 235 OTUs. Beta diversity analyses indicated that microbial communities vary in structure and cluster by sample origin. Pathogenic, Gram-negative, facultative and obligate anaerobic taxa were significantly detected, including Cetobacterium, Fusobacterium and Ureaplasma. Phocoenobacter and Arcobacter dominated the oral-type samples, while Cardiobacteriaceae and Vibrio were associated with the blowhole and Photobacterium were abundant in the gut. We report for the first time the association of Epulopiscium with a marine mammal gut.The striped dolphin microbiota shows variation in structure and diversity according to the organ type. The high dominance of Gram-negative anaerobic pathogens evidences a cetacean microbiome affected by human-related bacteria.     

Contributions of the early-life microbiome to childhood atopy and asthma development

The rapid rise in atopy and asthma in industrialized nations has led to the identification of early life environmental factors that promote these conditions and spurred research into how such exposures may mediate the trajectory to childhood disease development. Over the past decade, the human microbiome has emerged as a key determinant of human health. This is largely due to the increasing appreciation for the myriad of non-mutually exclusive mechanisms by which microbes tune and train host immunity. Microbiomes, particularly those in early life, are shaped by extrinsic and intrinsic factors, including many of the exposures known to influence allergy and asthma risk. This has led to the over-arching hypothesis that such exposures mediate their effect on childhood atopy and asthma by altering the functions and metabolic productivity of microbiomes that shape immune function during this critical developmental period. The capacity to study microbiomes at the genetic and molecular level in humans from the pre-natal period into childhood with well-defined clinical outcomes, offers an unprecedented opportunity to identify early-life and inter-generational determinants of atopy and asthma outcomes. Moreover, such studies provide an integrative microbiome research framework that can be applied to other chronic inflammatory conditions. This review attempts to capture key studies in the field that offer insights into the developmental origins of childhood atopy and asthma, providing novel insights into microbial mediators of maladaptive immunity and chronic inflammatory disease in childhood.     

Ageing of the gut microbiome: Potential influences on immune senescence and inflammageing

Advancing age is accompanied by changes in the gut microbiota characterised by a loss of beneficial commensal microbes that is driven by intrinsic and extrinsic factors such as diet, medications, sedentary behaviour and chronic health conditions. Concurrently, ageing is accompanied by an impaired ability to mount a robust immune response, termed immunesenescence, and age-associated inflammation, termed inflammaging. The microbiome has been proposed to impact the immune system and is a potential determinant of healthy aging. In this review we summarise the knowledge on the impact of ageing on microbial dysbiosis, intestinal permeability, inflammaging, and the immune system and investigate whether dysbiosis of the gut microbiota could be a potential mechanism underlying the decline in immune function, overall health and longevity with advancing age. Furthermore, we examine the potential of altering the gut microbiome composition as a novel intervention strategy to reverse the immune ageing clock and possibly support overall good health during old age.     

Smoothelin and caldesmon are reliable markers for distinguishing muscularis propria from desmoplasia: a critical distinction for accurate staging colorectal adenocarcinoma

An accurate distinction between deep muscularis propria invasion versus subserosal invasion by colonic adenocarcinoma is essential for the accurate staging of cancer and subsequent optimal patient management. However, problems may arise in pathologic staging when extensive desmoplasia blurs the junction between deep muscularis propria and subserosal fibroadipose tissue. To address this issue, forty-three (43) cases of colonic adenocarcinoma resections from 2007-2009 at The Methodist Hospital in Houston, TX were reviewed. These cases were selected to address possible challenges in differentiating deep muscularis propria invasion from superficial subserosal invasion based on H&E staining alone. Immunohistochemical staining using smooth muscle actin (SMA), smoothelin, and caldesmon were performed on 51 cases: 8 cases of pT1 tumors (used mainly as control); 12 pT2 tumors; and 31 pT3 tumors. All 51 (100%) had diffuse, strong (3+) immunoreactivity for caldesmon and smoothelin in the muscularis propria with a granular cytoplasmic staining pattern. However, the desmoplastic areas of these tumors, composed of spindled fibroblasts and myofibroblasts, showed negative immunostaining for caldesmon and smoothelin (0/35). SMA strongly stained the muscularis propria and weakly (1+) or moderately (2+) stained the spindled fibroblasts in the desmoplastic areas (the latter presumably because of myofibroblastic differentiation). Compared to SMA, caldesmon and smoothelin are more specific stains that allow better delineation of the muscularis propria from the desmoplastic stromal reaction which provides a critical aide for proper staging of colonic adenocarcinoma and subsequent patient care.     

一种直接评价HPV16L1抗体活性的新方法

目的 :以pcDNAL1质粒免疫啮齿类动物 (C5 7BL/ 6 )为模型 ,观察HPV16L1VLP细胞结合抑制实验是否可以用于检测免疫抗体的中和保护作用。方法 :实验组包括Ⅰ组pcDNAL1、Ⅱ组HPV16L1VLP ;Ⅲ组pcDNA3.1。每组动物均为 6只C5 7BL/ 6鼠。每组动物均肌肉注射免疫 3次 ,间隔 3w。末次免疫后 14d眼球后取血并拉颈处死动物 ,进行HPV16L1VLP结合抑制试验 :免疫血清中和HPV16L1VLP ;制备Hela ,EJ和RLC310细胞爬片 ,CS12 13细胞涂片 ;细胞免疫组化染色。结果 :实验组血清中和后Hela细胞呈染色阴性 ,而对照组、不共育组则细胞呈棕黄色。结论 :这说明实验组血清具有抑制VLP与Hela细胞粘附的活性效应。这一方法可能比HAI更能直接反映中和抗体的活性和保护作用。     

Use of a reliable PCR assay for the detection of Neisseria gonorrhoeae in Peruvian patients

Neisseria gonorrhoeae is the most common sexually transmitted disease-causing bacterium worldwide. An in-house PCR assay targeting the carbamoyl-phosphate synthase subunit A (carA) gene was developed for the specific detection of N. gonorrhoeae in clinical specimens. Samples from 605 patients were cultured on selective medium and assayed by PCR in a double-blind fashion. Of 605 urethral/cervical samples analysed, 13 were PCR-positive, of which 11 were culture-positive. The PCR showed a sensitivity and specificity of 100% and 99.7% with these samples. PCR targeting the carA gene appears to be a reliable method for the detection of N. gonorrhoeae in clinical specimens.     

Peripheral education of the immune system by the colonic microbiota

There is growing interest in understanding the effects of host–microbial interactions on host physiologic processes. Much of the work in this arena is logically focused on the interaction at mucosal surfaces as this is a primary site of interaction. However, there is ample evidence to suggest that the effects of the microbiota have a much farther reach including the systemic immune system. While there are some similarities to effects at mucosal surfaces (i.e. reduced numbers of adaptive immune cells, diminished innate responses), there are some important differences that we highlight such as the response to immunogens and bacterial antigens. We propose that understanding the details of how specific components of the microbiota influence the systemic immune system likely will have significant impact on our understanding the pathophysiology of a variety of autoimmune diseases.     

Analysis of the factors affecting the formation of the microbiome associated with chronic osteomyelitis of the jaw

Chronic osteomyelitis of the jaw (COMJ) is one of the most intractable diseases among head and neck infections. Antimicrobial agents are routinely administered for COMJ without sufficient bacterial information, resulting in frequent treatment failures. To improve our knowledge of the bacterial aetiology of COMJ and to assist in the development of effective treatments, we performed a comprehensive analysis of the microbiome. Sixteen patients with four clinical types of COMJ (four with suppurative osteomyelitis, three with osteoradionecrosis of the jaw, four with primary chronic osteomyelitis, and five with bisphosphonate-related osteonecrosis of the jaw) were enrolled in this study. Bone samples were subjected to bacterial community comparisons by 16S rRNA gene pyrosequencing. As a result, we clarified that COMJ was caused by a far greater range of bacterial species (12 phyla and 163 genera) than previously reported. Moreover, the bacterial structures in COMJ changed dramatically with disease stage and the condition of the affected bone. Multiple correlation analyses revealed that sequestration and bone exposure could affect the community structure. On the basis of these factors, we reclassified COMJ into three clinical stages: I, inflamed or sclerotic bone without exposure; II, sequestrum without exposure; and III, exposed sequestrum. In stage II, the bacterial diversity was significantly lower, and the anaerobe genera Fusobacterium, Tannerella (formerly Bacteroides) and Porphyromonas were more abundant, than observed during other stages. Because these bacteria habitually reside in any clinical stage, they were considered to constitute the core microbiome of COMJ. Targeting these bacteria should lead to the development of effective preventive measures and cures.     

Value of anal swabs for SARS-COV-2 detection: a literature review

Facing the unprecedented global public health crisis caused by coronavirus disease 2019 (COVID-19), nucleic acid tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the gold standard for diagnosing COVID-19. The asymptomatic carriers were not suspected of playing a significant role in the ongoing pandemic, and universal nucleic acid screening in close contacts of confirmed cases and asymptomatic carriers has been carried out in many medium- and high-risk areas for the spread of the virus. Recently, anal swabs for key population screening have been shown to not only reduce missed diagnoses but also facilitate the traceability of infectious sources. As a specimen for the detection of viruses, the goal of this paper is to briefly review the transmission route of SARS-CoV-2 and the necessity of using anal swabs for SARS-CoV-2 screening to minimize transmission and a threat to other people with COVID-19.     

The microbiological characteristics of lower respiratory tract infection in patients with neuromuscular disorders: An investigation based on a multiplex polymerase chain reaction to detect viruses and a clone library analysis of the bacterial 16S rRNA gene sequence in sputum samples

We performed gene amplification methods for the detections of bacteria and viruses using sputum samples to clarify the microbiological characteristics of lower respiratory tract infection in patients with neuromuscular disorders. The tendencies of higher proportion of respiratory virus detection and lower diversity of bacteria in sputum were observed.     

The international sinonasal microbiome study: A multicentre,multinational characterization of sinonasal bacterial ecology

The sinonasal microbiome remains poorly defined, with our current knowledge based on a few cohort studies whose findings are inconsistent. Furthermore, the variability of the sinus microbiome across geographical divides remains unexplored. We characterize the sinonasal microbiome and its geographical variations in both health and disease using 16S rRNA gene sequencing of 410 individuals from across the world. Although the sinus microbial ecology is highly variable between individuals, we identify a core microbiome comprised of Corynebacterium, Staphylococcus, Streptococcus, Haemophilus and Moraxella species in both healthy and chronic rhinosinusitis (CRS) cohorts. Corynebacterium (mean relative abundance = 44.02%) and Staphylococcus (mean relative abundance = 27.34%) appear particularly dominant in the majority of patients sampled. Amongst patients suffering from CRS with nasal polyps, a statistically significant reduction in relative abundance of Corynebacterium (40.29% vs 50.43%; P = .02) was identified. Despite some measured differences in microbiome composition and diversity between some of the participating centres in our cohort, these differences would not alter the general pattern of core organisms described. Nevertheless, atypical or unusual organisms reported in short-read amplicon sequencing studies and that are not part of the core microbiome should be interpreted with caution. The delineation of the sinonasal microbiome and standardized methodology described within our study will enable further characterization and translational application of the sinus microbiota.     

16SrRNA基因在快速诊断新生儿败血症的病原菌研究

目的探讨血液16SrRNA基因检测在新生儿败血症诊断中的应用价值。方法分析细菌16SrRNA基因保守区,设计一对通用引物扩增已知实验菌株,检测其特异性,用倍比稀释法检测其敏感性,同时进行血培养。结果已知实验菌株均获得920bp扩增产物,对照组中的人类基因组DNA、HBV—DNA和白色假丝酵母菌无相应产物。敏感性测试能达到lpg大肠杆菌DNA。PCR阳性率为31．7％（20／63）,血培养阳性率为14．3％（9／63）,两者比较有显著性差异（P〈0.05）。结论PCR检测血液细菌16SrRNA基因,具有特异性强,敏感度高等特点,能在临床推广应用。     

Quantitative detection of cell culture Mycoplasmas by a one step polymerase chain reaction method

A rapid, sensitive assay was developed that can detect the six species of Mycoplasmas that account for the vast majority of cell culture infections. This assay, a modification of the method published by Wong-Lee & Lovett [1], allows direct evaluation of culture medium by a single-step PCR method that utilizes primers complementary to conserved 16S rRNA sequences. Extensive testing of medium from uninfected cultures spiked with purified Mycoplasma DNAs showed that the method described in this report can detect the equivalent of one Mycoplasma in 15 l culture medium; thus, evaluation of a single culture sample allows detection of Mycoplasmas in cultures infected with the equivalent of 10 or more Mycoplasmas per 15 l (or 6.7×10² Mycoplasma equivalents/ml) with greater than 99.99% confidence. Comparison of results obtained with this PCR-based assay and a standard biological colony-forming assay revealed that the PCR assay is capable of detecting 0.0015-0.015 colony forming units, suggesting that the PCR assay may also be detecting nonviable Mycoplasmas. The high level of amplification achieved with this method allows direct detection of amplification products by ethidium bromide staining of agarose gels, and thus allows rapid screening of cell cultures.Abbreviations CFU colony forming unit - PCR polymerase chain reaction     

Rectal mucosal endometriosis primarily misinterpreted as adenocarcinoma: a case report and review of literature

Endometriosis involving intestinal mucosa is relatively uncommon. It poses a diagnostic challenge for clinicians and pathologists. We herein report a case of colonoscopic specimen revealing rectal mucosal endometriosis. A 39-year-old woman complained of red rectal bleeding and intermittent abdominal pain. Colonoscopic examination showed a rectal mass with ulceration and circum wall involvement. Biopsy was processed in the suspicious of carcinoma. Morphologically, irregular glands replaced residual colorectal ones, displayed mucin depletion, nuclear stratification and subtile subnuclear vacuoles. The stroma was full of spindle cells with abundant pink cytoplasm and unclear boundary. Due to subjectively interpreting as dysplastic glands in desmoplastic setting, primary rectal adenocarcinoma was firstly raised. Immunohistochemically, CK7, ER and CD10 identified the essence of ectopic endometrium. CK20 and CDX2 highlighted residual glands. In case of misdiagnosis, any pathologists should be aware of intestinal endometriosis for each female’s colorectal biopsy, especially for that morphology not typical for primary adenocarcinoma or endometriosis. Reading slides carefully combined with a panel of immunomarkers would solve the pitfall.     

The long leg radiograph is a reliable method of assessing alignment when compared to computer-assisted navigation and computer tomography

BackgroundThe mechanical alignment of the knee is an important factor in planning for, and subsequently assessing the success of a knee replacement. It is most commonly measured using a long-leg anteroposterior radiograph (LLR) encompassing the hip, knee and ankle. Other modalities of measuring alignment include computer tomography (CT) and intra-operative computer navigation (Cas). Recent studies comparing LLRs to Cas in measuring alignment have shown significant differences between the two and have hypothesized that Cas is a more accurate modality. This study aims to investigate the accuracy of the above mentioned modalities.MethodologyA prospective study was undertaken comparing alignment as measured by long-leg radiographs and computer tomography to intra-operative navigation measurements in 40 patients undergoing a primary total knee replacement to test this hypothesis. Alignment was measured three times by three observers. Intra- and inter-observer correlation was sought between modalities.ResultsIntra-observer correlation was excellent in all cases (> 0.98) with a coefficient of repeatability < 1.1°. Inter-observer correlation was also excellent measuring > 0.960 using LLRs and > 0.970 using CT with coefficient of repeatability < 2.8°. Inter-modality correlation proved to be higher when comparing LLRs and CT (> 0.893), than when comparing either of these modalities with Cas (> 0.643 and > 0.671 respectively). Pre-operative values had the greatest variability.ConclusionGiven its availability and reduced radiation dose when compared to CT, LLRs should remain the mainstay of measuring the mechanical alignment of the lower limb, especially post-operatively.Level of evidenceII     

Transport of amino-acids and sugars by the dog colonic mucosa

Summary Dog colon mucosa is able to concentrate amino-acids and sugars against a concentration gradient during incubation in vitro. Amongst other species tested, this property is only shared, to any reasonable extent, by the mouse colon.The properties of the dog colon transport systems have much in common with corresponding mechanisms in the small intestine (Na⁺-dependence, energy-dependeace, saturation kinetics), but their capacities are considerably lower.The difference between species (rats, guinea-pigs, mice and humans) is probably quantitative rather than qualitative in nature, since entry into rat colon slices is affected by the absence of Na⁺ ions and inhibited by homologues.     

Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer

Aberrant promoter hypermethylation of CpG dinucleotides is a frequent and significant mechanism of tumor suppressor gene (TSG) silencing in cancer. As increasing numbers of downregulated putative TSGs are emerging from large-scale expression profiling studies, high-throughput techniques are needed to screen for hypermethylation. DHPLC has been established as a reliable, highly sensitive technique for mutation analysis. In this study, the use of DHPLC as a prescreening method for the identification of CpG methylation was developed by analyzing DNA samples with different, well-characterized methylation patterns of the CDKN2A/p16 promoter. Bisulfite treatment of genomic DNA was followed by PCR-amplification of unmethylated as well as methylated CDKN2A/p16 promoter sequences. PCR products were denatured and renatured, permitting the formation of heteroduplex DNA detectable by DHPLC. Methylation of all CpG-sites results in a single peak (homoduplex) with a shift in retention time, whereas partial methylation can be recognized by additional signals representing diverse heteroduplex structures. After method development, 35 DNA samples from primary bladder and breast carcinomas were analyzed in a blinded fashion, revealing complete or partial methylation of the p16 promoter in eight cases and a heterozygous mutation in one case. In conclusion, DHPLC is a highly sensitive and convenient method for methylation screening.     

Repair of the surface epithelium after saponin-induced colonic mucosal injury in the rat

The superficial colonic epithelia of rats were exposed to 1.0% saponin solution for 3 min and fixed at various periods thereafter. The repair or restitution process was observed by light as well as by transmission and scanning electron microscopy. The exposure of the luminal surface to saponin resulted in uniform and extensive damage to the superficial epithelial cells without affecting the cells in the crypts. At 3 min after saponin treatment, the damaged epithelial cells exfoliated from the mucosa and the basal lamina was exposed. Within 15 min, most of the exposed basal lamina was covered by squamous to low-cuboidal epithelial cells, probably migrating from the crypts. These epithelial cells extended large lamellipodia over the denuded basal lamina. After 15 min the damaged surface was completely covered with epithelial cells, which became columnar at 1 h. Tight junction protein ZO-1 became positive along the restituted epithelium. Proliferating-cell nuclear antigen (PCNA) staining showed that proliferation of epithelial cells occurred after the restitution. These results suggest that saponin treatment serves as a good model system to study colonic restitution, which is carried out by rapid migration from the remaining crypt cells, followed by cellular proliferation. Rapid formation of tight junctions spanning the damaged regions allows rapid restoration of the barrier function of the colonic epithelium.