Microbial Screening Research Articles

Características epidemiológicas de pacientes com sepse em unidade de terapia intensiva

Justificativa e Objetivos: Apesar das novidades relacionadas ao diagnóstico precoce e ao rastreamento microbiano dos casos de sepse, faz-se necessário compreender os aspectos clínico-epidemiológicos dos serviços de saúde visando definir as especificidades dos pacientes acometidos em Unidades de Terapia Intensiva (UTI). Nesse contexto, objetiva-se descrever as características epidemiológicas de pacientes com sepse em UTI. Métodos: Trata-se de uma pesquisa exploratória-descritiva, retrospectiva, documental, com abordagem quantitativa, realizada entre maio e julho de 2018, em hospital de ensino da Zona Norte do Estado do Ceará. Utilizou-se como fonte de dados os prontuários de pacientes que apresentaram sepse nas UTI do referido hospital, alcançando-se uma amostra de 62 prontuários para análise. Os dados coletados foram armazenados nos programas Microsoft Excel e Origin Lab 8 para posterior construção de gráficos e tabelas para análise das informações. Resultados: Os casos tiveram predominância em indivíduos do sexo masculino, residentes em municípios interioranos circunvizinhos e adultos jovens com idade entre 19 e 39 anos. O traumatismo cranioencefálico e o politraumatismo foram os diagnósticos iniciais que prevaleceram. As bactérias grampositivas tiveram maior prevalência, superando os bacilos gram-negativos e os fungos. Os antimicrobianos mais utilizados foram Cefepime e Vancomicina. As evoluções das infecções estiveram associadas a fatores como: estado de saúde dos pacientes; utilização de dispositivos invasivos e longo período de internação. Conclusão: As informações obtidas nesta pesquisa indicam que os dados epidemiológicos desempenham papel crucial no que diz respeito à aplicação de novos recursos, tecnologias e tratamentos. Tais conhecimentos permitirão desenvolver estratégias e condutas direcionadas à qualidade na assistência intensiva.

Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain

Here, we describe the metagenome composition of a microbial community in a hot spring sediment as well as a sequence-based and function-based screening of the metagenome for identification of novel xylanases. The sediment was collected from the Lobios Hot Spring located in the province of Ourense (Spain). Environmental DNA was extracted and sequenced using Illumina technology, and a total of 3.6 Gbp of clean paired reads was produced. A taxonomic classification that was obtained by comparison to the NCBI protein nr database revealed a dominance of Bacteria (93%), followed by Archaea (6%). The most abundant bacterial phylum was Acidobacteria (25%), while Thaumarchaeota (5%) was the main archaeal phylum. Reads were assembled into contigs. Open reading frames (ORFs) predicted on these contigs were searched by BLAST against the CAZy database to retrieve xylanase encoding ORFs. A metagenomic fosmid library of approximately 150,000 clones was constructed to identify functional genes encoding thermostable xylanase enzymes. Function-based screening revealed a novel xylanase-encoding gene (XynA3), which was successfully expressed in E. coli BL21. The resulting protein (41 kDa), a member of glycoside hydrolase family 11 was purified and biochemically characterized. The highest activity was measured at 80 °C and pH 6.5. The protein was extremely thermostable and showed 94% remaining activity after incubation at 60 °C for 24 h and over 70% remaining activity after incubation at 70 °C for 24 h. Xylanolytic activity of the XynA3 enzyme was stimulated in the presence of β-mercaptoethanol, dithiothreitol and Fe3+ ions. HPLC analysis showed that XynA3 hydrolyzes xylan forming xylobiose with lower proportion of xylotriose and xylose. Specific activity of the enzyme was 9080 U/mg for oat arabinoxylan and 5080 U/mg for beechwood xylan, respectively, without cellulase activity.

Plant Growth Promotion Driven by a Novel Caulobacter Strain.

Soil microbial communities hold great potential for sustainable and ecologically compatible agriculture. Although numerous plant-beneficial bacterial strains from a wide range of taxonomic groups have been reported, very little evidence is available on the plant-beneficial role of bacteria from the genus Caulobacter. Here, the mode of action of a Caulobacter strain, designated RHG1, which had originally been identified through a microbial screen for plant growth-promoting (PGP) bacteria in maize (Zea mays), is investigated in Arabidopsis thaliana. RHG1 colonized both roots and shoots of Arabidopsis, promoted lateral root formation in the root, and increased leaf number and leaf size in the shoot. The genome of RHG1 was sequenced and was utilized to look for PGP factors. Our data revealed that the bacterial production of nitric oxide, auxins, cytokinins, or 1-aminocyclopropane-1-carboxylate deaminase as PGP factors could be excluded. However, the analysis of brassinosteroid mutants suggests that an unknown PGP mechanism is involved that impinges directly or indirectly on the pathway of this growth hormone.

Low frequency cyclical potentials for fine tuning insulator-based dielectrophoretic separations.

In this study, we demonstrate the use of cyclical low frequency signals with insulator-based dielectrophoresis (iDEP) devices for the separation of particles of similar characteristics and an experimental method for estimating particle DEP mobilities. A custom signal designer program was created using Matlab® and COMSOL Multiphysics® for the identification of specific low frequency signals aimed at separating particle mixtures by exploiting slight differences in surface charge (particle zeta potential) or particle size. For the separation by surface charge, a mixture of two types of 10 μm particles was analyzed and effectively separated employing both a custom step signal and a sawtooth left signal. Notably, these particles had the same shape, size, and surface functionalization as well as were made from the same substrate material. For the separation by size, a sample containing 2 μm and 5 μm particles was successfully separated using a custom step signal; these particles had the same shape, surface functionalization, were made from the same substrate materials, and had only a small difference in zeta potential (10 mV). Additionally, an experimental technique was developed to estimate the dielectrophoretic mobility of each particle type; this information was then utilized by the signal designer program. The technique developed in this study is readily applicable for designing signals capable of separating micron-sized particles of similar characteristics, such as microorganisms, where slight differences in cell size and the shape of surface charge could be effectively exploited. These findings open the possibility for applications in microbial screening using iDEP devices.

Status of Microbial Remediation Technology in Petroleum Contaminated Land

Leakage accidents occur frequently during the process of oil exploitation, storage and transportation. Effective management of the soil contaminated by it has become the focus of social attention. This paper introduces the oil pollution problem and physical, chemical and biological treatment methods. It focuses on the classification and application status of microbial remediation technology, and discusses the key factors that restrict bioremediation effects such as degrading microbial screening and colonization, and improving the bioavailability of petroleum hydrocarbons.

Isolation and Microbial Screening of 1-Methylcyclopentene from Bracystelma Togoense Schtlr

Isolation and Microbial Screening of 1-Methylcyclopentene from Bracystelma Togoense Schtlr

355 Establishing Ovine Model of Burn Wounds Grafted with Cadaver Skin

Shortage in autograft skin to cover burn wounds require a frequent use of cadaver skin as a temporary cover. Cadaver skin graft prevents infection and dehydration and prepares the wound bed for subsequent skin autograft or application of autologous tissue engineered skin constructs. The aim of this study was to establish an ovine model of burn wound healing using ovine cadaver skin. Quality and efficacy of fresh and frozen cadaver skins were evaluated comparing to skin autograph. Cadaver skin samples were harvested from sheep used for different studies at necropsy. The cadaver skin preparation process consisted of 4 steps: preparation for recovery by wool shaving and applying primary disinfection, collection, disinfection, and cryopreservation. First, to fully characterize fresh and frozen cadaver skin, the harvested skin was evaluated based on microbial screening, skin thickness, viability, and cell proliferation in in vitrocondition. The froze skin was evaluated after 10 and 40 days of freezing. Then we compared acceptance/rejection of autograft, freshly prepared and frozen cadaver skins overlaid onto burn ovine wounds. Full thickness skin burns (5X5cm) were induced in sheep dorsum. After 24 hrs, burned skins were excised and wounds were allocated to following groups: 1) Fresh ovine cadaver skin, 2) Frozen cadaver skin, 3) Autograft, 4) No cover. Pictorial record and skin biopsies were collected 7, 14 and 20 days after grafting cadaver skin. Prepared cadaver (fresh & frozen) skins were negative for bacterial growth. Thethickness was comparable between the freshly prepared and frozen skins for 10 or 40 days. Significant reduction in skin viability was detected in frozen skin after freezing for 40 days. Histological analysis of burn wounds biopsies showed that skin autograft maintains normal skin structure at different time points. However, fresh cadaver skin graft showed signs (vacuoles in epidermis) of rejection starting from day 7 as evidenced by microscopic analysis. At day 14, the epidermis was mostly rejected, and the rejection was completed by day 20. Histologically, signs of immunoreaction and presence of many immune cells were noted. Frozen Cadaver skin followed the same pattern of fresh cadaver skin rejection rate. Viability of fresh frozen cadaver skin before grafting was significantly higher vs. frozen (40 days). However, on average both fresh or frozen ovine cadaver skins were rejected within 8.5 days that mimics rejection time in humans (~8.4 days), suggesting that ovine model of burn wound grafted with cadaver skin can successfully be used in burn wound research mimicking clinical scenario. Establishing a clinically relevant ovine model for burn wounds grafted with cadaver skin is a potential contribution to the burn research.

Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering.

SummaryIndustrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.

The role of a sequencing-based clinical intestinal screening test in patients at high-risk for Clostridium difficile and other pathogens: a case report

BackgroundHospitalization and antibiotic treatment can put patients at high risk for Clostridium difficile infection, where a disturbance of the gut microbiome allows for Clostridium difficile proliferation and associated symptoms, including mild, moderate, or severe diarrhea. Clostridium difficile infection is challenging to treat, often recurrent, and leads to almost 30,000 annual deaths in the USA alone. Here we present a case where SmartGut™, an at-home, self-administered sequencing-based clinical intestinal screening test, was used to identify the presence of Clostridium difficile in a patient with worsening diarrhea. Identification of this pathogen and subsequent treatment led to a significant improvement in symptoms.Case presentationThe patient is a 29-year-old white woman with a history of severe irritable bowel syndrome with diarrhea, hemorrhoidectomy, and anal sphincterotomy complicated by a perianal fistula and perirectal abscesses that required extended courses of broad-spectrum antibiotics. In June 2016, she developed intermittent Clostridium difficile infections, which required continued antibiotic use. Months later she used an at-home, self-administered, intestinal microbial test, the first of which was negative for the presence of Clostridium difficile, but it detected the relative abundance of microbes associated with irritable bowel syndrome outside the healthy reference ranges. In the subsequent 2 months after the negative Clostridium difficile result, her gastrointestinal symptoms worsened dramatically. A second microbiome test resulted in a positive Clostridium difficile finding and continued abnormal microbial parameters, which led the treating physician to refer her to a gastroenterologist. Additional testing confirmed the presence of Clostridium difficile with a toxin-positive strain. She received treatment immediately and her gastrointestinal symptoms improved significantly over the next week.ConclusionsThis case report suggests that more frequent DNA testing for Clostridium difficile infections may be indicated in patients that are at high-risk for Clostridium difficile infection, especially for patients with irritable bowel syndrome, and those who undergo gastrointestinal surgery and/or an extended antibiotic treatment. This report also shows that such testing could be effectively performed using at-home, self-administered sequencing-based clinical intestinal microbial screening tests. Further research is needed to investigate whether the observations reported here extrapolate to a larger cohort of patients.

Domestic Wastewater Treatment and Lipid Accumulation for Biodiesel Production by an Isolated Heterotrophic Microalgae from an Arid Climate Zone

The isolation of microalgae that can use carbon-rich pollutant from wastewater and accumulate lipids is of great interest in biodiesel production. The heterotrophic cultivation of microalgae could overcome light dependency, and hence increase the yield of microalgae lipid per unit area. After several microbial screening and acclimation procedures, strains of microalgae were proved to be tolerant of wastewater and to grow heterotrophically. One top-performing strain (AE2) was identified through morphological observation as Chlorella sp and was isolated from an open pond of domestic wastewater. This microalgae is able to grow in raw wastewater at 20°C with no illumination and eliminates 100% of its COD (424 mg/L) in 9 days. The biomass produced in wastewater as growth medium comprises 53% of fats of the dry mass and 68% of fats of the dry mass in BG 11 culture medium supplemented with 5 g of glucose/L. The analysis of fatty acid methyl esters FAME composition was 32.5 % of the total biomass. The extracted microalgae oil was converted to good quality biodiesel. This heterotrophic isolated microalgae is a promising strain in terms of wastewater COD removal and cytoplasmic accumulation of high quality and quantity lipids for prospective biodiesel production.

CYTOTOXICITY ASSESSMENT AND ANTIMICROBIAL ACTION OF Caulerpa taxifolia (M. Vahl) C.

This study aims to determine the LC50 for Artemia salina Leach and perform a microbial screening two C. taxifolia extracts. In these biological assays have been used larvae of A. salina Leach ob-tained from the incubation of about 20 mg of A. salina cysts, under artificial light for 48 hours. Where groups of 10 metanauplius were exposed to different concentrations (50 mg / mL to 1000 ug / ml) of methanol extract and hydroalcoholic C. taxifolia . The antimicrobial activity of extracts of C. taxifolia was evaluated by diffusion method in paper disc. The percentage mortality deter-mined after 24 hours of contact. Through the data was calculated LC50. Behavioral analysis of Artemia salina Leach showed a decrease in handling compared to those observed in the control groups. The microbial activity of the extracts were evaluated by measuring the halo of inhibition for two species of gram-positive and two gram-negative bacteria. The extracts showed signifi-cant results for S. aureus and S. spp. This study demonstrates that C. taxifolia has a high cyto-toxicity suggested that its use in cell culture as a molluscicide and can be used in the production of production of antimicrobial drugs.

A programmable microscopic stage: Design and development.

Microscopes have become a significant part of pathological study. Currently, motorized microscopes are enabled with horizontal and vertical movements, with the facility of fast response in acquiring images or videos from the slide. During microbial screening, viewing the specimen needs following a directional path viz., zig-zag, inward spiral, meander, and so forth. Even though the motorized movements are built-in, human intervention is required while screening. This leads to time delay in the scanning process and may leave some portions of the specimen unattended. In this proposed system, a programmable framework to define the scanning direction for the specimen and a firmware to control the microscopic stage is implemented, to enable customization of the scanning pattern without any human intervention during complete course of screening. The user can define the customized scanning pattern using two-dimensional (2D) graphics drawing primitives. The final drawing is converted into preparatory codes through a micro-computer numeric controlled software, which extracts the address information relating to the movement and direction of the stage. These X, Y directional information are fed into the machine control unit for activating the linear driving system, which has servo drives and motors to power the spindle for precise microscopic stage movement. The proposed system is cost effective and reduces the reliance on technicians in examining the whole slide. The system is also portable and can be attached to any conventional or fluorescence microscope. Some pre-defined scanning patterns have been tested for the stage movements and validated in this work.

Limiting factors of saffron corm production from the perspective of microorganisms

BackgroundSaffron is a costly spice and a valuable medicine with high added value among agricultural crops. ObjectiveTo explore the role of bacteria and fungi in the limitation of saffron corm production from two aspects: the corms and soil. MethodsSoil samples and fifty corm samples that could not germinate or bloom were collected from South Tai Lake Agricultural Park. Next-generation sequencing technology was used to sequence bacterial 16S ribosomal RNA and fungal ITS ribosomal RNA. A bioinformatics analysis method was used to profile the microbial community structure and screen distinctive bacteria and fungi in the corms and soils. ResultsThere were large variations in fungal types in different soil samples, but no obvious differences were observed in the main bacterial types or bacterial abundance. The abundance of fungi such as Ilyonectria macrodidyma, Mortierella humilis and Chaetomiaceae was markedly higher in soil samples showing good corm growth. The fungi that affected corm germination and blooming mainly included Nectriaceae, Penicillium, Aspergillus and Saccharomycetales. Parts of corms that could germinate but did not bloom may have been infected by Serratia and Penicillium chrysogenum. ConclusionMicroorganisms may provide a direction for improving corm production in the two-stage cultivation of saffron.

Terminal restriction fragment length polymorphism is an \u201cold school\u201d reliable technique for swift microbial community screening in anaerobic digestion

The microbial community in anaerobic digestion has been analysed through microbial fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP), for decades. In the last decade, high-throughput 16S rRNA gene amplicon sequencing has replaced these techniques, but the time-consuming and complex nature of high-throughput techniques is a potential bottleneck for full-scale anaerobic digestion application, when monitoring community dynamics. Here, the bacterial and archaeal TRFLP profiles were compared with 16S rRNA gene amplicon profiles (Illumina platform) of 25 full-scale anaerobic digestion plants. The α-diversity analysis revealed a higher richness based on Illumina data, compared with the TRFLP data. This coincided with a clear difference in community organisation, Pareto distribution, and co-occurrence network statistics, i.e., betweenness centrality and normalised degree. The β-diversity analysis showed a similar clustering profile for the Illumina, bacterial TRFLP and archaeal TRFLP data, based on different distance measures and independent of phylogenetic identification, with pH and temperature as the two key operational parameters determining microbial community composition. The combined knowledge of temporal dynamics and projected clustering in the β-diversity profile, based on the TRFLP data, distinctly showed that TRFLP is a reliable technique for swift microbial community dynamics screening in full-scale anaerobic digestion plants.

Optimization of Media and Reaction Conditions for Production of Polyol Oils from Soybean Oil by Pseudomonas aeruginosa E03-12 NRRL B-59991

Abstract Soy-polyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. We reported methods for microbial screening and production of polyol oils from soybean oil through bioprocessing (Hou and Lin, 2013). Using this screening method, we screened 650 cultures isolated and found 50 cultures were positive for converting soybean oil to polyol oil. The two most active of these positive cultures are Acinetobacter haemolyticus A01-35 and Pseudomonas aeruginosa E03-12 (Hou et al., 2015). The polyol oil produced by strain A01-35 was a mixture of hydroxy fatty acids-containing DAG. Strain E03-12 produced more polyol oil than strain A01-35 and its product contains both hydroxy fatty acids-containing DAG and TAG. We employed one-factor-at-a-time method to develop an optimized culture medium composition and reaction conditions for polyol oils production from soybean oil by strain E03-12. We found that among the different carbon sources studied, glucose at 12.5 g/L is the best. A combination of both tryptone at 12.5 g/L and yeast extract at 10 g/L serves as the best nitrogen source. Both iron and magnesium were studied for the effect of metal on the production of polyol oils. MgSO4 7H2O at 0.75 g/L and FeSO4 7H2O at 0.30 g/L produced the best result. Using this optimum medium composition, the best pH for the polyol oils production was found at 6.8. The optimum temperature for polyol oil production is 28 C. The optimum substrate concentration is 183 mg/30 mL. In the time course studies with these optimum reaction conditions, the best yield is 18.5 mg/30 mL at 48 h. Longer incubation time leads to the decrease in product polyol oils and increase in DAGs. The information obtained from this study is important for developing scale up production of polyol oils directly from soybean oil.

Effects of synergistic fungal pretreatment on structure and thermal properties of lignin from corncob

Synergistic fungal pretreatment involving white-rot and brown-rot fugal pretreatment has shown great potential for enhancing the yield of sugars from biomass time-effectively and environmentally benign. In this work, the effects of this integrated fungal pretreatment on lignin characteristics and thermal behavior of corncob were examined in the view of whole plant valorization. The results show that an efficient deconstruction of lignin was achieved by white-rot fungus, and subsequent brown-rot fungus promoted the preferential breakdown of guaiacyl units, further enhancing lignin extraction efficiency (62.3%). Consequently, less phenolic hydroxyl, methoxyl, tricin, ester-linked p-coumaric acid, more carboxylic acid, ratio of syringyl to guaiacyl units, β-O-4′ linkage and molecular weight were found in W-BL. Thermal stability was improved and the increased phenol and alkyl-phenols contents in pyrolysis products demonstrated that synergistic fungal pretreatment definitely improved the lignin oil quality. These discoveries provide new insights into set strategy for microbial screening, pretreatment and lignin processing.

Microbial Genetic Screens for Monitoring Protein Misfolding Associated with Neurodegeneration: Tools for Identifying Disease-Relevant Genes and for Screening Synthetic and Natural Compound Libraries for the Discovery of Potential Therapeutics.

Neurodegenerative Diseases (ND) are a major threat to the aging population and the lack of a single preventive or disease-modifying agent only serves to increase their impact. In the past few years, protein misfolding and the subsequent formation of neurotoxic oligomeric/aggregated protein species have emerged as a unifying theme underlying the pathology of these complex diseases. Recently developed microbial genetic screens and selection systems for monitoring ND-associated protein misfolding have allowed the establishment of highthroughput assays for the identification of cellular factors and processes that are important mediators of NDassociated proteotoxicities. In addition, such systems have facilitated the discovery of synthetic and natural compounds with the ability to rescue the misfolding and the associated pathogenic effects of aggregation-prone proteins associated with NDs. This review outlines such available systems in bacteria and yeast, whose usage will likely accelerate the pre-clinical discovery process for effective drugs against a variety of NDs with high socioeconomic impact.

Intestinal microorganisms involved in colorectal cancer complicated with dyslipidosis

ABSTRACTBackground: Abnormal lipid metabolism is considered to be one of main promoters of colorectal cancer (CRC), and intestinal microorganisms may be involved in CRC in patients with abnormal lipid metabolism.Objective: To investigate lipid metabolism in CRC patients and explore the role of intestinal microorganisms in CRC complicated with abnormal lipid metabolism.Methods: Overall, 150 CRC patients in Huzhou Central Hospital from January 2016 to September 2017 were recruited in the present study. Basic patient information and clinical serological indicators were investigated and analyzed. Twenty-one stool samples were collected from patients after receiving informed consent. Next-generation sequencing technology was used to sequence bacterial 16S ribosomal RNA. Bioinformatics analysis was used to profile the microbial composition and screen distinctive bacteria in patients with CRC complicated with abnormal lipid metabolism.Results: Apo B and FFA levels were higher in patients with stage I disease than in patients with other stages. HDL, LDL, Apo B and FFA levels were higher in female patients than in male patients. FFA level was higher in rectal cancer patients than in colon cancer patients. These differences were statistically significant (p < 0.05). The proportion of Escherichia/Shigella was increased in CRC patients with hyperlipoidaemia and hypercholesteremia; the abundance of Streptococcus was increased in CRC patients with hyperlipoidaemia; the abundance of Clostridium XIVa was reduced in CRC patients with hyperlipoidaemia and hypercholesteremia; and the abundance of Ruminococcaceae was reduced in CRC patients with hypercholesteremia. Bilophila and Butyricicoccus were closely related to CRC patients without hyperlipoidaemia or hypercholesteremia, and Selenomonas, Clostridium, Bacteroidetes Slackia, Burkholderiales and Veillonellaceae were closely related to CRC patients with hyperlipoidaemia. Some pathways, including secretion system, chaperones and folding catalysts, amino sugar and nucleotide sugar metabolism, arginine and proline metabolism, glycine, serine and threonine metabolism, histidine metabolism, pores and ion channels, nitrogen metabolism and sporulation, may be involved in lipid metabolism abnormality in CRC patients.Conclusions: Many CRC patients have abnormal lipid metabolism, and the intestinal microbiota is altered in these CRC patients.

Abstract 2086: Comprehensive microbial and immunological screening of gastroesophageal biopsies: A multi-parametric analysis to study carcinogenesis

Abstract The development of gastric cancer in the context of chronic H. pylori infection is multifactorial, encompassing both bacterial factors and the altered immune microenvironment. We proposed an unbiased study to evaluate the relationships among microbiome composition, host immune response and genomic characterization from next generation sequencing of gastric biopsy samples. Endoscopic biopsies collected from patients with active H. pylori infection (n=21), prior infection (n=22) and no prior infection (n=26), and were sequenced at 10X to 30X coverage. In total, 77 gastric biopsies from 69 patients were freshly frozen for whole genome sequencing and RNASeq analysis. Detecting the microbiome from human biopsy sequencing data directly is challenging due to the low microbial content. A novel computational pipeline was developed to address this problem specifically. In our analysis population, in addition to the robust H. pylori signal, several bacteria associated with other cancers were also detected in several biopsy samples, such as Prevotella melaninogenica, Veillonella parvula and Fusobacterium nucleatum. H. pylori infection was associated with reduced microbial biodiversity compared to prior infection or control tissue (p=0.02). H. pylori active infection samples have a distinct non-H. pylori microbiome compared to prior infection and control samples. We also identified 5 patients with prior infection and 1 control patient with occult H. pylori infection. To characterize the immune infiltration in the mucosal biopsy samples, we define a 176-gene immune panel. The result of unsupervised clustering of gene expression revealed a much higher immune infiltration in H. pylori positive samples compared to uninfected samples, especially for CD8+, Th2 and Th17 cell populations. Two orthogonal experimental essays (ELISA and Flow Cytometry) confirmed the RNAseq-based expression profiling of inflammatory cytokines such as GRO, IL8, TNFa and SCD40L. Importantly, in 2 patients with prior H. pylori infection, the pro-inflammatory immune signature characteristic of H. pylori persisted. Finally, amongst H. pylori active infection patients, the biodiversity of the other bacteria present was inversely correlated with local immune infiltration (eg. greater the bacterial diversity, the less robust the pro-inflammatory immune signature). In summary, this study established a methodology for microbiome and immune profiling of gastric biopsy samples. We have identified a pro-inflammatory immune response signature associated with H. pylori infection, confirmed and validated by orthogonal assays. Notably, in several samples from previously eradicated H. pylori infection, the pro-inflammatory immune signature persisted, suggesting a potential long-term impact of H. pylori infection on mucosal immunity, that might contribute to gastric tumorigenesis. Citation Format: Chao Zhang, Prashant V. Thakkar, Felice Schnoll-Sussman, Bridget McClure, Greg Sonnenberg, Doron Betel, Manish A. Shah. Comprehensive microbial and immunological screening of gastroesophageal biopsies: A multi-parametric analysis to study carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2086.

Ligand Screening Systems for Human Glucose Transporters as Tools in Drug Discovery.

Hexoses are the major source of energy and carbon skeletons for biosynthetic processes in all kingdoms of life. Their cellular uptake is mediated by specialized transporters, including glucose transporters (GLUT, SLC2 gene family). Malfunction or altered expression pattern of GLUTs in humans is associated with several widespread diseases including cancer, diabetes and severe metabolic disorders. Their high relevance in the medical area makes these transporters valuable drug targets and potential biomarkers. Nevertheless, the lack of a suitable high-throughput screening system has impeded the determination of compounds that would enable specific manipulation of GLUTs so far. Availability of structural data on several GLUTs enabled in silico ligand screening, though limited by the fact that only two major conformations of the transporters can be tested. Recently, convenient high-throughput microbial and cell-free screening systems have been developed. These remarkable achievements set the foundation for further and detailed elucidation of the molecular mechanisms of glucose transport and will also lead to great progress in the discovery of GLUT effectors as therapeutic agents. In this mini-review, we focus on recent efforts to identify potential GLUT-targeting drugs, based on a combination of structural biology and different assay systems.