Clinical applications of sequencing take center stage

A report on the Advances in Genome Biology and Technology (AGBT) meeting, Marco Island, Florida, USA, February 20-23, 2013.This year''s Advances in Genome Biology and Technology (AGBT) meeting reflected the current state of ''next generation'' sequencing (NGS) technologies: significantly reduced competition and innovation, and a strong focus on standardization and application. Announcements of technological breakthroughs - a hallmark of previous AGBT meetings - were markedly absent, but existing technologies continued to improve following the now expected exponential curve. Although applications ranged widely, there was a strong emphasis on clinical diagnosis.     

The New Airborne Disease: Community Air Pollution

Community air pollution is the new airborne disease of our generation''s communities. It is caused by the increasing use of fuel, associated with both affluence and careless waste. Photochemical air pollution of the California type involves newly defined atmospheric reactions, is due mostly to motor vehicle exhaust, is oxidizing, and produces ozone, plant damage, impairment of visibility and eye and respiratory symptoms.Aggravation of asthma, impairment of lung function among persons with chronic respiratory disease and a possible causal role, along with cigarette smoking in emphysema and chronic bronchitis, are some of the effects of photochemical pollution.More subtle effects of pollution include impairment of oxygen transport by the blood due to carbon monoxide and interference with porphyrin metabolism due to lead. Carbon monoxide exposures may affect survival of patients who are in hospitals because of myocardial infarction.While many uncertainties in pollution-health reactions need to be resolved, a large number of people in California have health impairment due to airborne disease of this new type.     

Biodistribution and Therapeutic Utility of Liposomal Drug Carrier Systems

Abstract

Delivery of the drug at a specific site (drug targeting) or controlled and prolonged release of the liposome-bound drug are the two major considerations for adding liposomes to the existing arsenal of drug delivery systems. In particular the concept of liposomal drug targeting has been evolving rapidly in the past 10 years with the development of 'second generation' carriers such as immunoliposomes (liposomes bearing covalently coupled antibodies as homing device) and, more recently, the long-circulating liposomes. In this contribution novel approaches in the field of liposomal drug targeting will be briefly described: (1) immunoliposomes for chemotherapy of intraperitoneal malignancies, such as ovarian carcinoma, (2) a new type of immunoliposomes for mediating the targeting of enzymes to be used for site-specific prodrug activation (immuno-enzymosomes), (3) long-circulating liposomes for the targeting of antibiotics to sites of bacterial infection, and (4) polyethyleneglycol (PEG)-modified proteoliposomes with the homing device coupled to the ends of the long PEG chains for achieving effective target binding along with prolonged circulation times.     

The role of play objects and object play in human cognitive evolution and innovation

In this contribution, we address a major puzzle in the evolution of human material culture: If maturing individuals just learn their parental generation's material culture, then what is the origin of key innovations as documented in the archeological record? We approach this question by coupling a life‐history model of the costs and benefits of experimentation with a niche‐construction perspective. Niche‐construction theory suggests that the behavior of organisms and their modification of the world around them have important evolutionary ramifications by altering developmental settings and selection pressures. Part of Homo sapiens' niche is the active provisioning of children with play objects — sometimes functional miniatures of adult tools — and the encouragement of object play, such as playful knapping with stones. Our model suggests that salient material culture innovation may occur or be primed in a late childhood or adolescence sweet spot when cognitive and physical abilities are sufficiently mature but before the full onset of the concerns and costs associated with reproduction. We evaluate the model against a series of archeological cases and make suggestions for future research.     

Whole genome sequencing of marine organisms by Oxford Nanopore Technologies: Assessment and optimization of HMW‐DNA extraction protocols

Marine habitats are Earth''s largest aquatic ecosystems, yet little is known about marine organism''s genomes. Molecular studies can unravel their genetics print, thus shedding light on specie''s adaptation and speciation with precise authentication. However, extracting high molecular weight DNA from marine organisms and subsequent DNA library preparation for whole genome sequencing is challenging. The challenges can be explained by excessive metabolites secretion that co‐precipitates with DNA and barricades their sequencing. In this work, we sought to resolve this issue by describing an optimized isolation method and comparing its performance with the most commonly reported protocols or commercial kits: SDS/phenol–chloroform method, Qiagen Genomic Tips kit, Qiagen DNeasy Plant mini kit, a modified protocol of Qiagen DNeasy Plant kit, Qiagen DNeasy Blood and Tissue kit, and Qiagen Qiamp DNA Stool mini kit. Our method proved to work significantly better for different marine species regardless of their shape, consistency, and sample preservation, improving Oxford Nanopore Technologies sequencing yield by 39 folds for Spirobranchus sp. and enabling generation of almost 10 GB data per flow cell/run for Chrysaora sp. and Palaemon sp. samples.     

Adenosine accumulation causes metabolic disorders in testes and associates with lower testosterone level in obese mice

Overweight and obese men face numerous health problems, including type 2 diabetes, subfertility, and even infertility. However, few studies have focused on the effects of nutritional status and obesity‐related regulatory signals on fertility deficiency. Our previous observations have shown that the elevation of plasma 5'‐adenosine monophosphate (5'‐AMP) and the accumulation of adenosine in liver and muscle of obese diabetic db/db mice are related to insulin resistance. Here, we found that adenosine accumulation in testis is a common marker of both genetic obesity and high‐fat‐diet induced obese mice. An messenger RNA sequencing analysis indicated that 78 upregulated genes and 155 downregulated genes in the testis of 5'‐AMP‐treated mice overlapped with the same genes in the testis of ob/ob mice, and these genes belonged to the clusters of steroid metabolic process and regulation of hormone levels, respectively. Serum testosterone was reduced in ob/ob and 5'‐AMP‐treated mice. Metabolomic analysis based on ¹H nuclear magnetic resonance showed that the testicular metabolic profiles of ob/ob mice were similar to those of 5'‐AMP treated mice. Exogenous 5'‐AMP inhibited the phosphorylation of AKT and mammalian target of rapamycin signal transduction and reduced the proliferating cell nuclear antigen expressions in testes. Our results suggest that the accumulation of adenosine causes metabolic disorders in testes and associates lower testosterone level in obese mice.     

微生物组学研究的“淘金时代”

人体及动物肠道中生存着数量庞大的共生微生物;这些微生物无时无刻不参与着宿主的生命活动。揭示这些共生微生物在宿主体内的变化规律、与宿主之间的依存和博弈关系等,将使人类更加全面的认知高等生物体的生命本质。本专刊从肠道微生物与疾病、肠道微生物群落结构、肠道微生物与宿主互作、肠道微生物资源和肠道微生物研究方法 5个层面展示了我国科研工作者在肠道微生物研究领域的新进展及新观点。     

基于高通量测序的乐安江冬季细菌群落特征分析

【目的】分析乐安江从上游至下游水体细菌群落结构组成变化,揭示细菌群落结构组成变化的影响因素。【方法】分析不同河段水体中C、N、P、Cu、Zn、As和Pb等化学指标。对水体DNA的16S rRNA基因进行高通量测序确定细菌群落特征。基于Bray-Curtis距离的采样点非度量多维尺度(NMDS)分析和聚类分析研究乐安江水体细菌群落结构差异,基于冗余分析(RDA)研究环境因子与细菌群落的关系。【结果】乐安江水体中C、N、P、Cu、Zn、As和Pb等化学指标含量中下游偏高。中游河水受德兴铜矿废水影响,细菌群落多样性降低,下游受农业、生活废水影响,细菌群落丰富度和多样性升高。水体中优势菌群为β-变形菌纲(Beta-proteobacteria,53.03%)、放线菌门(Actinobacteria,20.24%)和拟杆菌门(Bacteroidetes,14.75%)。中游受德兴铜矿废水影响,Beta-proteobacteria丰度增大,而Actinobacteria丰度减小;下游受微生物间捕食影响,Bacteroidetes丰度下降。在细菌群落与环境因子的关系中,DO是解释乐安江细菌群落结构变化的最佳环境因子。【结论】乐安江中游德兴铜矿废水和中下游农业、生活废水明显改变了水体细菌群落结构组成,使水体细菌群落特征从上游到下游发生显著变化。本研究为揭示人类活动对乐安江水生态环境的影响提供了参考性数据。     

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA ''tags'', or ''barcodes,'' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.     

基于高通量测序的鄱阳湖典型湿地土壤细菌群落特征分析

采用高通量测序技术分析了鄱阳湖典型湿地土壤细菌群落特征。测序结果表明,不同植被土壤细菌群落丰度与多样性的排序相同:苔草带苔草-虉草带芦苇带泥滩带藜蒿带。沿湖面至坡地,空间位置相近的土壤细菌群落结构具有更大的相似性,苔草-虉草带、苔草带和芦苇带的细菌群落结构相近,泥滩带和藜蒿带的细菌群落结构差异较大。变形菌门(30.0%)是湿地土壤平均相对丰度最高的门,其次为酸杆菌门(16.7%)和绿弯菌门(16.5%);多数门分类细菌相对丰度沿湖面至坡地存在一定变化趋势。硝化螺菌属是第一大属分类水平细菌群落。在土壤化学指标中,与鄱阳湖湿地细菌群落相关性较大的是总磷、铵态氮和有机质含量。以上研究结果表明,鄱阳湖湿地不同植被土壤细菌群落具有结构性差异,但沿湖面至坡地存在规律性变化。     

中国东海微型鞭毛虫群落的分布特征及其环境影响因子

海洋微型鞭毛虫是海洋原生生物中一类高度异质化的类群,物种多样性高,具有多种营养方式,在全球海洋生态系统中占据广阔的生态位,在生物地球化学循环中发挥着关键作用。然而关于其生物多样性和群落结构的认识十分有限,特别是有关环境因子与其生物地理分布关系的研究更为罕见。为了探究微型鞭毛虫群落多样性、群落结构以及影响其生物地理分布格局的环境因素,将高通量测序技术与传统的显微镜观测方法相结合,全面调查了中国东海春季和秋季微型鞭毛虫的群落特征,并深入探讨了与环境因子之间的关系。结果表明：东海微型鞭毛虫的丰度平均为2.27×10~3个/mL,表现为近岸处较高、随离岸距离的增加而下降的趋势;Shannon多样性指数呈现表层低于底层、近岸区低于陆架区的特征,反映了生物群落的稳定程度以及对环境条件的适应程度;不同类群的鞭毛虫具有各自独特的营养模式和相对固定的粒级,表现出对温度、盐度、溶解氧等环境因素的不同响应,从而使群落的物种组成和分布模式呈现明显的季节变化和生境差异。研究结果可为深入认识东海海洋微型鞭毛虫的群落结构、分布格局以及环境影响因素提供理论依据。     

Recurrent gain-of-function USP8 mutations in Cushing's disease

Cushing''s disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing''s syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing''s disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing''s disease and provides insights into the therapeutics of this disease.     

Identification,cDNA Cloning,and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla

Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp.     

Serological and genetic analysis of a rare CisAB01/O01 blood group

The paper aims to analyze a rare blood sample in Ganzhou City Hospital with CisAB subtype and explore a feasible pattern for blood typing of rare blood type patients, so as to ensure clinical transfusion safety. The routine serological methods were used for ABO forward and reverse blood typing and the fluorescence real-time PCR technique was used for sample genotyping. A human ABO blood group 6-7 exon sequencing kit was used for sequence analysis. The nucleic acid sequence of the sample was compared with reference sequences. The forward typing results demonstrated that the sample was ABw, RhD positive. The sample exhibited 4+ agglutination with anti-H and anti-AB antibodies. Reverse typing by microcolumn gel method showed an AB result, but the serum sample demonstrated weak agglutination with B cell under room temperature, 4 °C and 37 °C in saline when tested with tube method respectively. The serological results matched with the A₂B₃ serotype. The fluorescent real-time PCR genotyping results displayed A/O01. The sequence analysis demonstrated deletion of guanine in 261-position 467C>T (heterozygote) and 803G>T (heterozygote) mutation respectively. The mutation caused the A glycosyltransferase peptide chain to change from proline to leucine (P156L) at 156 and from glutamate to alanine (G268A) at 268. The result demonstrated that the sample''s genotype was CisAB01/O01. The mutation of glycosyltransferase coding gene leads to an abnormal serological reaction pattern. Only by combining the results of genetic analysis can we get the true sample blood type and better ensure the safety of clinical blood transfusion.     

The evolution within us

The B-cell immune response is a remarkable evolutionary system found in jawed vertebrates. B-cell receptors, the membrane-bound form of antibodies, are capable of evolving high affinity to almost any foreign protein. High germline diversity and rapid evolution upon encounter with antigen explain the general adaptability of B-cell populations, but the dynamics of repertoires are less well understood. These dynamics are scientifically and clinically important. After highlighting the remarkable characteristics of naive and experienced B-cell repertoires, especially biased usage of genes encoding the B-cell receptors, we contrast methods of sequence analysis and their attempts to explain patterns of B-cell evolution. These phylogenetic approaches are currently unlinked to explicit models of B-cell competition, which analyse repertoire evolution at the level of phenotype, the affinities and specificities to particular antigenic sites. The models, in turn, suggest how chance, infection history and other factors contribute to different patterns of immunodominance and protection between people. Challenges in rational vaccine design, specifically vaccines to induce broadly neutralizing antibodies to HIV, underscore critical gaps in our understanding of B cells'' evolutionary and ecological dynamics.