Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA — including poly(rA) carrier and ribosomal RNA — from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based ''tagmentation'' step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.     

Evaluation of juvenile drought stress tolerance and genotyping by sequencing with wild barley introgression lines

Drought is a major stress which can seriously limit yield in many crops including barley. Wild barley introgression lines (ILs) like the S42IL library may enhance drought stress tolerance of barley cultivars through the introduction of exotic alleles. The S42IL library was already characterized with 636 Illumina SNPs. New approaches like genotyping by sequencing (GBS) are available for barley to enhance the characterization of ILs. We generated an improved genetic map of the S42IL library, consisting of 4,201 SNPs by adding GBS data. The new map with a total length of 989.2 cM confirmed the extent of wild barley introgressions. Adding GBS data increased the resolution of the S42IL map tenfold from 0.4 to 4.2 markers/cM. This may assist to select possible candidate genes that improve drought tolerance. In four greenhouse experiments, juvenile drought stress response of 52 barley S42ILs was tested to identify quantitative trait loci (QTL). Thirteen S42ILs showed effects for plant biomass and leaf senescence. Subsequently, two verification experiments were conducted with these S42ILs. Nine out of eleven QTL were verified, and 22 additional QTL were detected. For 21 QTL, the Hsp allele increased trait performance, indicating the value of wild barley introgressions. For example, S42IL-107 and S42IL-123 produced more biomass under drought. Two different water-saving strategies were observed. S42IL-143 and S42IL-129 both revealed increased relative water content under drought. While S42IL-143 reduced biomass under drought, S42IL-129 maintained a high biomass production. We recommend using S42IL-107, S42IL-123 and S42IL-129 in barley breeding programs to enhance drought tolerance.     

A Metabolic Profiling Strategy for the Dissection of Plant Defense against Fungal Pathogens

Here we present a metabolic profiling strategy employing direct infusion Orbitrap mass spectrometry (MS) and gas chromatography-mass spectrometry (GC/MS) for the monitoring of soybean''s (Glycine max L.) global metabolism regulation in response to Rhizoctonia solani infection in a time-course. Key elements in the approach are the construction of a comprehensive metabolite library for soybean, which accelerates the steps of metabolite identification and biological interpretation of results, and bioinformatics tools for the visualization and analysis of its metabolome. The study of metabolic networks revealed that infection results in the mobilization of carbohydrates, disturbance of the amino acid pool, and activation of isoflavonoid, α-linolenate, and phenylpropanoid biosynthetic pathways of the plant. Components of these pathways include phytoalexins, coumarins, flavonoids, signaling molecules, and hormones, many of which exhibit antioxidant properties and bioactivity helping the plant to counterattack the pathogen''s invasion. Unraveling the biochemical mechanism operating during soybean-Rhizoctonia interaction, in addition to its significance towards the understanding of the plant''s metabolism regulation under biotic stress, provides valuable insights with potential for applications in biotechnology, crop breeding, and agrochemical and food industries.     

Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells

A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1⁺ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3''UTR lengths while MEFs tend towards longer 3''UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3''UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3''UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis.     

干旱区绿洲城市生态文明建设公众参与意识研究——以“一带一路”沿线城市乌鲁木齐为例

生态文明是人类文明发展的新阶段,其强调人与自然的协调发展。基于公众科学理论框架,采用模型模拟方法、实地问卷调查、多元回归方法,分析了乌鲁木齐居民参与城市生态文明建设的自我感知、满意程度、参与意愿,并构建了结构方程模型(SEM),探讨了居民参与城市生态文明意愿的可行路径,分析了居民参与城市生态文明建设的意愿与其相关因素间的关系,揭示了居民参与城市生态文明建设主要限制因素。结果表明:(1)居民自我感知与城市生态文明建设参与意愿呈显著正相关,满意程度与参与意愿呈显著负相关;(2)45.36%的受访者表示非常愿意参与城市生态文明建设;(3)居民主体意识和支持度占比分别为72.92%、72.04%,生态认知、生态关注及参与信心占比分别为43.08%、64.33%和55.14%;(4)受访者对城市生态文明建设状况满意度的平均偏效应为2.32,各项满意度均值情况为:城市绿化状况(2.51)政府环境信息公开程度(2.41)城市空气状况(2.35)城市垃圾处理与分类状况(1.99)。研究成果以期为建立和推进城市生态文明建设提供科学参考。     

Handling class imbalance problem in miRNA dataset associated with cancer

MiRNAs are small (~22nt long) non-coding RNA sequences; binds to the complementarity target sites in 3'' Untranslated Region (UTR) of mRNA sequences but not restricted to other mRNA regions viz., 5'' UTR and Coding sequences (CDS). Complementarity binding of miRNA to mRNA target sites either results in complete degradation of the mRNA itself or it may regulate the mRNA as an oncogene or as a tumor suppressor gene. However, the exact mechanism involved in identifying a miRNA to be associated with cancer is still unclear. Further, with the outburst in the number of miRNAs sequences recorded every year in miRBase, the gap is still widening mainly due to the laborious and economically unfavorable experimental procedures associated with the functional annotation. Motivated by the fact, we constructed a two-step support vector machine-based predictive model - miRSEQ and miRINT. However, the major pitfall during the construction of the model is the class imbalance problem. Hence, in order to overcome class imbalance problem, in the present study we empirically compare the effectiveness of two different methods viz., Synthetic Minority Oversampling Technique (SMOTE) and cost-senstive learning method. Performance measures were evaluated in terms of Precision and Recall. Based on our result, it was observed that for miRNA dataset with high class imbalance utilized for predicting association of cancer, cost-sensitive method outperformed the oversampling method.     

Characterization of camel nanobodies specific for superfolder GFP fusion proteins

Superfolder green fluorescent protein (sfGFP) is a fusion tag which plays a dual role in monitoring and purifying the recombinant fusion proteins using specific binders. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies (HCAbs) occurring in camelids. They are produced as recombinant proteins in E. coli and have different biotechnological applications, including the detection and purification of their specific antigens. To produce anti-sfGFP specific nanobodies, an adult one-humped camel was successfully immunized and immune response was evaluated by ELISA, which showed an active participation of HCAbs in this response. A relatively large nanobody “immune” library of 5 × 10⁸ individual transformants, with 87.5 % positivity, was prepared from the blood of the immunized camel. Phage display biopanning on this nanobody library resulted in the isolation of seven anti-sfGFP specific nanobodies, referred to as NbsfGFP01, 02, 03, 04, 06, 07 and 08. These nanobodies were able to recognize sfGFP tag as free or in fusion with growth hormone in ELISA and immuno-blotting. Furthermore, they showed important apparent affinities in the detection and capture of sfGFP by ELISA, and they targeted three different epitopes on the surface of their antigen. The interesting characteristics of these molecular binders make them valuable tools for more in-depth structural and functional studies related to sfGFP fusion proteins.     

Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers

Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2–5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.     

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities¹. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a ''trunk'' fragment is cut from an intact worm, a new ''head'' will regenerate anteriorly and a ''tail'' will regenerate posteriorly restoring the original ''head-to-tail'' polarity of body structures prior to amputation (Figure 1A).Regeneration is driven by planarian stem cells, known as ''neoblasts'' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity^2-9.The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters¹⁰, represent an important system for studying evolution of the central nervous system^{11, 12} and have behavioral screening potential^{13, 14.} Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs^{8, 9, 15}. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals^16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.     

Nest shapes in paper wasps: can the variability of forms be deduced from the same construction algorithm?

We constructed a simple model to show that the variability of nest structures in the social wasp genus Polistes could result from a simple algorithm modulated by a single parameter. The interplay between the simple algorithm and the geometry of the nest structure resulted in isotropic growth. New construction on the sides of the comb was initiated according to the size of the sides. Due to the geometry of the comb a new rule emerged: if one side of the structure is extended, it decreases the chance that that side will be further extended in the next step (because the number of possible initiation positions decreases), but at the same time construction on one side increases the probability of the extension of that side''s neighbours (because their possible initiation positions increase). This emergent isotropic growth keeps the nest almost circular, regardless of nest size, and it ensures well-centred nests. Eccentric nests reflect differences in initiation preference towards particular sides of the previously built structure. Interplay between structural constraints and the simple behavioural algorithm, which is governed by both structural and ''external'' information, resulted in nests of different shapes. The model was a good predictor of the range of Polistes nest forms found in nature.     

MicroRNA Expression Profile during Aphid Feeding in Chrysanthemum (Chrysanthemum morifolium)

MicroRNAs (miRNAs) are important regulators of gene expression, affecting many biological processes. As yet, their roles in the response of chrysanthemum to aphid feeding have not been explored. Here, the identity and abundance of miRNAs induced by aphid infestation have been obtained using high-throughput Illumina sequencing platform. Three leaf small RNA libraries were generated, one from plants infested with the aphid Macrosiphoniella sanbourni (library A), one from plants with mock puncture treatment (library M), and the third from untreated control plants (library CK). A total of 7,944,797, 7,605,251 and 9,244,002 clean unique reads, ranging from 18 to 30 nucleotides (nt) in length, were obtained from library CK, A and M, respectively. As a result, 303 conserved miRNAs belonging to 276 miRNAs families and 234 potential novel miRNAs were detected in chrysanthemum leaf, out of which 80, 100 and 79 significantly differentially expressed miRNAs were identified in the comparison of CK-VS-A, CK-VS-M and M-VS-A, respectively. Several of the differentially abundant miRNAs (in particular miR159a, miR160a, miR393a) may be associated with the plant''s response to aphid infestation.     

Microbial diversity in formation water and enrichment cultures from the Gangxi bed of the Dagang terrigenous oilfield (PRC)

Microbial diversity and biogeochemical processes of the Gangxi bed with low-mineral water and a temperature gradient from 35 to 54°C were studied. The 16S rRNA gene clone libraries (over 800 clones) were obtained from microbial DNA isolated from formation water and from the primary enrichment cultures for fermenting, sulfate-reducing, methanogenic, and aerobic organotrophic prokaryotes. While both sulfate reduction and methanogenesis were registered in formation water by radioisotope techniques, the genes of sulfate-reducing prokaryotes were not revealed in the 16S rRNA gene clone library from formation water. The 16S rRNA genes of Methanobacterium congolense and Methanococcus vannielii predominated among archaeal sequences retrieved from formation water, while the genes of Methanothermobacter thermoautotrophicus, Methanomethylovorans thermophila, and Methanoculleus sp. predominated in the combined library from enrichment cultures. In the library of Bacteria 16S rRNA genes from formation water, the genes of thermophilic fermentative bacteria of the family Thermoanaerobacteriaceae predominated; the remaining sequences belonged to mesophiles (genera Brevundimonas, Sphingomonas, Oxalicibacterium, and Stenotrophomonas), the phylum Chloroflexi, and unidentified bacteria. The combined library from enrichment cultures, contained, apart from the sequences of the family Thermoanaerobacteriaceae, the genes of fermentative bacteria (genera Anaerobaculum, Coprothermobacter, Thermanaerovibrio, Soehngenia, Bacteroides, and Aminobacterium and the order Thermotogales), of aerobic hydrocarbon-oxidizing bacteria (genera Pannonibacter and Pseudomonas), and of sulfate reducers (genera Desulfomicrobium, Thermodesulfovibrio, and Desulfotomaculum). High coverage was shown for bacterial (97.6%) and archaeal (100%) clone libraries, indicating that a significant portion of the microbial diversity in the studied communities was revealed.     

Assessing the Underwater Acoustics of the World's Largest Vibration Hammer (OCTA-KONG) and Its Potential Effects on the Indo-Pacific Humpbacked Dolphin (Sousa chinensis)

Anthropogenic noise in aquatic environments is a worldwide concern due to its potential adverse effects on the environment and aquatic life. The Hongkong-Zhuhai-Macao Bridge is currently under construction in the Pearl River Estuary, a hot spot for the Indo-Pacific humpbacked dolphin (Sousa chinensis) in China. The OCTA-KONG, the world''s largest vibration hammer, is being used during this construction project to drive or extract steel shell piles 22 m in diameter. This activity poses a substantial threat to marine mammals, and an environmental assessment is critically needed. The underwater acoustic properties of the OCTA-KONG were analyzed, and the potential impacts of the underwater acoustic energy on Sousa, including auditory masking and physiological impacts, were assessed. The fundamental frequency of the OCTA-KONG vibration ranged from 15 Hz to 16 Hz, and the noise increments were below 20 kHz, with a dominant frequency and energy below 10 kHz. The resulting sounds are most likely detectable by Sousa over distances of up to 3.5 km from the source. Although Sousa clicks do not appear to be adversely affected, Sousa whistles are susceptible to auditory masking, which may negatively impact this species'' social life. Therefore, a safety zone with a radius of 500 m is proposed. Although the zero-to-peak source level (SL) of the OCTA-KONG was lower than the physiological damage level, the maximum root-mean-square SL exceeded the cetacean safety exposure level on several occasions. Moreover, the majority of the unweighted cumulative source sound exposure levels (SSELs) and the cetacean auditory weighted cumulative SSELs exceeded the acoustic threshold levels for the onset of temporary threshold shift, a type of potentially recoverable auditory damage resulting from prolonged sound exposure. These findings may aid in the identification and design of appropriate mitigation methods, such as the use of air bubble curtains, “soft start” and “power down” techniques.     

Comparison of large-insert,small-insert and pyrosequencing libraries for metagenomic analysis

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation'' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.     

US guidelines for treatment of schizophrenia expected next year.

The American Psychiatric Association (APA) is developing new guidelines for treating schizophrenia. The recommendations, which were previewed at the APA''s annual meeting earlier this year, are expected to be released in 1996.     

Construction and characterization of a BAC library from the Coffea arabica genotype Timor Hybrid CIFC 832/2

Most of the world’s coffee production originates from Coffea arabica, an allotetraploid species with low genetic diversity and for which few genomic resources are available. Genomic libraries with large DNA fragment inserts are useful tools for the study of plant genomes, including the production of physical maps, integration studies of physical and genetic maps, genome structure analysis and gene isolation by positional cloning. Here, we report the construction and characterization of a Bacterial Artificial Chromosome (BAC) library from C. arabica Timor Hybrid CIFC 832/2, a parental genotype for several modern coffee cultivars. The BAC library consists of 56,832 clones with an average insert size of 118 kb, which represents a dihaploid genome coverage of five to sixfold. The content of organellar DNA was estimated at 1.04 and 0.5 % for chloroplast and mitochondrial DNA, respectively. The BAC library was screened for the NADPH-dependent mannose-6-phosphate reductase gene (CaM6PR) with markers positioned on four linkage groups of a partial C. arabica genetic map. A mixed approach using PCR and membrane hybridization of BAC pools allowed for the discovery of nine BAC clones with the CaM6PR gene and 53 BAC clones that were anchored to the genetic map with simple sequence repeat markers. This library will be a useful tool for future studies on comparative genomics and the identification of genes and regulatory elements controlling major traits in this economically important crop species.