Detailed characterization of the mouse embryonic stem cell transcriptome reveals novel genes and intergenic splicing associated with pluripotency

<p>Abstract</p> <p>Background</p> <p>Transcriptional control of embryonic stem (ES) cell pluripotency has been a subject of intense study. Transcriptional regulators including Oct4 (Oct3/4 index), Sox2 and Nanog are fundamental for maintaining the undifferentiated state. However, the ES cell transcriptome is not limited to their targets, and exhibits considerable complexity when assayed with microarray, MPSS, cDNA/EST sequencing, and SAGE technologies. To identify novel genes associated with pluripotency, we globally searched for ES transcripts not corresponding to known genes, validated their sequences, determined their expression profiles, and employed RNAi to test their function.</p> <p>Results</p> <p>Gene Identification Signature (GIS) analysis, a SAGE derivative distinguished by paired 5' and 3' transcript end tags, identified 153 candidate novel transcriptional units (TUs) distinct from known genes in a mouse E14 ES mRNA library. We focused on 16 TUs free of artefacts and mapping discrepancies, five of which were validated by RTPCR product sequencing. Two of the TUs were revealed by annotation to represent novel protein-coding genes: a PRY-domain cluster member and a KRAB-domain zinc finger. The other three TUs represented intergenic splicing events involving adjacent, functionally unrelated protein-coding genes transcribed in the same orientation, with one event potentially encoding a fusion protein containing domains from both component genes (Clk2 and Scamp3). Expression profiling using embryonic samples and adult tissue panels confirmed that three of the TUs were unique to or most highly expressed in ES cells. Expression levels of all five TUs dropped dramatically during three distinct chemically induced differentiation treatments of ES cells in culture. However, siRNA knockdowns of the TUs did not alter mRNA levels of pluripotency or differentiation markers, and did not affect cell morphology.</p> <p>Conclusion</p> <p>Transcriptome libraries retain considerable potential for novel gene discovery despite massive recent cDNA and EST sequencing efforts; cDNA and EST evidence for these ES cell TUs had been limited or absent. RTPCR and full-length sequencing remain essential in resolving the bottleneck between numerous candidate novel transcripts inferred from high-throughput sequencing and the small fraction that can be validated. RNAi results indicate that, despite their strong association with pluripotency, these five transcriptomic novelties may not be required for maintaining it.</p

Plug-In Hybrid Electric Vehicle Value Proposition Study: Phase 1, Task 2: Select Value Propositions/Business Model for Further Study

The Plug-In Hybrid Electric Vehicle (PHEV) Value Propositions Workshop held in Washington, D.C. in December 2007 served as the Task 1 Milestone for this study. Feedback from all five Workshop breakout sessions has been documented in a Workshop Summary Report, which can be found at www.sentech.org/phev. In this report, the project team compiled and presented a comprehensive list of potential value propositions that would later serve as a 'grab bag' of business model components in Task 2. After convening with the Guidance and Evaluation Committee and other PHEV stakeholders during the Workshop, several improvements to the technical approach were identified and incorporated into the project plan to present a more realistic and accurate case study and evaluation. The assumptions and modifications that will have the greatest impact on the case study selection process in Task 2 are described in more detail in this deliverable. The objective of Task 2 is to identify the combination of value propositions that is believed to be achievable by 2030 and collectively hold promise for a sustainable PHEV market by 2030. This deliverable outlines what the project team (with input from the Committee) has defined as its primary scenario to be tested in depth for the remainder of Phase 1. Plans for the second and third highest priority/probability business scenarios are also described in this deliverable as proposed follow up case studies in Phase 2. As part of each case study description, the proposed utility system (or subsystem), PHEV market segment, and facilities/buildings are defined

Identification of two novel autism genes, TRPC4 and SCFD2, in Qatar simplex families through exome sequencing

This study investigated the genetic underpinnings of autism spectrum disorder (ASD) in a Middle Eastern cohort in Qatar using exome sequencing. The study identified six candidate autism genes in independent simplex families, including both four known and two novel autosomal dominant and autosomal recessive genes associated with ASD. The variants consisted primarily of de novo and homozygous missense and splice variants. Multiple individuals displayed more than one candidate variant, suggesting the potential involvement of digenic or oligogenic models. These variants were absent in the Genome Aggregation Database (gnomAD) and exhibited extremely low frequencies in the local control population dataset. Two novel autism genes, TRPC4 and SCFD2, were discovered in two Qatari autism individuals. Furthermore, the D651A substitution in CLCN3 and the splice acceptor variant in DHX30 were identified as likely deleterious mutations. Protein modeling was utilized to evaluate the potential impact of three missense variants in DEAF1, CLCN3, and SCFD2 on their respective structures and functions, which strongly supported the pathogenic natures of these variants. The presence of multiple de novo mutations across trios underscored the significant contribution of de novo mutations to the genetic etiology of ASD. Functional assays and further investigations are necessary to confirm the pathogenicity of the identified genes and determine their significance in ASD. Overall, this study sheds light on the genetic factors underlying ASD in Qatar and highlights the importance of considering diverse populations in ASD research

Plug-In Hybrid Electric Vehicle Value Proposition Study: Interim Report: Phase I Scenario Evaluation

Plug-in hybrid electric vehicles (PHEVs) offer significant improvements in fuel economy, convenient low-cost recharging capabilities, potential environmental benefits, and decreased reliance on imported petroleum. However, the cost associated with new components (e.g., advanced batteries) to be introduced in these vehicles will likely result in a price premium to the consumer. This study aims to overcome this market barrier by identifying and evaluating value propositions that will increase the qualitative value and/or decrease the overall cost of ownership relative to the competing conventional vehicles and hybrid electric vehicles (HEVs) of 2030 During this initial phase of this study, business scenarios were developed based on economic advantages that either increase the consumer value or reduce the consumer cost of PHEVs to assure a sustainable market that can thrive without the aid of state and Federal incentives or subsidies. Once the characteristics of a thriving PHEV market have been defined for this timeframe, market introduction steps, such as supportive policies, regulations and temporary incentives, needed to reach this level of sustainability will be determined. PHEVs have gained interest over the past decade for several reasons, including their high fuel economy, convenient low-cost recharging capabilities, potential environmental benefits and reduced use of imported petroleum, potentially contributing to President Bush's goal of a 20% reduction in gasoline use in ten years, or 'Twenty in Ten'. PHEVs and energy storage from advanced batteries have also been suggested as enabling technologies to improve the reliability and efficiency of the electric power grid. However, PHEVs will likely cost significantly more to purchase than conventional or other hybrid electric vehicles (HEVs), in large part because of the cost of batteries. Despite the potential long-term savings to consumers and value to stakeholders, the initial cost of PHEVs presents a major market barrier to their widespread commercialization. The purpose of this project is to identify and evaluate value-added propositions for PHEVs that will help overcome this market barrier. Candidate value propositions for the initial case study were chosen to enhance consumer acceptance of PHEVs and/or compatibility with the grid. Potential benefits of such grid-connected vehicles include the ability to supply peak load or emergency power requirements of the grid, enabling utilities to size their generation capacity and contingency resources at levels below peak. Different models for vehicle/battery ownership, leasing, financing and operation, as well as the grid, communications, and vehicle infrastructure needed to support the proposed value-added functions were explored during Phase 1. Rigorous power system, vehicle, financial and emissions modeling were utilized to help identify the most promising value propositions and market niches to focus PHEV deployment initiatives

Establishing criteria for human mesenchymal stem cell potency

This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age‐ and sex‐matched donors. Adherence to plastic was not indicative of potency, yet capacity for long‐term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high‐growth capacity or low‐growth capacity. Using this grouping strategy, high‐growth capacity MSCs were smaller in size, had greater colony‐forming efficiency, and had longer telomeres. Cell‐surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high‐growth capacity and low‐growth capacity MSCs, whereas STRO‐1 and platelet‐derived growth factor receptor alpha were preferentially expressed on high‐growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST‐1 and DERMO‐1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high‐growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low‐growth capacity MSCs when assessed for ectopic bone‐forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application

A Balanced Translocation in Kallmann Syndrome Implicates a Long Noncoding RNA, RMST, as a GnRH Neuronal Regulator.

CONTEXT: Kallmann syndrome (KS) is a rare, genetically heterogeneous Mendelian disorder. Structural defects in KS patients have helped define the genetic architecture of gonadotropin-releasing hormone (GnRH) neuronal development in this condition. OBJECTIVE: Examine the functional role a novel structural defect affecting a long noncoding RNA (lncRNA), RMST, found in a KS patient. DESIGN: Whole genome sequencing, induced pluripotent stem cells and derived neural crest cells (NCC) from the KS patient were contrasted with controls. SETTING: The Harvard Reproductive Sciences Center, Massachusetts General Hospital Center for Genomic Medicine, and Singapore Genome Institute. PATIENT: A KS patient with a unique translocation, t(7;12)(q22;q24). INTERVENTIONS/MAIN OUTCOME MEASURE/RESULTS: A novel translocation was detected affecting the lncRNA, RMST, on chromosome 12 in the absence of any other KS mutations. Compared with controls, the patient's induced pluripotent stem cells and NCC provided functional information regarding RMST. Whereas RMST expression increased during NCC differentiation in controls, it was substantially reduced in the KS patient's NCC coincident with abrogated NCC morphological development and abnormal expression of several "downstream" genes essential for GnRH ontogeny (SOX2, PAX3, CHD7, TUBB3, and MKRN3). Additionally, an intronic single nucleotide polymorphism in RMST was significantly implicated in a genome-wide association study associated with age of menarche. CONCLUSIONS: A novel deletion in RMST implicates the loss of function of a lncRNA as a unique cause of KS and suggests it plays a critical role in the ontogeny of GnRH neurons and puberty

A survey of root knot nematodes and resistance to Meloidogyne incognita in sweet potato varieties from Kenyan fields

AbstractThe root knot nematode, Meloidogyne is one of the most economically damaging plant parasitic nematode groups, and are widely distributed in Kenyan agro-ecosystems. The aim of this study was to determine the diversity of Meloidogyne species in Kenyan sweet potato fields and identify sweet potato varieties that exhibit resistance to M. incognita. Meloidogyne species were collected from Nyanza, Western, Eastern and Central Provinces of Kenya. Mitochondrial DNA was used to differentiate Meloidogyne species. The most common species in all sampled regions was M. incognita. Meloidogyne hapla was recorded for the first time in Kenyan sweet potato growing areas (Mosocho, Matayos, Teso South, Manyatta, and Nzaui sub-counties), while M. enterolobii was observed in Kiharu, Matayos and Mosocho sub-counties and a novel Meloidogyne sp. was identified in Kiharu sub-county. Seventy-two sweet potato varieties collected from both agricultural fields and research stations in Kenya were evaluated for resistance to M. incognita under greenhouse conditions in two separate trials. Known susceptible (Beauregard) and resistant (Tanzania) sweet potato varieties were included as controls. Responses of sweet potato varieties to M. incognita infection was assessed by the number of eggs present and level of galling on a scale of 1–5, where 0 = 0 galls and 5 ≥ 100 galls. The reproduction index (RI) was used to classify the varieties as resistant or susceptible. There was a significant difference (P < 0.001) in the number of eggs, GI and RI among the varieties tested. Forty nine sweet potato varieties were considered very resistant and may be used in breeding programs to incorporate resistance against M. incognita into commercial cultivars of sweet potato or to use them in crop rotation programmes for management of RKN. The results on Meloidogyne species diversity in Kenyan sweet potato fields will also be useful in nematode management programs

An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics

For a decade, The Cancer Genome Atlas (TCGA) program collected clinicopathologic annotation data along with multi-platform molecular profiles of more than 11,000 human tumors across 33 different cancer types. TCGA clinical data contain key features representing the democratized nature of the data collection process. To ensure proper use of this large clinical dataset associated with genomic features, we developed a standardized dataset named the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR), which includes four major clinical outcome endpoints. In addition to detailing major challenges and statistical limitations encountered during the effort of integrating the acquired clinical data, we present a summary that includes endpoint usage recommendations for each cancer type. These TCGA-CDR findings appear to be consistent with cancer genomics studies independent of the TCGA effort and provide opportunities for investigating cancer biology using clinical correlates at an unprecedented scale. Analysis of clinicopathologic annotations for over 11,000 cancer patients in the TCGA program leads to the generation of TCGA Clinical Data Resource, which provides recommendations of clinical outcome endpoint usage for 33 cancer types

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts