Functional annotation of novel lineage-specific genes using co-expression and promoter analysis

<p>Abstract</p> <p>Background</p> <p>The diversity of placental architectures within and among mammalian orders is believed to be the result of adaptive evolution. Although, the genetic basis for these differences is unknown, some may arise from rapidly diverging and lineage-specific genes. Previously, we identified 91 novel lineage-specific transcripts (LSTs) from a cow term-placenta cDNA library, which are excellent candidates for adaptive placental functions acquired by the ruminant lineage. The aim of the present study was to infer functions of previously uncharacterized lineage-specific genes (LSGs) using co-expression, promoter, pathway and network analysis.</p> <p>Results</p> <p>Clusters of co-expressed genes preferentially expressed in liver, placenta and thymus were found using 49 previously uncharacterized LSTs as seeds. Over-represented composite transcription factor binding sites (TFBS) in promoters of clustered LSGs and known genes were then identified computationally. Functions were inferred for nine previously uncharacterized LSGs using co-expression analysis and pathway analysis tools. Our results predict that these LSGs may function in cell signaling, glycerophospholipid/fatty acid metabolism, protein trafficking, regulatory processes in the nucleus, and processes that initiate parturition and immune system development.</p> <p>Conclusions</p> <p>The placenta is a rich source of lineage-specific genes that function in the adaptive evolution of placental architecture and functions. We have shown that co-expression, promoter, and gene network analyses are useful methods to infer functions of LSGs with heretofore unknown functions. Our results indicate that many LSGs are involved in cellular recognition and developmental processes. Furthermore, they provide guidance for experimental approaches to validate the functions of LSGs and to study their evolution.</p

Discovery and characterization of 91 novel transcripts expressed in cattle placenta

<p>Abstract</p> <p>Background</p> <p>Among the eutherian mammals, placental architecture varies to a greater extent than any other tissue. The diversity of placental types, even within a single mammalian order suggests that genes expressed in placenta are under strong Darwinian selection. Thus, the ruminant placenta may be a rich source of genes to explore adaptive evolutionary responses in mammals. The aim of our study was to identify novel transcripts expressed in ruminant placenta, and to characterize them with respect to their expression patterns, organization of coding sequences in the genome, and potential functions.</p> <p>Results</p> <p>A combination of bioinformatics, comparative genomics and transcript profiling was used to identify and characterize 91 novel transcripts (NTs) represented in a cattle placenta cDNA library. These NTs have no significant similarity to any non-ferungulate DNA or RNA sequence. Proteins longer than 100 aa were predicted for 29 NTs, and 21 are candidate non-coding RNAs. Eighty-six NTs were found to be expressed in one or more of 18 different tissues, with 39 (42%) showing tissue-preference, including six that were expressed exclusively in placentome. The authenticity of the NTs was confirmed by their alignment to cattle genome sequence, 42 of which showed evidence of mRNA splicing. Analysis of the genomic context where NT genes reside revealed 61 to be in intergenic regions, whereas 30 are within introns of known genes. The genes encoding the NTs were found to be significantly associated with subtelomeric regions.</p> <p>Conclusion</p> <p>The 91 lineage-specific transcripts are a useful resource for studying adaptive evolutionary responses of the ruminant placenta. The presence of so many genes encoding NTs in cattle but not primates or rodents suggests that gene loss and gain are important mechanisms of genome evolution in mammals. Furthermore, the clustering of NT genes within subtelomeric regions suggests that such regions are highly dynamic and may foster the birth of novel genes. The sequencing of additional vertebrate genomes with defined phylogenetic relationships will permit the search for lineage-specific genes to take on a more evolutionary context that is required to understand their origins and functions.</p

ESTIMA, a tool for EST management in a multi-project environment

BACKGROUND: Single-pass, partial sequencing of complementary DNA (cDNA) libraries generates thousands of chromatograms that are processed into high quality expressed sequence tags (ESTs), and then assembled into contigs representative of putative genes. Usually, to be of value, ESTs and contigs must be associated with meaningful annotations, and made available to end-users. RESULTS: A web application, Expressed Sequence Tag Information Management and Annotation (ESTIMA), has been created to meet the EST annotation and data management requirements of multiple high-throughput EST sequencing projects. It is anchored on individual ESTs and organized around different properties of ESTs including chromatograms, base-calling quality scores, structure of assembled transcripts, and multiple sources of comparison to infer functional annotation, Gene Ontology associations, and cDNA library information. ESTIMA consists of a relational database schema and a set of interactive query interfaces. These are integrated with a suite of web-based tools that allow a user to query and retrieve information. Further, query results are interconnected among the various EST properties. ESTIMA has several unique features. Users may run their own EST processing pipeline, search against preferred reference genomes, and use any clustering and assembly algorithm. The ESTIMA database schema is very flexible and accepts output from any EST processing and assembly pipeline. ESTIMA has been used for the management of EST projects of many species, including honeybee (Apis mellifera), cattle (Bos taurus), songbird (Taeniopygia guttata), corn rootworm (Diabrotica vergifera), catfish (Ictalurus punctatus, Ictalurus furcatus), and apple (Malus x domestica). The entire resource may be downloaded and used as is, or readily adapted to fit the unique needs of other cDNA sequencing projects. CONCLUSIONS: The scripts used to create the ESTIMA interface are freely available to academic users in an archived format from . The entity-relationship (E-R) diagrams and the programs used to generate the Oracle database tables are also available. We have also provided detailed installation instructions and a tutorial at the same website. Presently the chromatograms, EST databases and their annotations have been made available for cattle and honeybee brain EST projects. Non-academic users need to contact the W.M. Keck Center for Functional and Comparative Genomics, University of Illinois at Urbana-Champaign, Urbana, IL, for licensing information

Co-axial dual-core resonant leaky fibre for optical amplifiers

We present a co-axial dual-core resonant leaky optical fibre design, in which the outer core is made highly leaky. A suitable choice of parameters can enable us to resonantly couple power from the inner core to the outer core. In a large-core fibre, such a resonant coupling can considerably increase the differential leakage loss between the fundamental and the higher order modes and can result in effective single-mode operation. In a small-core single-mode fibre, such a coupling can lead to sharp increase in the wavelength dependent leakage loss near the resonant wavelength and can be utilized for the suppression of amplified spontaneous emission and thereby gain equalization of an optical amplifier. We study the propagation characteristics of the fibre using the transfer matrix method and present an example of each, the large-mode-area design for high power amplifiers and the wavelength tunable leakage loss design for inherent gain equalization of optical amplifiers.Comment: 6 page

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Usage and Perceived Side Effects of Personal Protective Measures against Mosquitoes among Current Users in Delhi

Background. Mosquito-borne diseases constitute an important cause of morbidity and mortality. The use of personal protective measures (PPM) like mats, bednets, screening, repellents, liquid vaporizers, mosquito coils, and so forth has been advocated as an effective tool in control of mosquito-borne diseases, but data about the safety profile of personal protective measures is still scarce. Objective. To study the usage and side effects of personal protective measures against mosquitoes among current users in Delhi. Materials and Methods. A community-based cross-sectional study among 350 adult individuals selected by systematic sampling method. Data was collected using pretested semistructured questionnaire after taking written informed consent. Data was analysed using SPSS version 17. Chi-square/Fisher’s Exact test was used for qualitative variables to find association and P value <0.05 was considered significant. Results. Out of 350 families selected, 210 belonged to rural area and 140 to urban area. Personal protective measures were used by 219 (62.5%) subjects. Liquid vaporizer was the most preferred method (41.4%). Most common perceived side effect of personal protective measures was headache (7.7%). Other perceived side effects were cough (3.2%), sore throat (2.7%), allergy (1.3%), and eye irritation (0.9%) predominantly among coil users. Conclusion. There is a need to have a close watch for side effects of personal protective measures among users. Further research is also needed to develop safe and effective personal protective measures against mosquitoes