A Biomedically Enriched Collection of 7000 Human ORF Clones

We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in ∼15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance

Effects of juvenile host density and food availability on adult immune response, parasite resistance and virulence in a Daphnia-parasite system

Host density can increase infection rates and reduce host fitness as increasing population density enhances the risk of becoming infected either through increased encounter rate or because host condition may decline. Conceivably, potential hosts could take high host density as a cue to up-regulate their defence systems. However, as host density usually covaries with food availability, it is difficult to examine the importance of host density in isolation. Thus, we performed two full-factorial experiments that varied juvenile densities of Daphnia magna (a freshwater crustacean) and food availability independently. We also included a simulated high-density treatment, where juvenile experimental animals were kept in filtered media that previously maintained Daphnia at high-density. Upon reaching adulthood, we exposed the Daphnia to their sterilizing bacterial parasite, Pasteuria ramosa, and examined how the juvenile treatments influenced the likelihood and severity of infection (Experiment I) and host immune investment (Experiment II). Neither juvenile density nor food treatments affected the likelihood of infection; however, well-fed hosts that were well-fed as juveniles produced more offspring prior to sterilization than their less well-fed counterparts. By contrast, parasite growth was independent of host juvenile resources or host density. Parasite-exposed hosts had a greater number of circulating haemocytes than controls (i.e., there was a cellular immune response), but the magnitude of immune response was not mediated by food availability or host density. These results suggest that density dependent effects on disease arise primarily through correlated changes in food availability: low food could limit parasitism and potentially curtail epidemics by reducing both the host's and parasite's reproduction as both depend on the same food

Persistence of single species of symbionts across multiple closelyrelated host species

Some symbiont species are highly host-specific, inhabiting only one or a very few host species, and typically have limited dispersal abilities. When they do occur on multiple host species, populations of such symbionts are expected to become genetically structured across these different host species, and this may eventually lead to new symbiont species over evolutionary timescales. However, a low number of dispersal events of symbionts between host species across time might be enough to prevent population structure and species divergence. Overall, processes of evolutionary divergence and the species status of most putative multi-host symbiont systems are yet to be investigated. Here, we used DNA metabarcoding data of 6,023 feather mites (a total of 2,225 OTU representative sequences) from 147 infracommunities (i.e., the assemblage consisting of all mites of different species collected from the same bird host individual) to investigate patterns of population genetic structure and species status of three different putative multi-host feather mite species Proctophyllodes macedo Vitzthum, 1922, Proctophyllodes motacillae Gaud, 1953, and Trouessartia jedliczkai (Zimmerman, 1894), each of which inhabits a variable number of different closely related wagtail host species (genus Motacilla). We show that mite populations from different host species represent a single species. This pattern was found in all the mite species, suggesting that each of these species is a multi-host species in which dispersal of mites among host species prevents species divergence. Also, we found evidence of limited evolutionary divergence manifested by a low but significant level of population genetic structure among symbiont populations inhabiting different host species. Our study agrees with previous studies showing a higher than expected colonization opportunities in host-specific symbionts. Indeed, our results support that these dispersal events would allow the persistence of multi-host species even in symbionts with limited dispersal capabilities, though additional factors such as the geographical structure of some bird populations may also play a role.This work was supported by the MINECO CGL2011-24466 to RJ and CGL2015-69650-P to RJ and DS

Consistent Pattern of Local Adaptation during an Experimental Heat Wave in a Pipefish-Trematode Host-Parasite System

Extreme climate events such as heat waves are expected to increase in frequency under global change. As one indirect effect, they can alter magnitude and direction of species interactions, for example those between hosts and parasites. We simulated a summer heat wave to investigate how a changing environment affects the interaction between the broad-nosed pipefish (Syngnathus typhle) as a host and its digenean trematode parasite (Cryptocotyle lingua). In a fully reciprocal laboratory infection experiment, pipefish from three different coastal locations were exposed to sympatric and allopatric trematode cercariae. In order to examine whether an extreme climatic event disrupts patterns of locally adapted host-parasite combinations we measured the parasite's transmission success as well as the host's adaptive and innate immune defence under control and heat wave conditions. Independent of temperature, sympatric cercariae were always more successful than allopatric ones, indicating that parasites are locally adapted to their hosts. Hosts suffered from heat stress as suggested by fewer cells of the adaptive immune system (lymphocytes) compared to the same groups that were kept at 18°C. However, the proportion of the innate immune cells (monocytes) was higher in the 18°C water. Contrary to our expectations, no interaction between host immune defence, parasite infectivity and temperature stress were found, nor did the pattern of local adaptation change due to increased water temperature. Thus, in this host-parasite interaction, the sympatric parasite keeps ahead of the coevolutionary dynamics across sites, even under increasing temperatures as expected under marine global warming

Genomic activation of the EGFR and HER2-neu genes in a significant proportion of invasive epithelial ovarian cancers

<p>Abstract</p> <p>Background</p> <p>The status of the EGFR and HER2-neu genes has not been fully defined in ovarian cancer. An integrated analysis of both genes could help define the proportion of patients that would potentially benefit from targeted therapies.</p> <p>Methods</p> <p>We determined the tumour mutation status of the entire tyrosine kinase (TK) domain of the EGFR and HER2-neu genes in a cohort of 52 patients with invasive epithelial ovarian cancer as well as the gene copy number and protein expression of both genes in 31 of these patients by DGGE and direct sequecing, immunohistochemistry and Fluorescent in Situ Hybridisation (FISH).</p> <p>Results</p> <p>The EGFR was expressed in 59% of the cases, with a 2+/3+ staining intensity in 38%. HER2-neu expression was found in 35%, with a 2/3+ staining in 18%. No mutations were found in exons 18–24 of the TK domains of EGFR and HER2-neu. High polysomy of the EGFR gene was observed in 13% of the invasive epthelial cancers and amplification of the HER2-neu gene was found in 10% and correlated with a high expression level by immunohistochemistry.</p> <p>Mutations within the tyrosine kinase domain were not found in the entire TK domain of both genes, but have been found in very rare cases by others.</p> <p>Conclusion</p> <p>Genomic alteration of the HER2-neu and EGFR genes is frequent (25%) in ovarian cancer. EGFR/HER2-neu targeted therapies should be investigated prospectively and specifically in that subset of patients.</p

A Defined, Feeder-Free, Serum-Free System to Generate In Vitro Hematopoietic Progenitors and Differentiated Blood Cells from hESCs and hiPSCs

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimentional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells

Susceptibility of Anopheles stephensi to Plasmodium gallinaceum: A Trait of the Mosquito, the Parasite, and the Environment

Vector susceptibility to Plasmodium infection is treated primarily as a vector trait, although it is a composite trait expressing the joint occurrence of the parasite and the vector with genetic contributions of both. A comprehensive approach to assess the specific contribution of genetic and environmental variation on "vector susceptibility" is lacking. Here we developed and implemented a simple scheme to assess the specific contributions of the vector, the parasite, and the environment to "vector susceptibility." To the best of our knowledge this is the first study that employs such an approach.We conducted selection experiments on the vector (while holding the parasite "constant") and on the parasite (while holding the vector "constant") to estimate the genetic contributions of the mosquito and the parasite to the susceptibility of Anopheles stephensi to Plasmodium gallinaceum. We separately estimated the realized heritability of (i) susceptibility to parasite infection by the mosquito vector and (ii) parasite compatibility (transmissibility) with the vector while controlling the other. The heritabilities of vector and the parasite were higher for the prevalence, i.e., fraction of infected mosquitoes, than the corresponding heritabilities of parasite load, i.e., the number of oocysts per mosquito.The vector's genetics (heritability) comprised 67% of "vector susceptibility" measured by the prevalence of mosquitoes infected with P. gallinaceum oocysts, whereas the specific contribution of parasite genetics (heritability) to this trait was only 5%. Our parasite source might possess minimal genetic diversity, which could explain its low heritability (and the high value of the vector). Notably, the environment contributed 28%. These estimates are relevant only to the particular system under study, but this experimental design could be useful for other parasite-host systems. The prospects and limitations of the genetic manipulation of vector populations to render the vector resistant to the parasite are better considered on the basis of this framework

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in Survival Motor Neuron 1 (SMN1), leading to degeneration of alpha motor neurons (MNs) but also affecting other cell types. Induced pluripotent stem cell (iPSC)-derived human MN models from severe SMA patients have shown relevant phenotypes. We have produced and fully characterized iPSCs from members of a discordant consanguineous family with chronic SMA. We differentiated the iPSC clones into ISL-1+/ChAT+ MNs and performed a comparative study during the differentiation process, observing significant differences in neurite length and number between family members. Analyses of samples from wild-type, severe SMA type I and the type IIIa/IV family showed a progressive decay in SMN protein levels during iPSC-MN differentiation, recapitulating previous observations in developmental studies. PLS3 underwent parallel reductions at both the transcriptional and translational levels. The underlying, progressive developmental decay in SMN and PLS3 levels may lead to the increased vulnerability of MNs in SMA disease. Measurements of SMN and PLS3 transcript and protein levels in iPSC-derived MNs show limited value as SMA biomarkers