To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600).
Target exon capture and next generation sequencing was ...used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed.
Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state-a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function.
We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.
Effective management of albatross populations requires understanding the impacts of environmental factors on albatross demographics. An integrated modelling approach incorporating multiple data ...sources can further the understanding of albatross demographics by incorporating error from all components of modeling, and help distinguish between variability related to one factor (e.g. environment) from that of another factor (e.g. density dependence). We applied such an integrated, spatially-explicit population model to quantify the impact of environmental conditions (sea surface temperature, SST), fisheries, and density dependence on a black-browed albatross Thalassarche melanophris population breeding on Kerguelen Island, southern Indian Ocean for the period 1950 to 2011. The model was structured by sex, age-class, and breeding stage, with a 5° × 5° spatial scale and monthly temporal scale. All parameters were estimated within a maximum likelihood framework. This includes estimation of seabird bycatch rates of each of 5 fishing super-fleets, grouped by gear type and reported bycatch rates: (1) Japanese pelagic longline, (2) other pelagic longline, (3) legal demersal longline, (4) trawl, and (5) illegal, unreported, and unregulated (IUU) demersal longline. A decline in the Kerguelen black-browed albatross population occurred between the mid-1990s through the mid-2000s. Our analysis attributes the majority of modelled bycatch to the IUU demersal longline super-fleet operating near Kerguelen Island for this period. Including SST during the incubation period indicated that warm SST favors high breeding success. These results indicate that effective management requires an integrated understanding of the impacts of the environment as well as illegal and legal fishing activities on vulnerable populations.
Climate change is projected to continue shifting the distribution of marine species, leading to changes in local assemblages and different interactions with human activities. With regard to ...fisheries, understanding the relationship between fishing fleets, target species catch per unit effort (CPUE), and the environment enhances our ability to anticipate fisher response and is an essential step towards proactive management. Here, we explore the potential impact of climate change in the southern Indian Ocean by modelling Japanese and Taiwanese pelagic longline fleet dynamics. We quantify the mean and variability of target species CPUE and the relative value and cost of fishing in different areas. Using linear mixed models, we identify fleet-specific effort allocation strategies most related to observed effort and predict the future distribution of effort and tuna catch under climate change for 2063–2068. The Japanese fleet's strategy targets high-value species and minimizes the variability in CPUE of the primary target species. Conversely, the Taiwanese strategy indicated flexible targeting of a broad range of species, fishing in areas of high and low variability in catch, and minimizing costs. The projected future mean and variability in CPUE across species suggest a slight increase in CPUE in currently high CPUE areas for most species. The corresponding effort projections suggest a slight increase in Japanese effort in the western and eastern study area, and Taiwanese effort increasing east of Madagascar. This approach provides a useful method for managers to explore the impacts of different fishing and fleet management strategies for the future.
Lineage Specificity of Gene Expression Patterns Kluger, Yuval; Tuck, David P.; Chang, Joseph T. ...
Proceedings of the National Academy of Sciences - PNAS,
04/2004, Letnik:
101, Številka:
17
Journal Article
Recenzirano
Odprti dostop
The hematopoietic system offers many advantages as a model for understanding general aspects of lineage choice and specification. Using oligonucleotide microarrays, we compared gene expression ...patterns of multiple purified hematopoietic cell populations, including neutrophils, monocytes, macrophages, resting, centrocytic, and centroblastic B lymphocytes, dendritic cells, and hematopoietic stem cells. Some of these cells were studied under both resting and stimulated conditions. We studied the collective behavior of subsets of genes derived from the Biocarta database of functional pathways, hand-tuned groupings of genes into broad functional categories based on the Gene Ontology database, and the metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes database. Principal component analysis revealed strikingly pervasive differences in relative levels of gene expression among cell lineages that involve most of the subsets examined. These results indicate that many processes in these cells behave differently in different lineages. Much of the variation among lineages was captured by the first few principal components. Principal components biplots were found to provide a convenient visual display of the contributions of the various genes within the subsets in lineage discrimination. Moreover, by applying tree-constructing methodologies borrowed from phylogenetics to the expression data from differentiated cells and stem cells, we reconstructed a tree of relationships that resembled the established hematopoietic program of lineage development. Thus, the mRNA expression data implicity contained information about developmental relationships among cell types.
The Columbia University Informatics for Diabetes Education and Telemedicine (IDEATel) Project is a four-year demonstration project funded by the Centers for Medicare and Medicaid Services with the ...overall goals of evaluating the feasibility, acceptability, effectiveness, and cost-effectiveness of telemedicine in the management of older patients with diabetes. The study is designed as a randomized controlled trial and is being conducted by a state-wide consortium in New York. Eligibility requires that participants have diabetes, are Medicare beneficiaries, and reside in federally designated medically underserved areas. A total of 1,500 participants will be randomized, half in New York City and half in other areas of the state. Intervention participants receive a home telemedicine unit that provides synchronous videoconferencing with a project-based nurse, electronic transmission of home fingerstick glucose and blood pressure data, and Web access to a project Web site. End points include glycosylated hemoglobin, blood pressure, and lipid levels; patient satisfaction; health care service utilization; and costs. The project is intended to provide data to help inform regulatory and reimbursement policies for electronically delivered health care services.
We have quantified postganglionic sweat output in human subjects resulting from axon reflex stimulation using acetylcholine electrophoresis. Dehumidified nitrogen of controlled temperature and flow ...rate was passed through an acrylic plastic chamber placed over a defined area of skin. Sweat droplets were evaporated; humidity change was sensed by a narrow-range humidity sensor housed in a temperature-controlled compartment and was plotted on a chart recorder. The time integral (area under the curve) was continuously integrated and converted to absolute units using a derived equation. Because stimulation and recording were simultaneous, an accurate determination of the latency of the sweat response was also possible. Quantitative sudomotor axon reflex tests were performed on the left forearm and foot of 33 female and 29 male normal subjects aged 11 to 69 years. Acetylcholine, 10%, was electrophoresed for 5 mA-minutes in the forearm and 10 mA-minutes in the foot, and recording was continued for an additional 5 minutes. The mean sweat output in males was 2.7 and 3.0 times that in females in forearm and foot, respectively (p less than 0.0001). Studies in selected autonomic neuropathies confirm that quantitative sudomotor axon reflex tests will detect postganglionic sudomotor abnormalities sensitively and reproducibly.
Cycloleucine was used to inhibit the formation of internal N
6-methyladenosine residues in the messenger ribonucleic acid transcripts from cultured methotrexate resistant mouse sarcoma cells. Cells ...cultured in cycloleucine produced transcripts deficient in N
6-methyladenosine residues and the 2′-O-methylated nucleosides of the cap structure; however, the formation of the 7-methylguanine nucleoside of the cap was not effected. Cytoplasmic polyadenylated transcripts were isolated from cells which had been pretreated with media containing cycloleucine and translated in an in vitro translation assay. The levels of translated dihydrofolate reductase were then analyzed by polyacrylamide gel electrophoresis. The amount of dihydrofolate reductase protein produced from the transcripts of the cycloleucine treated cells was 20% less than untreated transcripts. Ribonuclease protection assays demonstrated little difference in the cytoplasmic levels of dihydrofolate reductase transcripts between treated and untreated cells suggesting that the decrease in translation efficiency was not caused solely by an alteration in the processing or cytoplasmic transport of the transcripts. Translation of in vitro transcribed transcripts showed the presence of 2′-O-methylated nucleosides in the cap structure had a negative effect on translation efficiency, demonstrating that the results observed from cycloleucine treatment could not be due to the inhibition of 2′-O-methylation in the cap. These experiments therefore suggest that an inhibition of N
6-methyladenosine residues in dihydrofolate reductase transcripts significantly alters their rate of translation.
Heat shock proteins (HSPs) facilitate the folding or degradation of misfolded, damaged and aggregated proteins. Disruptions in HSP function may underlie the molecular basis of many degenerative ...disorders including some myopathies. The pathogenic mechanism of these chaperonopathies is unclear. We recently identified mutations in DNAJB6, an HSP40 co-chaperone, as the cause of a hereditary IBM also named LGMD1D. One feature of LGMD1D muscle is the accumulation of protein inclusions that contain TDP-43. TDP-43 is an RNA binding protein with a prion-like domain (PrLD) that is mutated in familial amyotrophic lateral sclerosis (ALS). LGMD1D mutations in DNAJB6 reside within the highly conserved G/F domain. Although the role of the G/F domain in DNAJB6 is unclear, studies in S.cerevisiae, have shown that the homologous G/F domain in Sis1 (a DNAJB6 ortholog) is required for the propagation of select yeast prions. Yeast prions contain Q/N rich PrLDs, a feature they share with TDP-43 and other RNA binding proteins. Consistent with this, homologous LGMD1D mutation in the G/F domain of Sis1 abrogate its ability to modulate yeast prion propagation. In mammalian cell culture DNAJB6 associates with TDP-43 in the nucleus upon heat shock suggesting that TDP-43 is indeed a DNAJB6 client protein. DNAJB6 expression reduces the formation and enhances the dissolution of TDP-43 positive nuclear bodies. LGMD1D mutant DNAJB6 expression increases TDP-43 granule formation and slows their dissolution upon heat shock recovery. This effect is more pronounced in cells expressing DNAJB6 that lacks the G/F domain. We hypothesize that LGMD1D mutant DNAJB6 affects localization, aggregation and toxicity of TDP43. Characterization of a transgenic mouse model of LGMD1D recently generated in our laboratory will help to elucidate the role of DNAJB6 and other HSPs in skeletal muscle disease and the complex interplay between RNA binding protein aggregation and disaggregation.
Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by ...single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.
We have used an approach using 2-dimensional gel electrophoresis with mass spectrometry analysis combined with oligonucleotide chip hybridization for a comprehensive and quantitative study of the ...temporal patterns of protein and mRNA expression during myeloid development in the MPRO murine cell line. This global analysis detected 123 known proteins and 29 “new” proteins out of 220 protein spots identified by tandem mass spectroscopy, including proteins in 12 functional categories such as transcription factors and cytokines. Bioinformatic analysis of these proteins revealed clusters with functional importance to myeloid differentiation. Previous analyses have found that for a substantial number of genes the absolute amount of protein in the cell is not strongly correlated to the amount of mRNA. These conclusions were based on simultaneous measurement of mRNA and protein at just a single time point. Here, however, we are able to investigate the relationship between mRNA and protein in terms of simultaneous changes in their levels over multiple time points. This is the first time such a relationship has been studied, and we find that it gives a much stronger correlation, consistent with the hypothesis that a substantial proportion of protein change is a consequence of changed mRNA levels, rather than posttranscriptional effects. Cycloheximide inhibition also showed that most of the proteins detected by gel electrophoresis were relatively stable. Specific investigation of transcription factor mRNA representation showed considerable similarity to those of mature human neutrophils and highlighted several transcription factors and other functional nuclear proteins whose mRNA levels change prominently during MPRO differentiation but which have not been investigated previously in the context of myeloid development. Data are available online athttp://bioinfo.mbb.yale.edu/expression/myelopoiesis.
PDF
