A functional genomic analysis of cell morphology using RNA interference

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology

The globin gene family of the cephalochordate amphioxus: implications for chordate globin evolution

<p>Abstract</p> <p>Background</p> <p>The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of <it>Branchiostoma floridae </it>now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire.</p> <p>Results</p> <p>We show that <it>B. floridae </it>harbors at least fifteen paralogous globin genes, all of which reveal evidence of gene expression. The protein sequences of twelve globins display the conserved characteristics of a functional globin fold. In phylogenetic analyses, the amphioxus globin BflGb4 forms a common clade with vertebrate neuroglobins, indicating the presence of this nerve globin in cephalochordates. Orthology is corroborated by conserved syntenic linkage of <it>BflGb4 </it>and flanking genes. The kinetics of ligand binding of recombinantly expressed BflGb4 reveals that this globin is hexacoordinated with a high oxygen association rate, thus strongly resembling vertebrate neuroglobin. In addition, possible amphioxus orthologs of the vertebrate globin X lineage and of the myoglobin/cytoglobin/hemoglobin lineage can be identified, including one gene as a candidate for being expressed in notochord tissue. Genomic analyses identify conserved synteny between amphioxus globin-containing regions and the vertebrate <it>β-globin </it>locus, possibly arguing against a late transpositional origin of the <it>β-globin </it>cluster in vertebrates. Some amphioxus globin gene structures exhibit minisatellite-like tandem duplications of intron-exon boundaries ("mirages"), which may serve to explain the creation of novel intron positions within the globin genes.</p> <p>Conclusions</p> <p>The identification of putative orthologs of vertebrate globin variants in the <it>B. floridae </it>genome underlines the importance of cephalochordates for elucidating vertebrate genome evolution. The present study facilitates detailed functional studies of the amphioxus globins in order to trace conserved properties and specific adaptations of respiratory proteins at the base of chordate evolution.</p

Reversible melting and equilibrium phase formation of (Bi,Pb)2Sr2Ca2Cu3O10+d

The decomposition and the reformation of the (Bi,Pb)2Sr2Ca2Cu3O10+d (?Bi,Pb(2223)?) phase have been investigated in-situ by means of High-Temperature Neutron Diffraction, both in sintered bulk samples and in Ag-sheathed monofilamentary tapes. Several decomposition experiments were performed at various temperatures and under various annealing atmospheres, under flowing gas as well as in sealed tubes, in order to study the appropriate conditions for Bi,Pb(2223) formation from the melt. The Bi,Pb(2223) phase was found to melt incongruently into (Ca,Sr)2CuO3, (Sr,Ca)14Cu24O41 and a Pb,Bi-rich liquid phase. Phase reformation after melting was successfully obtained both in bulk samples and Ag-sheathed tapes. The possibility of crystallising the Bi,Pb(2223) phase from the melt was found to be extremely sensitive to the temperature and strongly dependent on the Pb losses. The study of the mass losses due to Pb evaporation was complemented by thermogravimetric analysis which proved that Pb losses are responsible for moving away from equilibrium and therefore hinder the reformation of the Bi,Pb(2223) phase from the melt. Thanks to the full pattern profile refinement, a quantitative phase analysis was carried out as a function of time and temperature and the role of the secondary phases was investigated. Lattice distortions and/or transitions were found to occur at high temperature in Bi,Pb(2223), Bi,Pb(2212), (Ca,Sr)2CuO3 and (Sr,Ca)14Cu24O41, due to cation diffusion and stoichiometry changes. The results indicate that it is possible to form the Bi,Pb(2223) phase from a liquid close to equilibrium conditions, like Bi(2212) and Bi(2201), and open new unexplored perspectives for high-quality Ag-sheathed Bi,Pb(2223) tape processing.Comment: 45 pages (including references,figures and captions), 13 figures Submitted to Supercond. Sci. Techno

Case Growth analysis to inform Local Response to Covid-19 Epidemic in a Diverse Us Community

Early detection of new outbreak waves is critical for effective and sustained response to the COVID-19 pandemic. We conducted a growth rate analysis using local community and inpatient records from seven hospital systems to characterize distinct phases in SARS-CoV-2 outbreak waves in the Greater Houston area. We determined the transition times from rapid spread of infection in the community to surge in the number of inpatients in local hospitals. We identified 193,237 residents who tested positive for SARS-CoV-2 via molecular testing from April 8, 2020 to June 30, 2021, and 30,031 residents admitted within local healthcare institutions with a positive SARS-CoV-2 test, including emergency cases. We detected two distinct COVID-19 waves: May 12, 2020-September 6, 2020 and September 27, 2020-May 15, 2021; each encompassed four growth phases: lagging, exponential/rapid growth, deceleration, and stationary/linear. Our findings showed that, during early stages of the pandemic, the surge in the number of daily cases in the community preceded that of inpatients admitted to local hospitals by 12-36 days. Rapid decline in hospitalized cases was an early indicator of transition to deceleration in the community. Our real-time analysis informed local pandemic response in one of the largest U.S. metropolitan areas, providing an operationalized framework to support robust real-world surveillance for outbreak preparedness

Clinical peripherality: development of a peripherality index for rural health services

BACKGROUND: The configuration of rural health services is influenced by geography. Rural health practitioners provide a broader range of services to smaller populations scattered over wider areas or more difficult terrain than their urban counterparts. This has implications for training and quality assurance of outcomes. This exploratory study describes the development of a "clinical peripherality" indicator that has potential application to remote and rural general practice communities for planning and research purposes. METHODS: Profiles of general practice communities in Scotland were created from a variety of public data sources. Four candidate variables were chosen that described demographic and geographic characteristics of each practice: population density, number of patients on the practice list, travel time to nearest specialist led hospital and travel time to Health Board administrative headquarters. A clinical peripherality index, based on these variables, was derived using factor analysis. Relationships between the clinical peripherality index and services offered by the practices and the staff profile of the practices were explored in a series of univariate analyses. RESULTS: Factor analysis on the four candidate variables yielded a robust one-factor solution explaining 75% variance with factor loadings ranging from 0.83 to 0.89. Rural and remote areas had higher median values and a greater scatter of clinical peripherality indices among their practices than an urban comparison area. The range of services offered and the profile of staffing of practices was associated with the peripherality index. CONCLUSION: Clinical peripherality is determined by the nature of the practice and its location relative to secondary care and administrative and educational facilities. It has features of both gravity model-based and travel time/accessibility indicators and has the potential to be applied to training of staff for rural and remote locations and to other aspects of health policy and planning. It may assist planners in conceptualising the effects on general practices of centralising specialist clinical services or administrative and educational facilities

Efficiency of Spermatogonial Dedifferentiation during Aging

Adult stem cells are critical for tissue homeostasis; therefore, the mechanisms utilized to maintain an adequate stem cell pool are important for the survival of an individual. In Drosophila, one mechanism utilized to replace lost germline stem cells (GSCs) is dedifferentiation of early progenitor cells. However, the average number of male GSCs decreases with age, suggesting that stem cell replacement may become compromised in older flies.Using a temperature sensitive allelic combination of Stat92E to control dedifferentiation, we found that germline dedifferentiation is remarkably efficient in older males; somatic cells are also effectively replaced. Surprisingly, although the number of somatic cyst cells also declines with age, the proliferation rate of early somatic cells, including cyst stem cells (CySCs) increases.These data indicate that defects in spermatogonial dedifferentiation are not likely to contribute significantly to an aging-related decline in GSCs. In addition, our findings highlight differences in the ways GSCs and CySCs age. Strategies to initiate or enhance the ability of endogenous, differentiating progenitor cells to replace lost stem cells could provide a powerful and novel strategy for maintaining tissue homeostasis and an alternative to tissue replacement therapy in older individuals

The effect of periodontal therapy on lymphocyte blastogenesis to plaque associated microorganisms

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66105/1/j.1600-0765.1983.tb00340.x.pd

Centrosome misorientation reduces stem cell division during ageing

Asymmetric division of adult stem cells generates one self- renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. Here we show that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells ( GSCs) are oriented within their niche and this ensures asymmetric division. We found that GSCs containing misoriented centrosomes accumulate with age and that these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re- enter the cell cycle on correction of centrosome orientation. On the basis of these findings, we propose that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. We also show that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia.University of Michigan ; March of Dimes Basil O'Conner Starter Scholar Research Award ; Searle Scholar Program ; NIH [P01 DK53074, R01GM072006]We thank C. Gonzalez, D. McKearin, N. Rusan, M. Peifer and the Bloomington Stock Center for fly stocks; R. Lehmann, C. Field and the Developmental Studies Hybridoma Bank for antibodies; M. Kiel and D. Nakada for help with X-ray irradiation; and S. Morrison and T. Mahowald for comments on the manuscript. This research was supported by a University of Michigan start-up fund, March of Dimes Basil O'Conner Starter Scholar Research Award and the Searle Scholar Program (to Y.M.Y.), and NIH grants P01 DK53074 (to M.T.F.) and R01GM072006 (to A.J.H.).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62879/1/nature07386.pd

A Novel Pathway of TEF Regulation Mediated by MicroRNA-125b Contributes to the Control of Actin Distribution and Cell Shape in Fibroblasts

BACKGROUND: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF. PRINCIPAL FINDINGS: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts