Quantitative proteomic analyses of human cytomegalovirus-induced restructuring of endoplasmic reticulum-mitochondrial contacts at late times of infection

Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon continued cell survival. To gain insight into how human cytomegalovirus infection affects endoplasmic reticulum-mitochondrial contacts, we undertook quantitative proteomics of mitochondria-associated membranes using differential stable isotope labeling by amino acids in cell culture strategy and liquid chromatography-tandem MS analysis. This is the first reported quantitative proteomic analyses of a suborganelle during permissive human cytomegalovirus infection. Human fibroblasts were uninfected or human cytomegalovirus-infected for 72 h. Heavy mitochondria-associated membranes were isolated from paired unlabeled, uninfected cells and stable isotope labeling by amino acids in cell culture-labeled, infected cells and analyzed by liquid chromatography-tandem MS analysis. The results were verified by a reverse labeling experiment. Human cytomegalovirus infection dramatically altered endoplasmic reticulum-mitochondrial contacts by late times. Notable is the increased abundance of several fundamental networks in the mitochondria-associated membrane fraction of human cytomegalovirus-infected fibroblasts. Chaperones, including HSP60 and BiP, which is required for human cytomegalovirus assembly, were prominently increased at endoplasmic reticulum-mitochondrial contacts after infection. Minimal translational and translocation machineries were also associated with endoplasmic reticulum-mitochondrial contacts and increased after human cytomegalovirus infection as were glucose regulated protein 75 and the voltage dependent anion channel, which can form an endoplasmic reticulum-mitochondrial calcium signaling complex. Surprisingly, mitochondrial metabolic enzymes and cytosolic glycolytic enzymes were confidently detected in the mitochondria-associated membrane fraction and increased therein after infection. Finally, proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa1, were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. Together, these results suggest that human cytomegalovirus infection restructures the proteome of endoplasmic reticulum-mitochondrial contacts to bolster protein translation at these junctions, calcium signaling to mitochondria, cell survival, and bioenergetics and, thereby, allow for enhanced progeny production.     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

Testing for temporal variation in diversification rates when sampling is incomplete and nonrandom

A common pattern found in phylogeny-based empirical studies of diversification is a decrease in the rate of lineage accumulation toward the present. This early-burst pattern of cladogenesis is often interpreted as a signal of adaptive radiation or density-dependent processes of diversification. However, incomplete taxonomic sampling is also known to artifactually produce patterns of rapid initial diversification. The Monte Carlo constant rates (MCCR) test, based upon Pybus and Harvey's gamma (γ)-statistic, is commonly used to accommodate incomplete sampling, but this test assumes that missing taxa have been randomly pruned from the phylogeny. Here we use simulations to show that preferentially sampling disparate lineages within a clade can produce severely inflated type-I error rates of the MCCR test, especially when taxon sampling drops below 75%. We first propose two corrections for the standard MCCR test, the proportionally deeper splits that assumes missing taxa are more likely to be recently diverged, and the deepest splits only MCCR that assumes that all missing taxa are the youngest lineages in the clade, and assess their statistical properties. We then extend these two tests into a generalized form that allows the degree of nonrandom sampling (NRS)to be controlled by a scaling parameter, α. This generalized test is then applied to two recent studies. This new test allows systematists to account for nonrandom taxonomic sampling when assessing temporal patterns of lineage diversification in empirical trees. Given the dramatic affect NRS can have on the behavior of the MCCR test, we argue that evaluating the sensitivity of this test to NRS should become the norm when investigating patterns of cladogenesis in incompletely sampled phylogenies.     

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.     

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (～45%) of the 1,101 essential yeast genes, with ～30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.     

Assay development and high-throughput screening of small molecular c-Abl kinase activators

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC(50) <10 μM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.     

The seated medicine ball throw as a test of upper body power in older adults

Practitioners training the older adult may benefit from a low-cost, easy-to-administer field test of upper body power. This study evaluated validity and reliability of the seated medicine ball throw (SMBT) in older adults. Subjects (n = 33; age 72.4 ± 5.2 years) completed 6 trials of an SMBT in each of 2 testing days and 2 ball masses (1.5 and 3.0 kg). Subjects also completed 6 trials of an explosive push-up (EPU) on a force plate over 2 testing days. Validity was assessed via a Pearson Product-Moment correlation (PPM) between SMBT and EPU maximal vertical force. Reliability of the SMBT was determined using PPMs (r), Intraclass correlation (ICC, R) and Bland-Altman plots (BAPs). For validity, the association between the SMBT and the EPU revealed a PPM of r = 0.641 and r = 0.614 for the 1.5- and 3.0-kg medicine balls, respectively. Test-retest reliability of the 1.5- and 3.0-kg SMBT was r = 0.967 and r = 0.958, respectively. The ICC values of the 1.5- and 3.0-kg SMBT were R = 0.994 and 0.989, respectively. The BAPs revealed 94% of the differences between day 1 and 2 scores were within the 95% confidence interval of the mean difference. Test-retest reliability for the EPU was r = 0.944, R = 0.969. The BAPs showed 94% of the differences between day 1 and 2 scores were within the 95% confidence interval of the mean difference, for both medicine ball throws. In conclusion, for the older adult, the SMBT appears to be highly reliable test of upper body power. Its validity relative to the maximal force exerted during the EPU is modest. The SMBT is an inexpensive, safe, and repeatable measure of upper body power for the older adult.     

Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells

Human Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion during prophase and prevents premature sister-chromatid separation. Heterochromatin protein 1 (HP1) has been proposed to protect centromeric sister-chromatid cohesion by directly targeting Sgo1 to centromeres in mitosis. Here we show that HP1α is targeted to mitotic centromeres by INCENP, a subunit of the chromosome passenger complex (CPC). Biochemical and structural studies show that both HP1-INCENP and HP1-Sgo1 interactions require the binding of the HP1 chromo shadow domain to PXVXL/I motifs in INCENP or Sgo1, suggesting that the INCENP-bound, centromeric HP1α is incapable of recruiting Sgo1. Consistently, a Sgo1 mutant deficient in HP1 binding is functional in centromeric cohesion protection and localizes normally to centromeres in mitosis. By contrast, INCENP or Sgo1 mutants deficient in HP1 binding fail to localize to centromeres in interphase. Therefore, our results suggest that HP1 binding by INCENP or Sgo1 is dispensable for centromeric cohesion protection during mitosis of human cells, but might regulate yet uncharacterized interphase functions of CPC or Sgo1 at the centromeres.     

As we come to the end of 2011, several members of the Genome Biology Editorial Board give their views on the state of play in genomics

As we come to the end of 2011, Genome Biology has asked some members of our Editorial Board for their views on the state of play in genomics. What was their favorite paper of 2011? What are the challenges in their particular research area? Who has had the biggest influence on their careers? What advice would they give to young researchers embarking on a career in research?