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PubMed2025

PROLONG: penalized regression for outcome guided longitudinal omics analysis with network and group constraints.

MOTIVATION: There is a growing interest in longitudinal omics data paired with some longitudinal clinical outcome. Given a large set of continuous omics variables and some continuous clinical outcome, each measured for a few subjects at only a few time points, we seek to identify those variables that co-vary over time with the outcome. To motivate this problem we study a dataset with hundreds of urinary metabolites along with Tuberculosis mycobacterial load as our clinical outcome, with the objective of identifying potential biomarkers for disease progression. For such data clinicians usually apply simple linear mixed effects models which often lack power given the low number of replicates and time points. We propose a penalized regression approach on the first differences of the data that extends the lasso + Laplacian method [Li and Li (Network-constrained regularization and variable selection for analysis of genomic data. Bioinformatics 2008;24:1175-82.)] to a longitudinal group lasso + Laplacian approach. Our method, PROLONG, leverages the first differences of the data to increase power by pairing the consecutive time points. The Laplacian penalty incorporates the dependence structure of the variables, and the group lasso penalty induces sparsity while grouping together all contemporaneous and lag terms for each omic variable in the model. RESULTS: With an automated selection of model hyper-parameters, PROLONG correctly selects target metabolites with high specificity and sensitivity across a wide range of scenarios. PROLONG selects a set of metabolites from the real data that includes interesting targets identified during EDA. AVAILABILITY AND IMPLEMENTATION: An R package implementing described methods called "prolong" is available at https://github.com/stevebroll/prolong. Code snapshot available at 10.5281/zenodo.14804245.

Humans↗

PubMed1984

Identification of the replicative intermediates in SV40 DNA replication in vitro.

The soluble replication system is which the exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro, has been developed (Ariga and Sugano, J.Virol. 48, 481, 1983). This paper further characterized the in vitro products synthesized on the cloned DNA containing the origin of SV40 DNA replication. The time course and pluse-chase experiments showed that the in vitro products were converted from the open circle to closed circles having the various superhelical densities, and finally to the twisted formI DNA seen in vivo by the analysis of agarose gel electrophoresis, alkaline sucrose gradient centrifugation, and density-transfer in isopycnic centrifugation. The replicative intermediates isolated after the short term incubation had replicated strands of the size smaller than the full length, most of which correspond to that of the putative Okazaki fragment. These and the previous results indicate that this in vitro system should be useful to investigate the molecular mechanism of SV40 DNA replication.

DNA Replication↗

PubMed1983

Coreactivation of four inactive X genes in a hamster x human hybrid and persistence of late replication of reactivated X chromosome.

Hamster-human hybrids which contained an inactive human X chromosome were treated by 5-azacytidine. Hypoxanthine guanine phosphoribosyltransferase derepressed hybrids were selected and derepression of three other loci, phosphoglycerate kinase, alpha-galactosidase, and glucose-6-phosphate dehydrogenase were studied. Among 32 hybrids selected for hypoxanthine guanine phosphoribosyltransferase, two were found to be reactivated at four X loci. The independence or nonindependence of the reactivation events will be discussed. No correlation was found between the time of replication and the expression or nonexpression of the X chromosome genes: X chromosomes reactivated at four loci remained late replicating; conversely early replication can exist without the expression of some X genes.

Animals↗

PubMed2003

Lentivirus vector can integrate in the genome and exist and replicate in the cell as an episome.

Lethally irradiated mice were reconstituted with few purified primitive hemopoietic stem cells containing sequences of a gene encoding green fluorescent protein. The gene was transferred using a lentivirus vector. The presence of the marker gene in splenocyte colonies derived from the bone marrow of reconstituted mice and in cells of other hemopoietic and non-hemopoietic organs was studied during the life. It was shown that the lentivirus vector can persist for a long time and replicate in hemopoietic cells as an episome.

Animals↗

PubMed2003

Constitutive DNA damage is linked to DNA replication abnormalities in Bloom's syndrome cells.

Bloom's syndrome (BS) is an autosomal recessive disorder associated with an elevated incidence of cancers. The gene mutated in BS, BLM, encodes a RecQ helicase family member. BS cells exhibit genomic instability, including excessive homologous recombination and chromosomal aberrations. We reported previously that BS cells also demonstrate increased error-prone nonhomologous endjoining, which could contribute to genomic instability in these cells. Here, we show that BS cells display an abnormality in the timing of replication of both early-replicating genes and late-replicating loci such as chromosomal fragile sites. This delayed replication is associated with a constitutively increased frequency of sites of DNA damage and repair, as determined by the presence of DNA repair factors such as RAD51 and Ku86. In addition, another RecQ family helicase, WRN, also localizes to these repair sites. The presence of these repair sites correlates with the temporal appearance of cyclin B1 expression, indicative of the cells having progressed beyond mid-S phase in the cell division cycle. Critically, these defects in BS cells are the direct result of loss of BLM function, because BS cells phenotypically 'reverted' following transfection with the BLM cDNA no longer show such defects. Thus, our data indicate that constitutive DNA damage is coupled to delayed DNA replication in BS cells.

Aphidicolin↗

PubMed1984

Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

Adenoviridae↗

PubMed1994

Validation of a flow cytometric in vitro DNA repair (UDS) assay in rat hepatocytes.

An in vitro flow cytometric (FCM) DNA repair assay has been developed and validated by comparison to conventional autoradiography (ARG). Both assays measure unscheduled DNA synthesis (UDS). Cultures of hepatocytes from young male Sprague-Dawley rats were exposed to a battery of 26 chemicals plus bromodeoxyuridine (BrdUrd) or 3H-thymidine (3H-dT) for 18-20 h before harvest. Selection of test chemicals was based upon both their genotoxicity classifications and carcinogenicity bioassay results in male rats. DNA repair in chemically treated cultures was detected flow cytometrically by measuring the uptake of BrdUrd in non-replicating (G1, G2, mitotic and 4C) cells. Intracellular levels of incorporated BrdUrd were visualized by immunochemical labeling with fluorescein isothiocyanate (FITC), and total cellular DNA content was simultaneously estimated by counterstaining samples with the nucleic acid intercalator, propidium iodide (PI). Information was obtained from 10(4) cells/sample. Since repairing cells incorporate significantly less BrdUrd per unit of time than replicating cells, low intensity BrdUrd-FITC fluorescent signals from repairing cells are readily discriminated from high intensity signals from replicating cells when displayed on linear univariate histograms. Further distinction between repairing and replicating cells was achieved by displaying the DNA contents of all cells on linear bivariate histograms. Thus, repairing cells were resolved without subjecting these cultures to agents which suppress replicative synthesis (e.g., hydroxyurea). Results from these concurrent FCM and ARG investigations include the following: (1) conclusions (DNA repair positive or negative) were in agreement, with one exception, cinnamyl anthranilate, for which cytotoxic doses produced a positive FCM response, but lack of intact hepatocytes in parallel ARG preparations prevented analysis; (2) similar sensitivities for most of the positive chemicals were reported; (3) a high correlation (85%) exists between the reported genotoxicity classification and these DNA repair results in the absence of overt cytotoxicity; (4) a poor correlation exists between these DNA repair results and hepatocarcinogenesis (only 4/11 liver carcinogens tested positive) or overall carcinogenesis in the male rat (only 9/21 carcinogens tested positive). This FCM assay provides a rapid, sensitive, safe and reliable means of identifying agents which induce DNA repair in mammalian cells.

Animals↗

PubMed1975

Inhibition of DNA replication in Escherichia coli by cyanide and carbon monoxide.

The inhibition of DNA replication in aerobically growing Escherichia coli by cyanide or carbon monoxide occurs within about 20 s at 15 degrees, as previously reported by Cairns and Denhardt (Cairns, J., and Denhardt, D.T. (1968) J. Mol. Biol. 36, 335-342). This rapid inhibition can be explained by the nearly complete depletion of both intracellular ATP and deoxynucleoside triphosphates which occurs during the time that replication stops. There is probably no direct effect of carbon monoxide on any of the enzymes involved in replication because this reagent has no effect on replication rate or ATP level in anaerobic cells. These cells produce ATP by glycolysis. The inhibition of replication by cyanide, a highly reactive compound, appears to be more complex since anaerobically growing cells can still be completely inhibited, although higher concentrations are required than for aerobically growing cells. The sensitivity of anaerobic cells to cyanide is probably due to the ability of this highly reactive compound to react nonspecifically with many proteins and other molecules.

Adenosine Triphosphate↗

PubMed2005

Time may be compressed in sound representation as replicated in sensory memory.

The flow of time in the real world may differ from the one in the neural representation of auditory scene stored in the sensory memory, because the encoded information is free from the rule of actual time. The sensory memory underlying the automatic discriminative system is reflected by the mismatch negativity (MMN). The time-wise image of preceding sounds is integrated into the sensory memory as a 160-170 ms epoch. We measured the mismatch field (MMNm) and the reaction time responding to the omitted segments incorporated into a complex sound. The main result was that the encoded period from early to late omitted segment was shorter than the actual one, suggesting that time may be compressed in the sound representation.

Acoustic Stimulation↗

PubMed1979

Replication of porcine parvovirus in peripheral blood lymphocytes, monocytes, and peritoneal macrophages.

Porcine peripheral blood lymphocytes (PBL), peripheral blood monocytes, and peritoneal macrophages were examined for their ability to support porcine parvovirus (PPV) replication. The cell cultures were infected with the NADL-2 strain of PPV at 0.1 multiplicity of infection. PBL cultures were stimulated with the following phytomitogens: phytohemagglutinin M, concanavalin A, and pokeweed mitogen. Unstimulated PBL cultures infected with PPV and uninfected PBL stimulated with phytomitogens served as controls. All cultures were examined daily for PPV-specific immunofluorescence and hemagglutinin. PPV replicated in PBL cultures stimulated with all phytomitogens. Both viral hemagglutinin in culture fluids and nuclear immunofluorescence in cells were detected. In contrast, unstimulated PBL did not support viral replication; however, PPV antigen was detected in the cytoplasm. PPV persisted in unstimulated PBL for 21 days (duration of the experiment) without replication, but replicated each time with the addition of phytohemagglutinin M at 0, 3, 7, 14, and 21 days after infection. Uninfected PBL stimulated with phytomitogens lacked both viral hemagglutinin and immunofluorescence. Simultaneous detection of lymphocyte surface marker and viral antigens in pokeweed mitogen-stimulated PBL revealed that both T and non-T cells (B and null cells) are able to support PPV replication. Peripheral blood monocytes and peritoneal macrophages phagocytized PPV but did not support virus replication.

Animals↗

PubMedDate not supplied

Effect of age of maternal flock and strain on the incidence of tibial dyschondroplasia in growing chicks.

Two experiments were conducted to study the effects of age of the maternal flock (27 vs. 57 weeks of age at time the eggs were set) and strain of broiler chicks (Cobb 500, Ross, Arbor Acres, and Avian) on the incidence of tibial dyschondroplasia (TD) in broiler cockerels grown to 3 weeks of age. The chicks were fed a corn/soybean meal-based diet containing 3145 kcal metabolizable energy corrected for nitrogen retention (MEn)/kg, 24% protein, and 0.50% available phosphorus. Each treatment was replicated four times with eight chicks per pen. In Expt. 1, chicks from the old parent flock had significantly higher body weights (P < or = 0.05) than those from the young parent flock. There were no significant differences in feed efficiency or incidence of TD. In Expt. 2, body weights, feed efficiency, and TD were unaffected by strain.

Animal Feed↗

PubMed1972

Deoxyribonucleic acid-deoxyribonucleic acid hybridization assay for replication origin deoxyribonucleic acid of Escherichia coli.

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.

Amino Acid Sequence↗

PubMed1985

Controlled study of withdrawal symptoms and rebound anxiety after six week course of diazepam for generalised anxiety.

A group of patients suffering from anxiety, as assessed by general practitioners and psychologists using research criteria for generalised anxiety, were treated with either diazepam or placebo double blind for six weeks. This active treatment period was preceded by a one week single blind placebo "wash in" period and followed by a two week single blind placebo "wash out" period. The results suggest that diazepam can produce rebound anxiety and withdrawal symptoms when used in moderate doses and for what has previously been regarded as a safe length of time. If replicated these results have implications for the therapeutic use of benzodiazepines.

Adult↗

PubMed1968

Replicating units (replicons) of DNA in cultured mammalian cells.

Exponentially growing L5178Y mouse leukemic cells were incubated in the presence of 5'-bromodeoxyuridine (BUdR) for about 4 hr, transferred to the nonBUdR-containing medium for a certain period (t hours), and then pulse-labeled with TdR-(3)H for 10 min. When DNA isolated from these cells was subjected to CsCl gradient centrifugation, the (3)H-activity was found to shift gradually from the heavy BUdR-containing peak to the light nonBUdR-containing peak with increasing time t. The average time required for the complete shift of (3)H-activity from the heavy to the light DNA fraction was 2.76 hr. Taking this as the average replicating time and the size of DNA fragments in the present preparation as 1.3 x 10(7) daltons, the rate of replication was found to be 2.1 nucleotides per strand per replicon per sec. By taking the upper limit of the average replicating time as the S period (7.3 hr), various characteristics of the replicating units, such as the lower and upper limits of average size, the average replicating time, the average number of replicating units, etc., were calculated (see Table I).

Animals↗

PubMed2006

Identification of genes whose expression is associated with cisplatin resistance in human ovarian carcinoma cells.

The goal of this study was to identify genes consistently differentially expressed in multiple pairs of isogenic cisplatin (DDP)-sensitive and resistant human ovarian carcinoma cell lines using microarray-based expression profiling. Expression profiling was carried out on six pairs of ovarian carcinoma cells lines growing under identical conditions; each cell expression profile was independently replicated six times. No genes were differentially expressed in all six pairs of cells or even in even in any five of the six pairs. Eighteen genes and 1 EST were upregulated, and four genes and 1 EST were downregulated, in at least four cell pairs. Of these, only metallothionein 2A has previously been implicated in DDP resistance. Among the genes identified on the basis of six replicates, an average of 24.8% would have been missed if only five replicates had been performed, and 38.3% would have been missed with only four replicates. The genes did not identify a dominant biochemical pathway or ontology category as being linked to DDP resistance; however, hierarchical clustering provided evidence for two classes DDP-resistant phenotypes within which there are additional cell pair-specific alterations. Many of the genes identified in this study play important roles in cell surface interactions and trafficking pathways not previously linked to DDP resistance. The genes discovered by this extensively replicated analysis are candidates for prediction of DDP responsiveness in ovarian cancer patients.

Antineoplastic Agents↗

PubMed1995

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.

Adenoviridae↗

PubMed1998

The acetylation patterns of histones H3 and H4 along Vicia faba chromosomes are different.

The acetylation pattern of H3 was studied on field bean chromosomes by means of indirect immunofluorescence using polyclonal antibodies recognizing H3 isoforms acetylated at lysine positions 9/18, 14 and 23. H3 was found to be hypoacetylated at lysine residues 9/18 and 14 within the heterochromatic regions composed of tandem repetitive Fok-I elements. Hyperacetylation of these residues was observed at the nucleolar organizing region (NOR) and in heterochromatic regions composed of repeats other than Fok-I elements. In contrast, H4 was underacetylated (H4.Ac5, 8, 12) or uniformly acetylated (H4.Ac16) at all heterochromatic regions, and acetylated above the average at all four lysines only within the NOR. Acetylation of lysine-23 of H3 was uniform, except for the NOR that showed no fluorescence. Inhibition of deacetylase during and after replication of heterochromatin by trichostatin A had no influence on the acetylation status of H3 but mediated an increase in acetylation of lysines 5, 12 and 16 of H4 above the average in the field bean heterochromatin. Thus, the chromosomal acetylation patterns of H4 and H3 of this species revealed common and divergent features. Whereas the acetylation level of H4 correlates well with the potential transcriptional activity and inversely with the time of replication of defined chromatin domains of Vicia faba, this is not generally true for H3.

Acetylation↗

PubMed2004

Does a sentinel or a subset of short telomeres determine replicative senescence?

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

Cellular Senescence↗