Biomedical subjects

Ravi S Kamath

Publications and source records attributed to Ravi S Kamath.

15 recordsLinked to original sources

PubMed2005

Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.

Caenorhabditis elegans homologues of the retinoblastoma (Rb) tumour suppressor complex specify cell lineage during development. Here we show that mutations in Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes and elaborate perinuclear structures normally limited to germline-specific P granules. Furthermore, particular gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act by a similar mechanism to transform mammalian cells.

Alleles↗

PubMed2005

Functional genomic analysis of RNA interference in C. elegans.

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.

Amino Acid Motifs↗

PubMed2004

Radiation from "extra" images acquired with abdominal and/or pelvic CT: effect of automatic tube current modulation.

PURPOSE: To retrospectively determine the number and usefulness of images acquired beyond the intended anatomic area of interest with abdominal and/or pelvic computed tomography (CT) and to assess the effect of automatic tube current modulation (ATCM) on associated radiation. MATERIALS AND METHODS: Superior and inferior levels at routine abdominal and/or pelvic CT were defined as the dome of the diaphragm and the inferior margin of the pubic symphysis, respectively. Records of 106 consecutive examinations (male-to-female ratio, 45:61; age range, 21-86 years) performed from June 1 to June 30, 2003, were reviewed to determine the number of "extra" images. Sixty-two abdominal and/or pelvic CT examinations performed concurrently with chest or thigh CT or for trauma were not included in the 106. Abdominal and/or pelvic CT was performed with either ATCM (n = 44) or manual selection of tube current (n = 62). CT parameters recorded for each extra image included tube current, peak kilovoltage, and gantry rotation time. Mean and median tube current-time products were calculated for extra images. Extra images were analyzed for pathologic findings. Statistical analysis was performed with the Student t test. RESULTS: Extra images were acquired above the dome of the diaphragm in 103 (97%) of 106 examinations and below the pubic symphysis in 100 (94%) of 106. A total of 1,280 extra images were acquired in 106 examinations (mean, 12 images per examination). Nineteen additional findings were observed on extra images. With ATCM, mean tube current-time product was 74.5 and 120.6 mAs for extra images acquired above the diaphragm and below the pubic symphysis, respectively; with manual selection, mean tube current-time products were 167.5 and 168.3 mAs (P <.05). CONCLUSION: Most extra images acquired at abdominal and/or pelvic CT contributed no additional information. With ATCM, the radiation dose was reduced by a mean of 56% (median, 72%) for extra images above the diaphragm and 29% (median, 36%) for images below the pubic symphysis, compared with dose levels with manual selection.

Adult↗

PubMed2004

Comparison of Z-axis automatic tube current modulation technique with fixed tube current CT scanning of abdomen and pelvis.

PURPOSE: To compare image quality, diagnostic acceptability, and radiation exposure associated with 16-section multi-detector row computed tomographic (CT) examinations of abdomen and pelvis performed with z-axis modulation technique of automatic tube current modulation and with manual selection of fixed tube current. MATERIALS AND METHODS: Sixty-two consecutive subjects (mean age, 60 years; age range, 19-84 years; male-to-female ratio, 35:27) underwent follow-up CT of abdomen and pelvis with use of a 16-section multi-detector row scanner and z-axis modulation technique (10.5-12.0-HU noise index, 10-380 mA). Scanning parameters included 140 kVp, 0.5-1.0-second gantry rotation time, 0.938:1 beam pitch, and 5-mm reconstructed section thickness. For each subject, images obtained with z-axis modulation were compared with previous images obtained with fixed tube current (200-300 mA) and with other parameters identical. Images were compared for noise and diagnostic acceptability by two subspecialty radiologists using a five-point scale (1, unacceptable; 3, acceptable; 5, excellent) at five levels: upper liver at diaphragm, porta hepatis, right kidney hilum, iliac crest, and upper margin of acetabulum. Tube current and gantry rotation time used for acquisitions at these levels were recorded. Data were analyzed with parametric and nonparametric statistical tests. RESULTS: Although no significant differences were found (P =.34), images acquired with z-axis modulation at the levels of the upper liver (diaphragm) and acetabulum had a higher noise and lower diagnostic quality, compared with images acquired with fixed tube current. Compared with fixed tube current, z-axis modulation resulted in tube current-time product reduction in 54 (87%) of 62 examinations (mean reduction, 71.2 mAs) and increase in eight (13%) (mean increase, 17.0 mAs). CONCLUSION: Compared with manually selected fixed tube current, z-axis automatic tube current modulation resulted in reduced tube current-time product and similar image noise and diagnostic acceptability at CT of abdomen and pelvis.

Abdominal Neoplasms↗

PubMed2004

Sixteen-detector row CT of abdomen and pelvis: study for optimization of Z-axis modulation technique performed in 153 patients.

PURPOSE: To retrospectively determine the optimal noise indexes required to obtain diagnostically acceptable computed tomographic (CT) images of the abdomen and pelvis with z-axis modulation. MATERIALS AND METHODS: Ninety-five patients underwent 16-section multi-detector row CT of the abdomen and pelvis with z-axis modulation at noise indexes of 10.5, 11.0, 11.5, and 12.0 HU with 10-380 mA. Subsequently, 58 patients were scanned at noise indexes of 12.5 and 15.0 HU with 75-380 mA. The weights of all subjects were recorded, and transverse and anteroposterior diameters were measured. The CT images were evaluated for abnormalities and graded for image quality in terms of noise and diagnostic acceptability by using a five-point scale. Objective noise in the liver parenchyma was measured, and the tube current was recorded at each section in all 153 patients. Statistical analyses were performed to determine the appropriate noise index and to assess the effect of patient weight and abdominal diameters on image noise and diagnostic acceptability at different noise indexes. Tube current-time products (in milliampere seconds) at various noise indexes were compared with those at CT previously performed without z-axis modulation. RESULTS: No significant difference in subjective image noise or diagnostic acceptability was found at noise indexes of 10.5-15.0 HU (P =.14), and objective noise was significantly inferior only at a noise index of 15.0 HU (P =.009). Compared with CT scanning at a 10.5-HU noise index, CT scanning at 12.5- and 15.0-HU noise indexes yielded, respectively, 10.0% and 41.3% reductions in radiation exposure. Patient weight and abdominal diameters affected subjective image quality. CONCLUSION: Use of a 15.0-HU noise index at 75-380 mA results in acceptable subjective image noise and diagnostic acceptability but significantly greater objective image noise at routine abdominal-pelvic CT. For greater image quality demands, a noise index of 12.5 HU results in acceptable image quality and a 19.6% reduction in radiation exposure.

Abdomen↗

PubMed2003

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions.

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.

Animals↗

PubMed2003

A genome-wide screen identifies 27 genes involved in transposon silencing in C. elegans.

Transposon jumps are a major cause of genome instability. In the C. elegans strain Bristol N2, transposons are active in somatic cells, but they are silenced in the germline, presumably to protect the germline from mutations. Interestingly, the transposon-silencing mechanism shares factors with the RNAi machinery. To better understand the mechanism of transposon silencing, we performed a genome-wide RNAi screen for genes that, when silenced, cause transposition of Tc1 in the C. elegans germline. We identified 27 such genes, among which are mut-16, a mutator that was previously found but not identified at the molecular level, ppw-2, a member of the argonaute family, and several factors that indicate a role for chromatin structure in the regulation of transposition. Some of the newly identified genes are also required for cosuppression and therefore represent the shared components of the two pathways. Since most of the newly identified genes have clear homologs in other species, and since transposons are found from protozoa to human, it seems likely that they also protect other genomes against transposon activity in the germline.

Animals↗

PubMed2003

Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans.

Ageing is a fundamental, unsolved mystery in biology. DAF-16, a FOXO-family transcription factor, influences the rate of ageing of Caenorhabditis elegans in response to insulin/insulin-like growth factor 1 (IGF-I) signalling. Using DNA microarray analysis, we have found that DAF-16 affects expression of a set of genes during early adulthood, the time at which this pathway is known to control ageing. Here we find that many of these genes influence the ageing process. The insulin/IGF-I pathway functions cell non-autonomously to regulate lifespan, and our findings suggest that it signals other cells, at least in part, by feedback regulation of an insulin/IGF-I homologue. Furthermore, our findings suggest that the insulin/IGF-I pathway ultimately exerts its effect on lifespan by upregulating a wide variety of genes, including cellular stress-response, antimicrobial and metabolic genes, and by downregulating specific life-shortening genes.

Aging↗

PubMed2003

Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi.

An RNA interference (RNAi)-based genome-wide screen was performed to detect genes that contribute to genome stability in somatic cells of Caenorhabditis elegans. We identified 61 such genes; these also affect spontaneous mutagenesis in the germ line. Their sequence suggests a role in DNA repair and/or replication, in chromatin remodeling, or in cell cycle control; there are also many novel genes that are highly conserved from yeast to human. Because known mutator genes are causally involved in many hereditary and sporadic human cancers, it is likely that some of these new mutators are equally relevant in cancer etiology.

Animals↗

PubMed2003

Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.

A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of approximately 86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.

Animals↗

PubMed2003

Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes.

Regulation of body fat storage involves signalling between centres that regulate feeding in the brain and sites of fat storage and use in the body. Here we describe an assay for analysing fat storage and mobilization in living Caenorhabditis elegans. By using RNA-mediated interference (RNAi) to disrupt the expression of each of the 16,757 worm genes, we have systematically screened the C. elegans genome for genes necessary for normal fat storage. We identify 305 gene inactivations that cause reduced body fat and 112 gene inactivations that cause increased fat storage. Analysis of the fat-reducing gene inactivations in insulin, serotonin and tubby signalling mutants of C. elegans, which have increased body fat, identifies a core set of fat regulatory genes as well as pathway-specific fat regulators. Many of the newly identified worm fat regulatory genes have mammalian homologues, some of which are known to function in fat regulation. Other C. elegans fat regulatory genes that are conserved across animal phylogeny, but have not previously been implicated in fat storage, may point to ancient and universal features of fat storage regulation, and identify targets for treating obesity and its associated diseases.

Adipose Tissue↗

PubMed2003

Genome-wide RNAi screening in Caenorhabditis elegans.

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.

Animals↗

PubMed2002

Rates of behavior and aging specified by mitochondrial function during development.

To explore the role of mitochondrial activity in the aging process, we have lowered the activity of the electron transport chain and adenosine 5'-triphosphate (ATP) synthase with RNA interference (RNAi) in Caenorhabditis elegans. These perturbations reduced body size and behavioral rates and extended adult life-span. Restoring messenger RNA to near-normal levels during adulthood did not elevate ATP levels and did not correct any of these phenotypes. Conversely, inhibiting respiratory-chain components during adulthood only did not reset behavioral rates and did not affect life-span. Thus, the developing animal appears to contain a regulatory system that monitors mitochondrial activity early in life and, in response, establishes rates of respiration, behavior, and aging that persist during adulthood.

Aging↗

PubMed2002

A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.

We report a systematic RNA interference (RNAi) screen of 5,690 Caenorhabditis elegans genes for gene inactivations that increase lifespan. We found that genes important for mitochondrial function stand out as a principal group of genes affecting C. elegans lifespan. A classical genetic screen identified a mutation in the mitochondrial leucyl-tRNA synthetase gene (lrs-2) that impaired mitochondrial function and was associated with longer-lifespan. The long-lived worms with impaired mitochondria had lower ATP content and oxygen consumption, but differential responses to free-radical and other stresses. These data suggest that the longer lifespan of C. elegans with compromised mitochrondria cannot simply be assigned to lower free radical production and suggest a more complex coupling of metabolism and longevity.

Adenosine Triphosphate↗

PubMed2002

Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.

The genetic analysis of life span has revealed many interesting genes and pathways; however, our understanding of aging has been limited by the lack of a way to assay the aging process itself. Here we show that the tissues of aging worms have a characteristic appearance that is easy to recognize and quantify using Nomarski optics. We have used this assay to determine whether life-span mutations affect the rate of aging, to identify animals that age more rapidly than normal, and to infer the cause of death in C. elegans. Mutations that reduce insulin/IGF-1 signaling double the life span of C. elegans, and we find that tissue decline is slowed in these mutants. Thus this endocrine system appears to influence the rate at which tissues age. This effect extends even to the germline, which is the only mitotically active tissue in the adult. We find that Nomarski microscopy also allows a ready distinction between short-lived mutants that age more rapidly than normal and those that are simply sick, and we have identified an RNAi clone that confers a dramatic rapid-aging phenotype. This clone encodes the C. elegans heat-shock factor (HSF), a transcription factor that regulates the response to heat and oxidative stress. This suggests that heat-shock proteins, many of which act as chaperones, may function in normal animals to slow the rate of aging. Finally, we have identified a cause of death of C. elegans: namely, proliferating bacteria. This suggests that increased susceptibility to bacterial infections contributes to mortality in these animals, just as it does in humans.

Aging↗