Differences in segmental fat accumulation patterns by sex and ethnicity: An international approach.

Excess fat on the body impacts obesity-related co-morbidity risk; however, the location of fat stores affects the severity of these risks. The purpose of this study was to examine segmental fat accumulation patterns by sex and ethnicity using international datasets. An amalgamated and cross-calibrated dataset of dual x-ray absorptiometry (DXA)-measured variables compiled segmental mass for bone mineral content (BMC), lean mass (LM), and fat mass (FM) for each participant; percentage of segment fat (PSF) was calculated as PSFsegment = (FMsegment/(BMCsegment + LMsegment + FMsegment)) × 100. A total of 30 587 adults (N = 16 490 females) from 13 datasets were included. A regression model was used to examine differences in regional fat mass and PSF. All populations followed the same segmental fat mass accumulation in the ascending order with statistical significance (arms < legs < trunk), except for Hispanic/Latinx males (arms < [legs = trunk]). Relative fat accumulation patterns differed between those with greater PSF in the appendages (Arab, Mexican, Asian, Black, American Caucasian, European Caucasian, and Australasian Caucasian females; Black males) and those with greater PSF in the trunk (Mexican, Asian, American Caucasian, European Caucasian, and Australasian Caucasian males). Greater absolute and relative fat accumulation in the trunk could place males of most ethnicities in this study at a higher risk of visceral fat deposition and associated co-morbidities.

A novel duplex real-time PCR for HPIV-4 detects co-circulation of both viral subtypes among ill children during 2008.

The two subtypes of the human parainfluenzavirus type 4 (HPIV-4) are rarely sought in testing for acute respiratory illness (ARI) and this may be confounding our understanding of its role. This study presents a novel duplex real-time RT-PCR assay targeting the P gene that can detect and differentiate the two subtypes in a single reaction. Subtype-specific synthetic RNA positive controls were prepared and used to determine an analytical sensitivity of 10 copies per reaction with an 8log(10) dynamic range. The assays were validated using 1140 clinical specimens mostly nasopharyngeal aspirates collected from children during 2008. These included specimens previously determined to be positive for all commonly considered respiratory viruses. The novel assay did not cross-reaction with any other virus. Fourteen HPIV-4 positives, ten detected in the absence of any co-detections (four with rhinovirus), were identified in 2008 and their subtype confirmed by conventional RT-PCR and sequencing of P gene fragments. Most detections were in children two years of age or younger. Our assay proved suitably sensitive and specific for inclusion in future studies seeking to better understand the role HPIV-4 and other respiratory viruses in children with ARI.

Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough and wheeze during 2003.

BACKGROUND: Human rhinoviruses (HRVs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified HRVs. OBJECTIVES: To determine whether HRVC-QCE was a distinct HRV-C strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients. STUDY DESIGN: Novel real-time RT-PCRs and retrospective chart reviews were used to investigate a well-defined population of 1247 specimen extracts to observe the prevalence and the clinical features of each HRV-QCE positive case from an in- and out-patient pediatric, hospital-based population during 2003. An objective illness severity score was determined for each HRVC-QCE positive patient. RESULTS: Differences in overall polyprotein and VP1 binding pocket residues and the predicted presence of a cis-acting replication element in 1B defined HRVC-QCE as a novel HRV-C strain. Twelve additional HRVC-QCE detections (1.0% prevalence) occurred among infants and toddlers (1-24 months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. HRVC-QCE was frequently detected in the absence of another virus and was the only virus detected in three (23% of HRVC-QCE positives) children with asthma exacerbation and in two (15%) toddlers with febrile convulsion. CONCLUSIONS: HRVC-QCE is a newly identified, genetically distinct HRV strain detected in hospitalized children with a range of clinical features. HRV strains should be independently considered to ensure we do not overestimate the HRVs in asymptomatic illness.

Do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections?

BACKGROUND: Human rhinoviruses (HRVs) are often concurrently detected with other viruses found in the respiratory tract because of the high total number of HRV infections occurring throughout the year. This feature has previously relegated HRVs to being considered passengers in acute respiratory infections. HRVs remain poorly characterized and are seldom included as a target in diagnostic panels despite their pathogenic potential, infection-associated healthcare expenditure and relatively unmoderated elicitation of an antiviral state. OBJECTIVES: To test the hypothesis that respiratory viruses are proportionately more or less likely to co-occur, particularly the HRVs. STUDY DESIGN: Retrospective PCR-based analyses of 1247 specimens for 17 viruses, including HRV strains, identified 131 specimens containing two or more targets. We investigated the proportions of co-detections and compared the proportion of upper vs. lower respiratory tract presentations in the HRV positive group. Both univariate contingency table and multivariate logistic regression analyses were conducted to identify trends of association among the viruses present in co-detections. RESULTS: Many of the co-detections occurred in patterns. In particular, HRV detection was associated with a reduced probability of detecting human adenoviruses, coronaviruses, bocavirus, metapneumovirus, respiratory syncytial virus, parainfluenza virus, influenza A virus, and the polyomaviruses KIPyV and WUPyV (p < or = 0.05). No single HRV species nor cluster of particular strains predominated. CONCLUSIONS: HRVs were proportionately under-represented among viral co-detections. For some period, HRVs may render the host less likely to be infected by other viruses.

FOXO animal models reveal a variety of diverse roles for FOXO transcription factors.

The Foxo subfamily of FOX transcription factors plays a variety of roles in a broad assortment of diverse physiological processes including cellular differentiation, tumor suppression, metabolism, cell cycle arrest, cell death and protection from stress. Animal models have proved to be invaluable tools in furthering our understanding of the role of particular genes in complex organismal processes. Multiple animal models in diverse species, including Caenorhabditis elegans, Drosophila. melanogaster and the laboratory mouse, exist for the Foxo family of transcription factors. Foxo genes are highly conserved throughout the evolution and each of these model systems has provided valuable insight into the roles of Foxo factors. Many roles are conserved among the different model organisms. Several Foxo-related animal model systems are reviewed here along with the knowledge gleaned to date from each model system.

A novel mutation in ferroportin1 is associated with haemochromatosis in a Solomon Islands patient.

BACKGROUND: A severe form of iron overload with the clinicopathological features of haemochromatosis inherited in an autosomal dominant manner has been described in the Solomon Islands. The genetic basis of the disorder has not been identified. The disorder has similarities to type 4 haemochromatosis, which is caused by mutations in ferroportin1. AIMS: The aims of this study were to identify the genetic basis of iron overload in a patient from the Solomon Islands. PATIENT AND METHODS: Genomic DNA was isolated from peripheral blood leucocytes of a Solomon Islands man with severe iron overload. The entire coding region and splice sites of the ferroportin1 gene was sequenced. RESULTS AND CONCLUSIONS: A novel missense mutation (431A>C; N144T) was identified in exon 5 of the ferroportin1 gene. A novel restriction endonuclease based assay which identifies both the N144T and N144H mutations was developed which will simplify the diagnosis and screening of patients for iron overload in the Solomon Islands and other populations. This is the first identified mutation associated with haemochromatosis in the Solomon Islands population.

cDNA and protein sequence, genomic organization, and analysis of cis regulatory elements of mouse and human PLVAP genes.

PV-1 is a novel protein component of the endothelial fenestral and stomatal diaphragms. PV-1 cDNA and protein sequences are highly conserved across species. The conserved extracellular domain features found in rat, mouse, and human PV-1 protein are four N-glycosylation sites, two coiled-coil domains, a proline-rich region, and even cysteine spacing. No consensus site in the intracellular domain was found. Northern blotting of mouse and human tissues is in agreement with and expands those performed in rat and correlated well with the postulated presence of PV-1 in the endothelial diaphragms. The genomic organization of the human and mouse genes (HGMW-approved symbol PLVAP) has been determined, and the analysis of their 5' flanking regions has found a highly conserved classical TATA-driven promoter that shows several transcription factor consensus binding sites. Radiation hybrid panel mapping has localized the human and mouse PLVAP genes to chromosomes 19p13.2 and 8B3-C1, respectively.

Identification and characterization of members of the FKHR (FOX O) subclass of winged-helix transcription factors in the mouse.

Genetic alterations of FKHR (FOXO1), AF6q21 (FOXO2), and AFX (FOXO4), closely related members of the forkhead family of DNA binding proteins, in human cancers has suggested they play a role in the regulation of cellular differentiation or proliferation. In order to elucidate the function of this gene subfamily during mammalian development, we have identified and characterized three novel mouse genes; Fkhr1 (Foxo1), Fkhr2 (Foxo3), and Afxh (Foxo4), which are closely related to the human FKHR (Foxo1), AF6q21 (FOXO2), and AFX (FOXO4) genes, respectively. The genes are each expressed both during development and in the adult with distinct patterns ranging from ubiquitous [Fkhr2 (Foxo3)] to tissue-specific [Afxh (Foxo4)]. Selection of high-affinity DNA-binding sites from a pool of degenerate oligonucleotides demonstrated that the proteins encoded by these genes recognize a core sequence [(T/A) (A/T) A A C A] similar to that recognized by other forkhead domain-containing proteins. We have also identified additional FKHR-related genes expressed during development in both the chick and zebrafish. Further characterization will provide insight into the roles of members of the FKHR subfamily of forkhead-related genes during both normal and neoplastic development.

Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3.

A unique chromosomal translocation involving the genes PAX3 and FKHR is characteristic of most human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses the PAX3 DNA-binding domains to the transactivation domain of FKHR, suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas through dysregulation of PAX3-specific target genes. Here, we have produced transgenic mice in which PAX3-FKHR expression was driven by mouse Pax3 promoter/enhancer sequences. Five independent lines expressed PAX3-FKHR in the dorsal neural tube and lateral dermomyotome. Each line exhibited phenotypes that correlated with PAX3-FKHR expression levels and predominantly involved pigmentary disturbances of the abdomen, hindpaws, and tail, with additional neurological related alterations. Phenotypic severity could be increased by reducing Pax3 levels through matings with Pax3-defective Splotch mice, and interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal Pax3 developmental functions as a prelude to transformation.

p190-A, a human tumor suppressor gene, maps to the chromosomal region 19q13.3 that is reportedly deleted in some gliomas.

To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains. Here we report the cloning of the full-length human p190-A cDNA and its first exon covering more than 80% of this protein, as well as its chromosomal mapping. Human p190-A encodes a protein of 1514 amino acids, and shares overall 97% sequence identity with rat p190-A. Like the p190-B exon, the first exon of p190-A is extremely large (3.7 kb in length), encoding both the GTPase and middle domains (residues 1-1228), but not the remaining GAP domain, suggesting a high conservation of genomic structure between two p190 genes. Using a well characterized monochromosome somatic cell hybrid panel, fluorescent in situ hybridization (FISH) and other complementary approaches, we have mapped the p190-A gene between the markers D19S241E and STD (500 kb region) of human chromosome 19q13.3. Interestingly, this chromosomal region is known to be rearranged in a variety of human solid tumors including pancreatic carcinomas and gliomas. Moreover, at least 40% glioblastoma/astrocytoma cases with breakpoints in this region were previously reported to show loss of the chromosomal region encompassing p190-A, suggesting the possibility that loss or mutations of this gene might be in part responsible for the development of these tumors.

ADAMTS9, a novel member of the ADAM-TS/ metallospondin gene family.

ADAM-TS/metallospondin genes encode a new family of proteins with structural homology to the ADAM metalloprotease-disintegrin family. However, unlike other ADAMs, these proteins contain thrombospondin type 1 (TSP1) repeats at the carboxy-terminal end and are secreted proteins instead of being membrane bound. Members of the ADAM-TS family have been implicated in the cleavage of proteoglycans, the control of organ shape during development, and the inhibition of angiogenesis. We have cloned a new member of the ADAM-TS/metallospondin family designated here as ADAMTS9. This protein has a metalloprotease domain, a disintegrin-like domain, one internal TSP1 motif, and three carboxy-terminal TSP1-like submotifs. In contrast to other ADAM-TS family members, ADAMTS9 is expressed in all fetal tissues examined as well as some adult tissues. Using FISH and radiation hybrid analysis, we have localized ADAMTS9 to chromosome 3p14.2-p14.3, an area known to be lost in hereditary renal tumors.

CT10: a new cancer-testis (CT) antigen homologous to CT7 and the MAGE family, identified by representational-difference analysis.

Assays relying on humoral or T-cell-based recognition of tumor antigens to identify potential targets for immunotherapy have led to the discovery of a significant number of immunogenic gene products, including cancer-testis (CT) antigens predominantly expressed in cancer cells and male germ cells. The search for cancer-specific antigens has been extended via the technique of representational-difference analysis and SK-MEL-37, a melanoma cell line expressing a broad range of CT antigens. Using this approach, we have isolated CT antigen genes, genes over-expressed in cancer, e. g., PRAME and KOC, and genes encoding neuro-ectodermal markers. The identified CT antigen genes include the previously defined MAGE-A6, MAGE-A4a, MAGE-A10, CT7/MAGE-C1, as well as a novel gene designated CT10, which shows strong homology to CT7/MAGE-C1 both at cDNA and at genomic levels. Chromosome mapping localized CT10 to Xq27, in close proximity to CT7/MAGE-C1 and MAGE-A genes. CT10 mRNA is expressed in testis and in 20 to 30% of various human cancers. A serological survey identified 2 melanoma patients with anti-CT10 antibody, demonstrating the immunogenicity of CT10 in humans.

Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.

In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.12-14.2. The DICE1 gene is highly conserved in evolution and its mRNA is expressed in a wide variety of fetal and adult tissues. The DICE1 cDNA encodes a predicted protein of 887 amino acids corresponding to an 100 kD protein that shows 92.9% identity to the carboxy-terminal half of the mouse EGF repeat transmembrane protein DBI-1. The DBI-1 protein interferes with the mitogenic response to insulin-like growth factor 1 (IGF-I) and is presumably involved in anchorage-dependent growth. When compared to normal lung tissue expression of the DICE1 mRNA was reduced or undetectable in the majority of non-small cell lung carcinomas analysed. The location of the DICE1 gene in the region of allelic loss, its high evolutionary conservation and the downregulation of expression in carcinoma cells suggests that DICE1 is a candidate tumor suppressor gene in non-small cell lung carcinomas and possibly in other sporadic carcinomas.

The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death.

Studies aimed at examining the precise function(s) of the retinoblastoma tumor suppressor protein, RB, have been hindered by the rapid phosphorylation and inactivation of ectopically expressed RB which occurs in the majority of cell types. Therefore, ectopically expressed RB is a poor inhibitor of cellular proliferation. We have designed constitutively active RB proteins, PSM-RB, that cannot be inactivated by phosphorylation. Using these proteins, we show that unlike wild-type RB, PSM-RB proteins inhibit cell cycle progression in a broad range of tumor cell types. Furthermore, unlike p16ink4a, PSM-RB is also a potent inhibitor of cell cycle progression in RB-deficient tumor cells. Surprisingly, we identified a tumor cell line that is resistant to the cell cycle inhibitory effects of PSM-RB. This finding challenges the hypothesis that RB must be inactivated in all cells for cell cycle progression to occur. Further characterization of this 'resistant' tumor line revealed that proliferation of these cells is still inhibited by PSM-RB. We show that this is due to PSM-RB-induced cell death. As such, these studies are the first to show that RB inhibits cellular proliferation through at least two distinct mechanisms - inhibition of cell cycle progression and induction of cell death.

Characterization of the GAGE genes that are expressed in various human cancers and in normal testis.

The GAGE-1 gene was identified previously as a gene that codes for an antigenic peptide, YRPRPRRY, which was presented on a human melanoma by HLA-Cw6 molecules and recognized by a clone of CTLs derived from the patient bearing the tumor. By screening a cDNA library from this melanoma, we identified five additional, closely related genes named GAGE-2-6. We report here that further screening of this library led to the identification of two more genes, GAGE-7B and -8. GAGE-1, -2, and -8 code for peptide YRPRPRRY. Using another antitumor CTL clone isolated from the same melanoma patient, we identified antigenic peptide, YYWPRPRRY, which is encoded by GAGE-3, -4, -5, -6, and -7B and which is presented by HLA-A29 molecules. Genomic cloning of GAGE-7B showed that it is composed of five exons. Sequence alignment showed that an additional exon, which is present only in the mRNA of GAGE-1, has been disrupted in gene GAGE-7B by the insertion of a long interspersed repeated element retroposon. These GAGE genes are located in the p11.2-p11.4 region of chromosome X. They are not expressed in normal tissues, except in testis, but a large proportion of tumors of various histological origins express at least one of these genes. Treatment of normal and tumor cultured cells with a demethylating agent, azadeoxycytidine, resulted in the transcriptional activation of GAGE genes, suggesting that their expression in tumors results from a demethylation process.

Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1.

Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the mechanisms underlying their regulation. Here we show that the activation of phosphatidylinositol 3 (PI3) kinase by extracellular growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction, phosphorylated recombinant FKHR1 in vitro at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and FKHR1(T24A/S253A) were resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus.