Evaluation of the Accuracy of Anthropometric Clinical Indicators of Visceral Fat in Adults and Elderly

BackgroundVisceral obesity is associated with higher occurrence of cardiovascular events. There are few studies about the accuracy of anthropometric clinical indicators, using Computed Tomography (CT) as the gold standard. We aimed to determine the accuracy of anthropometric clinical indicators for discrimination of visceral obesity.MethodsCross-sectional study with 191 adults and elderly of both sexes. Variables: area of visceral adipose tissue (VAT) identified by CT, Waist-to-Height Ratio (WHtR), Conicity index (C index), Lipid Accumulation Product (LAP) and Visceral Adiposity Index (VAI). ROC analyzes.ResultsThere were a strong correlation between adiposity indicators and VAT area. Higher accuracy of C index and WHtR (AUC≥0.81) than the LAP and the VAI was observed. The higher AUC of LAP and VAI were observed among elderly with areas of 0.88 (CI: 0.766–0.944) and 0.83 (CI: 0.705–0.955) in men and 0.80 (CI: 0.672–0.930) and 0.71 (CI: 0.566–0.856) in women, respectively. The cutoffs of C index were 1.30 in elderly, in both sexes, with sensitivity ≥92%, the LAP ranged from 26.4 to 37.4 in men and from 40.6 to 44.0 in women and the VAI was 1.24 to 1.45 (sens≥76.9%) in men and 1.46 to 1.84 in women.ConclusionBoth the anthropometric indicators, C Index and WHtR, as well as LAP and VAI had high accuracy in visceral obesity discrimination. So, they are effective in cardiovascular risk assessment and in the follow-up for individual and collective clinical practice.     

EGFR and FGFR Pathways Have Distinct Roles in Drosophila Mushroom Body Development and Ethanol-Induced Behavior

Epidermal Growth Factor Receptor (EGFR) signaling has a conserved role in ethanol-induced behavior in flies and mice, affecting ethanol-induced sedation in both species. However it is not known what other effects EGFR signaling may have on ethanol-induced behavior, or what roles other Receptor Tyrosine Kinase (RTK) pathways may play in ethanol induced behaviors. We examined the effects of both the EGFR and Fibroblast Growth Factor Receptor (FGFR) RTK signaling pathways on ethanol-induced enhancement of locomotion, a behavior distinct from sedation that may be associated with the rewarding effects of ethanol. We find that both EGFR and FGFR genes influence ethanol-induced locomotion, though their effects are opposite – EGFR signaling suppresses this behavior, while FGFR signaling promotes it. EGFR signaling affects development of the Drosophila mushroom bodies in conjunction with the JNK MAP kinase basket (bsk), and with the Ste20 kinase tao, and we hypothesize that the EGFR pathway affects ethanol-induced locomotion through its effects on neuronal development. We find, however, that FGFR signaling most likely affects ethanol-induced behavior through a different mechanism, possibly through acute action in adult neurons.     

Overexpression of MEOX2 and TWIST1 Is Associated with H3K27me3 Levels and Determines Lung Cancer Chemoresistance and Prognosis

Lung cancer is the leading cause of death from malignant diseases worldwide, with the non-small cell (NSCLC) subtype accounting for the majority of cases. NSCLC is characterized by frequent genomic imbalances and copy number variations (CNVs), but the epigenetic aberrations that are associated with clinical prognosis and therapeutic failure remain not completely identify. In the present study, a total of 55 lung cancer patients were included and we conducted genomic and genetic expression analyses, immunohistochemical protein detection, DNA methylation and chromatin immunoprecipitation assays to obtain genetic and epigenetic profiles associated to prognosis and chemoresponse of NSCLC patients. Finally, siRNA transfection-mediated genetic silencing and cisplatinum cellular cytotoxicity assays in NSCLC cell lines A-427 and INER-37 were assessed to describe chemoresistance mechanisms involved. Our results identified high frequencies of CNVs (66–51% of cases) in the 7p22.3–p21.1 and 7p15.3–p15.2 cytogenetic regions. However, overexpression of genes, such as MEOX2, HDAC9, TWIST1 and AhR, at 7p21.2–p21.1 locus occurred despite the absence of CNVs and little changes in DNA methylation. In contrast, the promoter sequences of MEOX2 and TWIST1 displayed significantly lower/decrease in the repressive histone mark H3K27me3 and increased in the active histone mark H3K4me3 levels. Finally these results correlate with poor survival in NSCLC patients and cellular chemoresistance to oncologic drugs in NSCLC cell lines in a MEOX2 and TWIST1 overexpression dependent-manner. In conclusion, we report for the first time that MEOX2 participates in chemoresistance irrespective of high CNV, but it is significantly dependent upon H3K27me3 enrichment probably associated with aggressiveness and chemotherapy failure in NSCLC patients, however additional clinical studies must be performed to confirm our findings as new probable clinical markers in NSCLC patients.     

Antarctic strict anaerobic microbiota from <Emphasis Type="Italic">Deschampsia antarctica</Emphasis> vascular plants rhizosphere reveals high ecology and biotechnology relevance

The Antarctic soil microbial community has a crucial role in the growth and stabilization of higher organisms, such as vascular plants. Analysis of the soil microbiota composition in that extreme environmental condition is crucial to understand the ecological importance and biotechnological potential. We evaluated the efficiency of isolation and abundance of strict anaerobes in the vascular plant Deschampsia antarctica rhizosphere collected in the Antarctic’s Admiralty Bay and associated biodiversity to metabolic perspective and enzymatic activity. Using anaerobic cultivation methods, we identified and isolated a range of microbial taxa whose abundance was associated with Plant Growth-Promoting Bacteria (PGPB) and presences were exclusively endemic to the Antarctic continent. Firmicutes was the most abundant phylum (73 %), with the genus Clostridium found as the most isolated taxa. Here, we describe two soil treatments (oxygen gradient and heat shock) and 27 physicochemical culture conditions were able to increase the diversity of anaerobic bacteria isolates. Heat shock treatment allowed to isolate a high percentage of new species (63.63 %), as well as isolation of species with high enzymatic activity (80.77 %), which would have potential industry application. Our findings contribute to the understanding of the role of anaerobic microbes regarding ecology, evolutionary, and biotechnological features essential to the Antarctic ecosystem.     

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.     

Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex

The Pam/Highwire/RPM-1 (PHR) proteins include: Caenorhabditis elegans RPM-1 (Regulator of Presynaptic Morphology 1), Drosophila Highwire, and murine Phr1. These important regulators of neuronal development function in synapse formation, axon guidance, and axon termination. In mature neurons the PHR proteins also regulate axon degeneration and regeneration. PHR proteins function, in part, through an ubiquitin ligase complex that includes the F-box protein FSN-1 in C. elegans and Fbxo45 in mammals. At present, the structure-function relationships that govern formation of this complex are poorly understood. We cloned 9 individual domains that compose the entire RPM-1 protein sequence and found a single domain centrally located in RPM-1 that is sufficient for binding to FSN-1. Deletion analysis further refined FSN-1 binding to a conserved 97-amino acid region of RPM-1. Mutagenesis identified several conserved motifs and individual amino acids that mediate this interaction. Transgenic overexpression of this recombinant peptide, which we refer to as the RPM-1·FSN-1 complex inhibitory peptide (RIP), yields similar phenotypes and enhancer effects to loss of function in fsn-1. Defects caused by transgenic RIP were suppressed by loss of function in the dlk-1 MAP3K and were alleviated by point mutations that reduce binding to FSN-1. These findings suggest that RIP specifically inhibits the interaction between RPM-1 and FSN-1 in vivo, thereby blocking formation of a functional ubiquitin ligase complex. Our results are consistent with the FSN-1 binding domain of RPM-1 recruiting FSN-1 and a target protein, such as DLK-1, whereas the RING-H2 domain of RPM-1 ubiquitinates the target.     

Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP₆). In this study, we demonstrated that InsP₆ is not simply an allosteric cofactor, but rather binding of InsP₆ stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP₆-bound unprocessed form of CPD was determined and revealed the scissile bond Leu³⁴²⁸–Ala³⁴²⁹ captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP₆, but was reactivated for high affinity binding of InsP₆ by cooperative binding of both a new substrate and InsP₆. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.Multifunctional-autoprocessing repeats-in-toxin (MARTX)³ toxins are a family of large bacterial protein toxins with conserved repeat regions at the N and C termini that are predicted to transfer effector domains located between the repeats across the eukaryotic cell plasma membrane (1). The best characterized MARTX is the >450-kDa secreted virulence-associated MARTX of Vibrio cholerae. This toxin causes disassembly of the actin cytoskeleton and enhances V. cholerae colonization of the small intestine, possibly by facilitating evasion of phagocytic cells (2, 3). The central region of the V. cholerae MARTX toxin contains four discrete domains: the actin cross-linking domain (ACD) that introduces lysine-glutamate cross-links between actin protomers (4, 5), the Rho-inactivating domain (RID) that disables small Rho GTPases (6), an αβ hydrolase of unknown function (1), and an autoprocessing cysteine protease domain (CPD) (7, 8).The CPD is a 25-kDa domain found in all MARTX toxins located just before the start of the C-terminal repeats (7, 8). This domain is activated for autoproteolysis upon binding inositol hexakisphosphate (InsP₆) (7), a molecule ubiquitously present in eukaryotic cell cytosol (9–11), but absent in extracellular spaces and bacteria. Thus, autocatalytic processing would not occur until after translocation of the CPD and effector domains is completed. In the context of the holotoxin, catalytic residue Cys³⁵⁶⁸ was found to be essential for the toxin to induce efficient actin cross-linking by the ACD and Rho inactivation by the RID, demonstrating that autoprocessing is essential for MARTX to induce cell rounding (8).While it is clear that InsP₆ activates the CPD and that autoprocessing is essential for MARTX function (7), the mechanism by which InsP₆ activates CPD is not well understood. Furthermore, only one processing site at Leu³⁴²⁸–Ala³⁴²⁹ has been identified, although multiple processing events would be required to release each effector independently. In fact, after autoprocessing at Leu³⁴²⁸–Ala³⁴²⁹, CPD is reported to adopt a conformation with reduced affinity for InsP₆ (7), raising questions as to how the protease might process MARTX at other sites.We present here the structure of the pre-processed form of the V. cholerae MARTX CPD bound to InsP₆. Our results demonstrate that autoprocessing is activated by rearrangement of a β-hairpin loop upon InsP₆ binding that locks the N terminus of the CPD in the active site, facilitating hydrolysis of the Leu³⁴²⁸–Ala³⁴²⁹ peptide bond. After autoprocessing, CPD adopts a post-processing form that has poor affinity for InsP₆ and thus must be cooperatively reactivated for high affinity binding of InsP₆ by association of a new substrate. As a consequence, we are able to demonstrate how CPD cleaves MARTX toxin between effector domains and releases them from the large toxin resulting in increased catalytic activity of the effectors.     

Altered chromatin modifications in AML1/RUNX1 breakpoint regions involved in (8;21) translocation

The RUNX1/AML1 gene is the most frequent target for chromosomal translocation, and often identified as a site for reciprocal rearrangement of chromosomes 8 and 21 in patients with acute myelogenous leukemia. Virtually all chromosome translocations in leukemia show no consistent homologous sequences at the breakpoint regions. However, specific chromatin elements (DNase I and topoisomerase II cleavage) have been found at the breakpoints of some genes suggesting that structural motifs are determinant for the double strand DNA-breaks. We analyzed the chromatin organization at intron 5 of the RUNX1 gene where all the sequenced breakpoints involved in t(8;21) have been mapped. Using chromatin immunoprecipitation assays we show that chromatin organization at intron 5 of the RUNX1 gene is different in HL-60 and HeLa cells. Two distinct features mark the intron 5 in cells expressing RUNX1: a complete lack or significantly reduced levels of Histone H1 and enrichment of hyperacetylated histone H3. Strikingly, induction of DNA damage resulted in formation of t(8;21) in HL-60 but not in HeLa cells. Taken together, our results suggest that H1 depletion and/or histone H3 hyperacetylation may have a linkage with an increase susceptibility of specific chromosomal regions to undergo translocations.     

Andes Virus Antigens Are Shed in Urine of Patients with Acute Hantavirus Cardiopulmonary Syndrome

Hantavirus cardiopulmonary syndrome (HCPS) is a highly pathogenic emerging disease (40% case fatality rate) caused by New World hantaviruses. Hantavirus infections are transmitted to humans mainly by inhalation of virus-contaminated aerosol particles of rodent excreta and secretions. At present, there are no antiviral drugs or immunotherapeutic agents available for the treatment of hantaviral infection, and the survival rates for infected patients hinge largely on early virus recognition and hospital admission and aggressive pulmonary and hemodynamic support. In this study, we show that Andes virus (ANDV) interacts with human apolipoprotein H (ApoH) and that ApoH-coated magnetic beads or ApoH-coated enzyme-linked immunosorbent assay plates can be used to capture and concentrate the virus from complex biological mixtures, such as serum and urine, allowing it to be detected by both immunological and molecular approaches. In addition, we report that ANDV-antigens and infectious virus are shed in urine of HCPS patients.Hantaviruses are segmented RNA viruses belonging to the genus Hantavirus in the family Bunyaviridae (43). Hantaviral genomes are tripartite, consisting of three different single-stranded RNA segments, designated large (L), medium (M), and small (S), that are packed into helical nucleocapsids (39, 42). These segments encode the RNA polymerase, a glycoprotein precursor that is cotranslationally processed to yield two envelope glycoproteins (Gc and Gn) and a nucleocapsid (N) protein. Hantaviruses are maintained in various rodent reservoirs, in which the hosts are persistently infected but lack disease symptoms (28, 32). Virus transmission to humans does not require direct human-to-rodent contact. Instead, human hantaviral infections are acquired by the respiratory route, most commonly through inhalation of virus-contaminated aerosol particles of rodent excreta (feces, saliva, or urine). Hantaviruses are known to cause two serious and often fatal human diseases, hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome (HCPS) (19, 31). Of the two diseases, HCPS is more severe, with a mortality rate of approximately 40% (19). Hemorrhagic fever with renal syndrome is a mild-to-severe disease characterized by the development of an acute influenza-like febrile illness that may lead to hemorrhagic manifestations, and renal failure is caused by pathogenic Old World hantaviruses, which include Seoul, Hantaan, Dobrava, Tula, and Puumala viruses (28, 32, 42). The New World hantaviruses are responsible for HCPS, which is characterized by a febrile phase (prodrome) and pulmonary infection, cardiac depression, and hematologic manifestations (18, 31). HCPS pathogenesis generally includes capillary leak syndrome, which selectively involves the pulmonary bed, noncardiogenic pulmonary edema, thrombocytopenia, hypotension, and/or cardiogenic shock (19, 31). The pathogenesis of HCPS, like that of many other viral hemorrhagic fevers, is poorly understood. However, the long incubation period for illness, the generally well-advanced adaptive immune response at the time of onset of the disease, and the apparent absence of direct lytic damage to vascular endothelium, all characteristics shared with other hemorrhagic fevers, are among the findings that strongly suggest that HCPS pathogenesis is largely immune mediated (22, 27). The lack of an FDA-approved vaccine for HCPS, the absence of specific antiviral drugs or immunotherapeutic agents, and the high overall mortality rate for hantavirus infection highlight the medical significance of New World hantavirus (5, 8, 19, 28, 32).Survival rates for patients with hantaviral infection hinge largely on early virus recognition and hospital admission and aggressive pulmonary and hemodynamic support (19, 31). The diagnosis, clinical course, and supportive care of patients with New World hantaviral infections have recently been reviewed (19, 31). Unfortunately, early diagnosis of New World hantaviral infections is complex, as the prodrome leading to acute cardiopulmonary deterioration in HCPS can be confused with febrile phases produced by, for example, mycoplasmas and chlamydophilial infections (52).Human apolipoprotein H (ApoH), also known as beta 2-glycoprotein I, is a constituent of human plasma (0.2 mg/ml) notorious for binding to negatively charged surfaces (3, 7, 14, 17, 44, 45). Several reports show that ApoH also interacts with viral proteins, such as the hepatitis B virus (HBV) antigen and proteins p18, p26, and gp160 of the human immunodeficiency virus (12, 30, 46, 47). Interestingly, studies involving binding to the HBV antigen suggest that ApoH specifically binds DNA-containing HBV particles, thus discriminating, through an undefined mechanism, between active replicating virus and empty noninfectious particles (47). These findings prompted us to assess a possible interaction between ApoH and Andes virus (ANDV), which is the major etiological agent of HCPS in South America and is unique among hantaviruses in its reported ability to be transmitted from person to person (19, 29, 34). The mechanism of person-to-person dissemination of ANDV remains to be elucidated, yet it is likely that person-to-person transmission of ANDV could be explained by mechanisms similar to those described for rodent-to-rodent and rodent-to-human transmission. If so, a compulsory condition for ANDV dissemination among humans is that the infected host must shed the pathogen in, for example, urine.In this study, we show that when fixed to a solid matrix, ApoH can be used to capture and concentrate ANDV from complex biological samples, including serum and urine, allowing virus detection by both immunological and molecular approaches. Furthermore, we took advantage of the ApoH-ANDV interaction to develop a high-throughput screening assay and show for the first time ANDV-antigen shedding in the urine of patients with acute HCPS. We also report the presence of infectious viral particles in the urine of two patients with HCPS.