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Cardiac-Specific Disruption of GH Receptor Alters Glucose Homeostasis While Maintaining Normal Cardiac Performance in Adult Male Mice

Wed, 01/11/2017 - 10:16am

GH is considered necessary for the proper development and maintenance of several tissues, including the heart. Studies conducted in both GH receptor null and bovine GH transgenic mice have demonstrated specific cardiac structural and functional changes. In each of these mouse lines, however, GH-induced signaling is altered systemically, being decreased in GH receptor null mice and increased in bovine GH transgenic mice. Therefore, to clarify the direct effects GH has on cardiac tissue, we developed a tamoxifen-inducible, cardiac-specific GHR disrupted (iC-GHRKO) mouse line. Cardiac GH receptor was disrupted in 4-month-old iC-GHRKO mice to avoid developmental effects due to perinatal GHR gene disruption. Surprisingly, iC-GHRKO mice showed no difference vs controls in baseline or postdobutamine stress test echocardiography measurements, nor did iC-GHRKO mice show differences in longitudinal systolic blood pressure measurements. Interestingly, iC-GHRKO mice had decreased fat mass and improved insulin sensitivity at 6.5 months of age. By 12.5 months of age, however, iC-GHRKO mice no longer had significant decreases in fat mass and had developed glucose intolerance and insulin resistance. Furthermore, investigation via immunoblot analysis demonstrated that iC-GHRKO mice had appreciably decreased insulin stimulated Akt phosphorylation, specifically in heart and liver, but not in epididymal white adipose tissue. These changes were accompanied by a decrease in circulating IGF-1 levels in 12.5-month-old iC-GHRKO mice. These data indicate that whereas the disruption of cardiomyocyte GH-induced signaling in adult mice does not affect cardiac function, it does play a role in systemic glucose homeostasis, in part through modulation of circulating IGF-1.

Role of MicroRNAs in NAFLD/NASH

Wed, 01/11/2017 - 10:16am

MicroRNAs (miRNAs) are highly conserved, small, 18-25 nucleotide, non-coding RNAs that regulate gene expression at the post-transcriptional level. Each miRNA can regulate hundreds of target genes, and vice versa each target gene can be regulated by numerous miRNAs, suggesting a very complex network and explaining how miRNAs play pivotal roles in fine-tuning essentially all biological processes in all cell types in the liver. Here, we summarize the current knowledge on the role of miRNAs in the pathogenesis and diagnosis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) with an outlook to the broader aspects of metabolic syndrome. Furthermore, we discuss the role of miRNAs as potential biomarkers and therapeutic targets in NAFLD/NASH.

Activation of mTORC1 is essential for beta-adrenergic stimulation of adipose browning

Wed, 01/11/2017 - 10:16am

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through beta-adrenergic receptor (betaARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known betaAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted betaAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from betaARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.

Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

Wed, 01/11/2017 - 10:16am

The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate.

The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis

Wed, 01/11/2017 - 10:15am

BACKGROUND and AIMS: Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in vivo role of miR-155 in ALD.

METHODS: Wild-type (WT) (C57/BL6J) or miR-155 knockout (KO) and TLR4 KO mice received Lieber DeCarli diet for 5weeks. Some mice received corn oil or CCl4 for 2 or 9weeks.

RESULTS: We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased peroxisome proliferator-activated receptor response element (PPRE) and peroxisome proliferator-activated receptors (PPAR)alpha (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPARgamma expression in naive and alcohol treated RAW macrophages. Alcohol increased lipid metabolism gene expression (FABP4, LXRalpha, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet caused an increase in the number of CD163(+) CD206(+) infiltrating macrophages and neutrophils in WT mice, which was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPbeta. Pro-fibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT mice, attenuation in CCl4 induced hydroxyproline and alpha-SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet.

CONCLUSIONS: Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo.

Map4k4 Signaling Nodes in Metabolic and Cardiovascular Diseases

Wed, 01/11/2017 - 10:15am

Mitogen-activated kinase kinase kinase kinase 4 (Map4k4), originally identified in small interfering (si)RNA screens and characterized by tissue-specific gene deletions, is emerging as a regulator of glucose homeostasis and cardiovascular health. Recent studies have shown that Map4k4 gene ablation or inhibition of its kinase activity attenuates hyperglycemia and plaque formation in mouse models of insulin resistance and atherosclerosis, and suggest roles for Map4k4 in multiple signaling systems, including NFkappaB activation, small GTPase regulation, the Hippo cascade, and regulation of cell dynamics by FERM domain proteins. This new and promising area of inquiry raises key questions that need to be addressed, such as defining which of the above or other effectors mediate Map4k4 control of metabolic and vascular functions, and identifying upstream activators of Map4k4.

Therapeutic Benefits of Spleen Tyrosine Kinase Inhibitor Administration on Binge Drinking-Induced Alcoholic Liver Injury, Steatosis, and Inflammation in Mice

Wed, 01/11/2017 - 10:15am

BACKGROUND: Binge drinking is increasingly recognized as an important cause of liver disease with limited therapeutic options for patients. Binge alcohol use, similar to chronic alcohol consumption, induces numerous deregulated signaling events that drive liver damage, steatosis, and inflammation. In this article, we evaluated the role of spleen tyrosine kinase (SYK), which modulates numerous signaling events previously identified linked in the development alcohol-induced liver pathology.

METHODS: A 3-day alcohol binge was administered to C57BL/6 female mice, and features of alcoholic liver disease were assessed. Some mice were treated daily with intraperitoneal injections of a SYK inhibitor (R406; 5 to 10 mg/kg body weight) or drug vehicle control. Liver and serum samples were collected and were assessed by Western blotting, biochemical, ELISA, electrophoretic mobility shift assays, real-time quantitative polymerase chain reaction, and histopathological analysis.

RESULTS: We found that binge drinking induced significant SYK activation (SYK(Y525/526) ) with no change in total SYK expression in the liver. Functional inhibition of SYK activation using a potent SYK inhibitor, R406, was associated with a significant decrease in alcohol-induced hepatic inflammation as demonstrated by decreased phospho-nuclear factor kappa beta (NF-kappaB) p65, NF-kappaB nuclear binding, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 mRNA in the liver. Compared to vehicle controls, SYK inhibitor treatment decreased alcohol binge-induced hepatocyte injury indicated by histology and serum alanine aminotransferase. Strikingly, SYK inhibitor treatment also resulted in a significant reduction in alcohol-induced liver steatosis.

CONCLUSIONS: Our novel observations demonstrate the role of SYK, activation in the pathomechanism of binge drinking-induced liver disease highlighting SYK a potential multifaceted therapeutic target.

Interleukin-1 and inflammasomes in alcoholic liver disease/acute alcoholic hepatitis and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

Wed, 01/11/2017 - 10:15am

Both alcoholic liver disease (ALD) and nonalcoholic fatty liver disease are characterized by massive lipid accumulation in the liver accompanied by inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma in a substantial subgroup of patients. At several stages in these diseases, mediators of the immune system, such as cytokines or inflammasomes, are crucially involved. In ALD, chronic ethanol exposure sensitizes Kupffer cells to activation by lipopolysaccharides through Toll-like receptors, e.g., Toll-like receptor 4. This sensitization enhances the production of various proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha, thereby contributing to hepatocyte dysfunction, necrosis, and apoptosis and the generation of extracellular matrix proteins leading to fibrosis/cirrhosis. Indeed, neutralization of IL-1 by IL-1 receptor antagonist has recently been shown to potently prevent liver injury in murine models of ALD. As IL-1 is clearly linked to key clinical symptoms of acute alcoholic hepatitis such as fever, neutrophilia, and wasting, interfering with the IL-1 pathway might be an attractive treatment strategy in the future. An important role for IL-1-type cytokines and certain inflammasomes has also been demonstrated in murine models of nonalcoholic fatty liver disease. IL-1-type cytokines can regulate hepatic steatosis; the NLR family pyrin domain containing 3 inflammasome is critically involved in metabolic dysregulation.

CONCLUSION: IL-1 cytokine family members and various inflammasomes mediate different aspects of both ALD and nonalcoholic fatty liver disease.

Effects of zinc and multivitamin supplementation on hematologic status during infancy

Fri, 01/06/2017 - 12:01pm

OBJECTIVE: To determine the separate effects of zinc and multivitamin supplementation on infant hematologic status.

METHODS: In a double-blind RCT, infants born to HIV-negative women (n=2400) in Dar es Salaam, Tanzania were randomized to daily oral supplementation with zinc (Zn), multivitamins (MV), Zn and MV, or placebo at age 6 wk. Hemoglobin concentration (Hb) and red cell indices were measured at baseline and ages 6, 12, and 18 mo. A subset of infants (n = 589) was examined for iron deficiency at 6 mo, defined as ferritin < 12 μg/L or, where α-glycoprotein 蠅 1.0 g/L, serum transferrin receptor > 8.3 mg/L.

RESULTS: Infants treated with MV had higher mean Hb than those given placebo or Zn alone at 18 mo (9.9 vs. 9.6 g/dL, p = .0009). Infants treated with Zn had RR of 1.71 (95% CI 1.01 – 2.89) for iron deficiency at 6 mo, while those treated with MV had RR 0.52 (95% CI 0.30 – 0.90). In Cox models, MV were associated with a 28% reduction in risk of severe anemia (HR = 0.72 [95%CI 0.56 – 0.94]) and a 26% reduction in risk of severe microcytic anemia (HR = 0.74 [95% CI 0.56 – 0.96]) through age 18 mo. No effects of Zn on risk of anemia were seen.

CONCLUSIONS: MV were associated with reduced risk of both iron deficiency and severe microcytic anemia, while Zn was associated with increased risk of iron deficiency but no longer term increase in risk of anemia.

Funding: NICHD (R01 HD048969; K24HD058795); NIAAA (K23 AA020516); Bill and Melinda Gates Foundation (OPP1066203)

Serum citrulline as a biomarker of gastrointestinal function during hematopoietic cell transplantation in children

Fri, 01/06/2017 - 12:01pm

OBJECTIVES: We sought to determine whether serum citrulline (CIT), an amino acid produced by small bowel enterocytes, was associated with clinical and biochemical markers of gastrointestinal function in children undergoing hematopoietic cell transplantation (HCT).

METHODS: We conducted a multicenter, prospective cohort study of 26 children to define time-related changes in serum CIT during the course of HCT. Markers of gastrointestinal function including oral energy intake, emesis, stool volume, presence of graft-versus-host disease (GVHD), oral mucositis severity, and cytokine and neurohormone levels were measured. Weekly serum CIT concentrations were obtained from 10 days prior until 30 days after HCT.

RESULTS: Mean baseline CIT concentration was 22.7 mumol/L (95% confidence interval [CI] 17.7-27.6) on day -10, which decreased to a nadir of 7.5 mumol/L (95% CI 3.1-18.0, P = 0.017) on day 8 following HCT before returning to baseline by day 30. After adjustment for IL-6 level (1.0% lower CIT per 10% increase in interleukin-6, P = 0.004), presence of acute GVHD (27% lower CIT, P = 0.025), and oral energy intake (2.1% lower CIT per 10% decrease in energy intake, P = 0.018), the nadir shifted to day 10, when mean CIT concentration was lower in patients with severe oral mucositis (6.7 mumol/L, 95% CI 3.4-13.1) than in those without severe mucositis (11.9 mumol/L, 95% CI 5.8-24.4, P = 0.003). Change in CIT was not correlated with stool volume, C-reactive protein, tumor necrosis factor-alpha, leptin, or ghrelin.

CONCLUSIONS: In children undergoing HCT, serum CIT correlates with measures of gastrointestinal function (oral mucositis severity, dietary intake, acute GVHD) and may reflect mucosal injury to the gastrointestinal tract.

Enteral nutrition in the management of pediatric intestinal failure

Fri, 01/06/2017 - 12:01pm

Intestinal failure is an uncommon but devastating condition whose natural history has dramatically improved over the past two decades. Infants with intestinal failure due to severe short bowel syndrome or other diagnoses previously considered incompatible with life are now routinely being saved and cared for in cutting edge, multidisciplinary programs. Enteral nutrition plays a central role in the management of children with intestinal failure. This review provides an overview of enteral nutrition in pediatric intestinal failure, with specific emphasis on recent advances in clinical management, patient outcomes, and emerging therapies.

Elevations in serum anti-flagellin and anti-LPS Igs are related to growth faltering in young Tanzanian children

Fri, 01/06/2017 - 12:01pm

BACKGROUND: Antibodies to LPS and flagellin have been described as indirect measures of increased gastrointestinal permeability and may be markers of environmental enteric dysfunction (EED), which is a condition associated with poor child growth.

OBJECTIVE: We assessed whether LPS- and flagellin-specific immunoglobulin (Ig) concentrations were associated with poor growth in young Tanzanian children at risk of EED. DESIGN: Blood samples were obtained from 590 childre

RESULTS: Anti-LPS and anti-flagellin IgA and IgG concentrations increased over the first year of life and were higher than concentrations (measured at 9 mo of age) in healthy controls. Children with anti-flagellin IgA, anti-LPS IgA, anti-flagellin IgG, and anti-LPS IgG concentrations in the highest quartile at 6 wk of age were 2.02 (95% CI: 1.11, 3.67), 1.84 (95% CI: 1.03, 3.27), 1.94 (95% CI: 1.04, 3.62), and 2.31 (95% CI: 1.25, 4.27) times, respectively, more likely to become underweight (weight-for-age z score < -2) after adjustment for covariates (P-trend < 0.05) than were children with Ig concentrations in the lowest quartile. Children with increased concentrations of anti-flagellin IgA were also more likely to become wasted; however, there was no association between any of the markers and subsequent stunting.

CONCLUSION: Serologic measures of increased intestinal permeability to bacterial components are associated with subsequent poor growth and could help identify children who may benefit most from preventive interventions. This trial was registered at clinicaltrials.gov as NCT00421668.

Etiology of Diarrhea, Nutritional Outcomes, and Novel Intestinal Biomarkers in Tanzanian Infants

Fri, 01/06/2017 - 12:01pm

OBJECTIVE: Diarrheal diseases are a leading cause of morbidity and mortality worldwide, but the etiology of diarrhea and its relation to nutritional outcomes in resource-limited settings is poorly defined. We sought to determine the etiology of community-acquired diarrhea in Tanzanian infants and to assess the association with anthropometrics and novel intestinal biomarkers.

METHODS: A convenience sample of infants in a trial of zinc and/or multivitamin supplementation in Tanzania was selected. Subjects were enrolled at age 6 weeks and studied for 18 months. Stool samples were obtained from children with acute diarrhea. A novel, polymerase chain reaction-based TaqMan array was used to screen stool for 15 enteropathogens. A subset of subjects had serum gastrointestinal biomarkers measured.

RESULTS: One hundred twenty-three subjects with diarrhea were enrolled. The mean +/- SD age at stool sample collection was 12.4 +/- 3.9 months. Thirty-five enteropathogens were identified in 34 (27.6%) subjects: 11 rotavirus, 9 Cryptosporidium spp, 7 Shigella spp, 3 Campylobacter jejuni/coli, 3 heat stable-enterotoxigenic Escherichia coli, and 2 enteropathogenic E coli. Subjects with any identified enteropathogen had significantly lower weight-for-length z scores (-0.55 +/- 1.10 vs 0.03 +/- 1.30, P = 0.03) at the final clinic visit than those without an identified pathogen. Fifty of the 123 subjects (40.7%) had serum analyzed for antibodies to lipopolysaccharide (LPS) and flagellin. Subjects with any identified enteropathogen had lower immunoglobulin (IgA) antibodies to LPS (0.75 +/- 0.27 vs 1.13 +/- 0.77, P = 0.01) and flagellin (0.52 +/- 0.16 vs 0.73 +/- 0.47, P = 0.02) than those without an identified pathogen.

CONCLUSIONS: This quantitative polymerase chain reaction method may allow identification of enteropathogens that place children at higher risk for suboptimal growth. IgA anti-LPS and flagellin antibodies hold promise as emerging intestinal biomarkers.

A Model to Explain How the Bacille Calmette Guerin (BCG) Vaccine Drives Interleukin-12 Production in Neonates

Thu, 01/05/2017 - 5:09pm

The Bacille Calmette Guerin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-alpha (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-gamma (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins.

The unmet need in rheumatology: reports from the Targeted Therapies meeting 2016

Thu, 01/05/2017 - 5:09pm

The 18th annual international Targeted Therapies meeting brought together over 100 leading scientists and clinicians from around the world in the field of rheumatology. During the meeting, breakout sessions were held consisting of 5 disease-specific groups each with 20-40 experts assigned to each group based on clinical or scientific expertise. Specific groups included: rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis/spondyloarthritis, systemic lupus erythematous, and other connective tissue diseases (e.g. Sjogren's, Behcet's, others). In each group, experts were asked to identify unmet needs in 3 categorical areas: basic/translational science, clinical science and therapeutic development, and clinical care. Needs were prioritised as primary or secondary. Overall, similar primary unmet needs were identified within each disease foci. Within translational science, these included the need for better understanding the heterogeneity within each disease, such that predictive tools for therapeutic response could be developed. Within clinical science and therapeutic trials, the ability to prevent progression to disease onset in those at risk, and the ability to cure disease were identified. A further unmet need was to develop new and accessible therapeutics, as well as to conduct strategic trials of currently approved therapies. Within the clinical care realm, improved co-morbidity management and patient-centered care were identified as unmet needs. Lastly, it was strongly felt there was a need to develop a scientific infrastructure for well-characterised, longitudinal cohorts married with biobanks and mechanisms to support data-sharing. This infrastructure could facilitate many of the unmet needs identified within each disease area.

PD-L1 expression in EBV-negative diffuse large B-cell lymphoma: clinicopathologic features and prognostic implications

Thu, 01/05/2017 - 5:09pm

Programmed cell death ligand 1 (PD-L1) is a cell surface glycoprotein that regulates the cellular immune response and serves as a targetable immune checkpoint molecule. PD-L1 is expressed on tumor cells and the immune microenvironment of several human malignancies, including a subset of aggressive lymphomas. We sought to investigate further the clinical and pathologic features of EBV-negative diffuse large B-cell lymphoma (DLBCL) cases that express PD-L1. Immunohistochemical staining using an anti-PD-L1 monoclonal antibody was performed on DLBCL cases from 86 patients. These patients received standard chemotherapy treatment and were followed for up to 175 months. Overall, 14 cases (16%) were considered positive for PD-L1 in tumor cells. In comparison with PD-L1 negative cases, PD-L1 positive cases had a higher rate of non-GCB type (71% vs. 30%, P=0.0060), and higher Ann Arbor stage (II-IV) (100% vs. 73%, P=0.0327). No significant differences were seen in the immunohistochemical expression of BCL2, MYC, or Ki67. Patients with tumors expressing PD-L1 demonstrated inferior overall survival (OS) upon long term follow up (P=0.0447). Both age/sex-adjusted and multivariate analyses identified PD-L1 as an independent predictor for OS (P=0.0101 and P=0.0424). There was no significant difference, however, in terms of remission rates after first treatment, relapse rates, and progression free survival between the groups. Identification of DLBCL cases that express PD-L1 may serve to select a subset of patients that could further benefit from targeted immunotherapy.

Forward genetic screen of human transposase genomic rearrangements

Thu, 01/05/2017 - 5:09pm

BACKGROUND: Numerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates.

RESULTS: To enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates.

CONCLUSIONS: The discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases.

Insulin-like Signaling Promotes Glial Phagocytic Clearance of Degenerating Axons through Regulation of Draper

Thu, 01/05/2017 - 5:09pm

Neuronal injury triggers robust responses from glial cells, including altered gene expression and enhanced phagocytic activity to ensure prompt removal of damaged neurons. The molecular underpinnings of glial responses to trauma remain unclear. Here, we find that the evolutionarily conserved insulin-like signaling (ILS) pathway promotes glial phagocytic clearance of degenerating axons in adult Drosophila. We find that the insulin-like receptor (InR) and downstream effector Akt1 are acutely activated in local ensheathing glia after axotomy and are required for proper clearance of axonal debris. InR/Akt1 activity, it is also essential for injury-induced activation of STAT92E and its transcriptional target draper, which encodes a conserved receptor essential for glial engulfment of degenerating axons. Increasing Draper levels in adult glia partially rescues delayed clearance of severed axons in glial InR-inhibited flies. We propose that ILS functions as a key post-injury communication relay to activate glial responses, including phagocytic activity.

External validation of the DHAKA score and comparison with the current IMCI algorithm for the assessment of dehydration in children with diarrhoea: a prospective cohort study

Thu, 01/05/2017 - 5:09pm

BACKGROUND: Dehydration due to diarrhoea is a leading cause of child death worldwide, yet no clinical tools for assessing dehydration have been validated in resource-limited settings. The Dehydration: Assessing Kids Accurately (DHAKA) score was derived for assessing dehydration in children with diarrhoea in a low-income country setting. In this study, we aimed to externally validate the DHAKA score in a new population of children and compare its accuracy and reliability to the current Integrated Management of Childhood Illness (IMCI) algorithm.

METHODS: DHAKA was a prospective cohort study done in children younger than 60 months presenting to the International Centre for Diarrhoeal Disease Research, Bangladesh, with acute diarrhoea (defined by WHO as three or more loose stools per day for less than 14 days). Local nurses assessed children and classified their dehydration status using both the DHAKA score and the IMCI algorithm. Serial weights were obtained and dehydration status was established by percentage weight change with rehydration. We did regression analyses to validate the DHAKA score and compared the accuracy and reliability of the DHAKA score and IMCI algorithm with receiver operator characteristic (ROC) curves and the weighted kappa statistic. This study was registered with ClinicalTrials.gov, number NCT02007733.

FINDINGS: Between March 22, 2015, and May 15, 2015, 496 patients were included in our primary analyses. On the basis of our criterion standard, 242 (49%) of 496 children had no dehydration, 184 (37%) of 496 had some dehydration, and 70 (14%) of 496 had severe dehydration. In multivariable regression analyses, each 1-point increase in the DHAKA score predicted an increase of 0.6% in the percentage dehydration of the child and increased the odds of both some and severe dehydration by a factor of 1.4. Both the accuracy and reliability of the DHAKA score were significantly greater than those of the IMCI algorithm.

INTERPRETATION: The DHAKA score is the first clinical tool for assessing dehydration in children with acute diarrhoea to be externally validated in a low-income country. Further validation studies in a diverse range of settings and paediatric populations are warranted.

FUNDING: National Institutes of Health Fogarty International Center.

State-of-the-art monitoring in treatment of dengue shock syndrome: a case series

Thu, 01/05/2017 - 5:09pm

BACKGROUND: Early recognition and treatment of circulatory volume loss is essential in the clinical management of dengue viral infection. We hypothesized that a novel computational algorithm, originally developed for noninvasive monitoring of blood loss in combat casualties, could: (1) indicate the central volume status of children with dengue during the early stages of "shock"; and (2) track fluid resuscitation status.

METHODS: Continuous noninvasive photoplethysmographic waveforms were collected over a 5-month period from three children of Thai ethnicity with clinical suspicion of dengue. Waveform data were processed by the algorithm to calculate each child's Compensatory Reserve Index, where 1 represents supine normovolemia and 0 represents the circulatory volume at which hemodynamic decompensation occurs. Values between 1 and 0 indicate the proportion of reserve remaining before hemodynamic decompensation.

RESULTS: This case report describes a 7-year-old Thai boy, another 7-year-old Thai boy, and a 9-year-old Thai boy who exhibited signs and symptoms of dengue shock syndrome; all the children had secondary dengue virus infections, documented by serology and reverse transcriptase polymerase chain reaction. The three boys experienced substantial plasma leakage demonstrated by pleural effusion index > 25, ascites, and > 20 % hemoconcentration. They received fluid administered intravenously; one received a blood transfusion. All three boys showed a significantly low initial Compensatory Reserve Index ( > /=0.20), indicating a clinical diagnosis of "near shock". Following 5 days with fluid resuscitation treatment, their Compensatory Reserve Index increased towards "normovolemia" (that is, Compensatory Reserve Index > 0.75).

CONCLUSIONS: The results from these cases demonstrate a new variation in the diagnostic capability to manage patients with dengue shock syndrome. The findings shed new light on a method that can avoid possible adverse effects of shock by noninvasive measurement of a patient's compensatory reserve rather than standard vital signs or invasive diagnostic methods.