New insights into adipocyte-specific leptin gene expression

The adipocyte-derived hormone leptin is a critical regulator of many physiological functions, ranging from satiety to immunity. Surprisingly, very little is known about the transcriptional pathways that regulate adipocyte-specific expression of leptin. In a recent published study, we pursued a strategy integrating BAC transgenic reporter mice, in vitro reporter assays, and chromatin state mapping to locate an adipocyte-specific cis-element upstream of the LEP gene in human fat cells. Quantitative proteomics (stable isotope labeling by amino acids in cell culture, SILAC) with affinity enrichment of protein-DNA complexes identified the transcription factor FOSL2 as a specific binder to the identified region. We confirmed that FOSL2 is an important regulator of LEP gene expression in vitro and in vivo using cell culture models and genetic mouse models. In this commentary, we discuss the transcriptional regulation of LEP gene expression, our strategy to identify an adipocyte-specific cis-regulatory element and the transcription factor(s) responsible for LEP gene expression. We also discuss our data on FOSL2 and leptin levels in physiology and pathophysiology. We speculate on unanswered questions and future directions

Impaired DNA double-strand break repair contributes to chemoresistance in HIF-1α-deficient mouse embryonic fibroblasts

A mismatch between metabolic demand and oxygen delivery leads to microenvironmental changes in solid tumors. The resulting tumor hypoxia is associated with malignant progression, therapy resistance and poor prognosis. However, the molecular mechanisms underlying therapy resistance in hypoxic tumors are not fully understood. The hypoxia-inducible factor (HIF) is a master transcriptional activator of oxygen-regulated gene expression. Transformed mouse embryonic fibroblasts (MEFs) derived from HIF-1α-deficient mice are a popular model to study HIF function in tumor progression. We previously found increased chemotherapy and irradiation susceptibility in the absence of HIF-1α. Here, we show by single-cell electrophoresis, histone 2AX phosphorylation and nuclear foci formation of γH2AX and 53BP1, that the number of DNA double-strand breaks (DSB) is increased in untreated and etoposide-treated HIF-deficient MEFs. In etoposide-treated cells, cell cycle control and p53-dependent gene expression were not affected by the absence of HIF-1α. Using a candidate gene approach to screen 17 genes involved in DNA repair, messenger RNA (mRNA) and protein of three members of the DNA-dependent protein kinase complex were found to be decreased in HIF-deficient MEFs. Of note, residual HIF-1α protein in cancer cells with a partial HIF-1α mRNA knockdown was sufficient to confer chemoresistance. In summary, these data establish a novel molecular link between HIF and DNA DSB repair. We suggest that selection of early, non-hypoxic tumor cells expressing low levels of HIF-1α might contribute to HIF-dependent tumor therapy resistanc

Perforated Peptic Ulcer Repair: Factors Predicting Conversion in Laparoscopy and Postoperative Septic Complications.

The surgical treatment for perforated peptic ulcers can be safely performed laparoscopically. The aim of the study was to define simple predictive factors for conversion and septic complications. This retrospective case-control study analyzed patients treated with either laparoscopic surgery or laparotomy for perforated peptic ulcers. A total of 71 patients were analyzed. Laparoscopically operated patients had a shorter hospital stay (13.7 vs. 15.1 days). In an intention-to-treat analysis, patients with conversion to open surgery (analyzed as subgroup from laparoscopic approach group) showed no prolonged hospital stay (15.3 days) compared to patients with a primary open approach. Complication and mortality rates were not different between the groups. The statistical analysis identified four intraoperative risk factors for conversion: Mannheim peritonitis index (MPI) > 21 (p = 0.02), generalized peritonitis (p = 0.04), adhesions, and perforations located in a region other than the duodenal anterior wall. We found seven predictive factors for septic complications: age >70 (p = 0.02), cardiopulmonary disease (p = 0.04), ASA > 3 (p = 0.002), CRP > 100 (p = 0.005), duration of symptoms >24 h (p = 0.02), MPI > 21(p = 0.008), and generalized peritonitis (p = 0.02). Our data suggest that a primary laparoscopic approach has no disadvantages. Factors necessitating conversions emerged during the procedure inhibiting a preoperative selection. Factors suggesting imminent septic complications can be assessed preoperatively. An assessment of the proposed parameters may help optimize the management of possible septic complications

HIF mediated and DNA damage independent histone H2AX phosphorylation in chronic hypoxia

The histone variant 2AX (H2AX) is phosphorylated at Serine 139 by the PI3K-like kinase family members ATM, ATR and DNA-PK. Genotoxic stress, such as tumor radio- and chemotherapy, is considered to be the main inducer of phosphorylated H2AX (γH2AX), which forms distinct foci at sites of DNA damage where DNA repair factors accumulate. γH2AX accumulation under severe hypoxic/anoxic (0.02% oxygen) conditions has recently been reported to follow replication fork stalling in the absence of detectable DNA damage. In this study, we found HIF-dependent accumulation of γH2AX in several cancer cell lines and mouse embryonic fibroblasts exposed to physiologically relevant chronic hypoxia (0.2% oxygen), which did not induce detectable levels of DNA strand breaks. The hypoxic accumulation of γH2AX was delayed by the RNAi-mediated knockdown of HIF-1α or HIF-2α and further decreased when both HIF-αs were absent. Conversely, basal phosphorylation of H2AX was increased in cells with constitutively stabilized HIF-2α. These results suggest that both HIF-1 and HIF-2 are involved in γH2AX accumulation by tumor hypoxia, which might increase a cancer cell's capacity to repair DNA damage, contributing to tumor therapy resistanc

Human NK Cell Subset Functions Are Differentially Affected by Adipokines

Background: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host’s primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). Results: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56dim NK cells. The production of GzmA in CD56bright NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. Conclusion: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Perfil psicomotor y lenguaje en niños/as con Trastorno Específico del Lenguaje mixto escolarizados

El diagnóstico de trastorno específico del lenguaje (TEL) suele abordarse desde los saberes del profesional fonoaudiólogo con escaso aporte de otras disciplinas. Se ha demostrado que el desarrollo infantil es un proceso complejo influido por diferentes factores que se retroalimentan y participan en la evolución de habilidades, aptitudes y destrezas durante la niñez. Debido a la imbricación de distintas dimensiones, resulta de interés visualizar las interrelaciones que puedan darse entre la adquisición patológica del lenguaje y otras esferas del desarrollo. En este estudio se observaron los perfiles psicomotores de 22 niños/as diagnosticados con TEL mixto entre 5 y 7 años y 11 meses, que asistían a un establecimiento educacional gratuito de la Región Metropolitana de Chile. A continuación, se correlacionaron estos perfiles con algunas variables lingüísticas: vocabulario, gramática receptiva, gramática expresiva y fonología. Los resultados muestran que los niños/as con diagnóstico de TEL mixto presentan un perfil psicomotor normal o dispráxico, sin diferencias entre los tres rangos etarios estudiados. Todos los factores psicomotores correlacionan con al menos una variable lingüística, a excepción de lateralidad. El vocabulario fue la variable lingüística que correlacionó con más factores psicomotores. Los factores psicomotores que aparecieron más descendidos fueron noción de cuerpo, estructuración espaciotemporal y praxia finaDiagnosing Specific language Impairment (SLI) is usually done by speech therapists who resort exclusively to their own professional knowledge as clinician, with not much input from other fields. It has been shown that children’s development is a complex process which may be impacted by different factors which influence each other and play a role in how skills, abilities, and capabilities evolve during childhood. Since dimensions are interwoven, it might be of interest to inspect the possible relation between the pathological acquisition of language and some other developmental aspects. In this study, the psychomotor profile of 22 children with mixed SLI was observed (age range 5.0 – 7.11). They all attended a public school in Santiago, Chile. Scores obtained on psychomotor measures were correlated with some linguistic variables: vocabulary, receptive grammar, expressive grammar and phonology. Results show that the observed children with SLI had an either normal or a dyspraxic profile, with no difference per age group. All inspected psychomotor factors correlated with one or more linguistic variable, except for laterality, which correlated with no linguistic variable. Vocabulary was the language variable that more frequently correlated with psychomotor factors. Children scored the lowest on three psychomotor factors: notion of body, spatiotemporal structuring and fine-grained praxi

Exercise-induced CITED4 expression is necessary for regional remodeling of cardiac microstructural tissue helicity

Both exercise-induced molecular mechanisms and physiological cardiac remodeling have been previously studied on a whole heart level. However, the regional microstructural tissue effects of these molecular mechanisms in the heart have yet to be spatially linked and further elucidated. We show in exercised mice that the expression of CITED4, a transcriptional co-regulator necessary for cardioprotection, is regionally heterogenous in the heart with preferential significant increases in the lateral wall compared with sedentary mice. Concordantly in this same region, the heart's local microstructural tissue helicity is also selectively increased in exercised mice. Quantification of CITED4 expression and microstructural tissue helicity reveals a significant correlation across both sedentary and exercise mouse cohorts. Furthermore, genetic deletion of CITED4 in the heart prohibits regional exercise-induced microstructural helicity remodeling. Taken together, CITED4 expression is necessary for exercise-induced regional remodeling of the heart's microstructural helicity revealing how a key molecular regulator of cardiac remodeling manifests into downstream local tissue-level changes. Expression of transcription factor CITED4 is necessary for exercise-induced regional remodeling of the heart's microstructural helicity, revealing how a key molecular regulator of cardiac remodeling mediates local tissue-level changes

Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging

SummaryEver since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible

A BAC-Based Transgenic Mouse Specifically Expresses an Inducible Cre in the Urothelium

Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC)-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported

p38MAPK, ERK and PI3K Signaling Pathways Are Involved in C5a-Primed Neutrophils for ANCA-Mediated Activation

BACKGROUND: The complement system is one of the important contributing factors in the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. The current study further investigated the signaling pathways of C5a-mediated priming of human neutrophils for ANCA-induced neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: The effects of the p38 mitogen-activated protein kinase (p38MAPK) inhibitor (SB202190), extracellular signal-regulated kinase (ERK) inhibitor (PD98059), c-Jun N-terminal kinase (JNK) inhibitor (6o) and phosphoinositol 3-kinase (PI3K) inhibitor (LY294002) were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of membrane-bound PR3 (mPR3) on neutrophils. For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI) value was 254.8±67.1, which decreased to 203.6±60.3, 204.4±36.7, 202.4±49.9 and 188±47.9 upon pre-incubation with SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0.05, P<0.01 and P<0.05), respectively. For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with above-mentioned inhibitors. The lactoferrin concentration increased in C5a-primed neutrophils induced by MPO or PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with above-mentioned three inhibitors. mPR3 expression increased from 923.3±182.4 in untreated cells to 1278.3±299.3 after C5a treatment and decreased to 1069.9±188.9, 1100±238.2, 1092.3±231.8 and 1053.9±200.3 by SB202190, PD98059, LY294002 and the mixture of above-mentioned three inhibitors (compared with that without inhibitors, P<0.01, P<0.05, P<0.01 and P<0.01), respectively. CONCLUSIONS/SIGNIFICANCE: Activation of p38MAPK, ERK and PI3K are important steps in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA