Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor), an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis

CONTEXT Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. OBJECTIVE To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. PATIENTS 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. METHODS SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). RESULTS Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. CONCLUSIONS Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations.

Comparison of energy expenditure, eating pattern and physical activity of grazing and zero-grazing dairy cows at different time points during lactation

An experiment was conducted to determine the effect of grazing versus zero-grazing on energy expenditure (EE), feeding behaviour and physical activity in dairy cows at different stages of lactation. Fourteen Holstein cows were subjected to two treatments in a repeated crossover design with three experimental series (S1, S2, and S3) reflecting increased days in milk (DIM). At the beginning of each series, cows were on average at 38, 94 and 171 (standard deviation (SD) 10.8) DIM, respectively. Each series consisted of two periods containing a 7-d adaptation and a 7-d collection period each. Cows either grazed on pasture for 16–18.5 h per day or were kept in a freestall barn and had ad libitum access to herbage harvested from the same paddock. Herbage intake was estimated using the double alkane technique. On each day of the collection period, EE of one cow in the barn and of one cow on pasture was determined for 6 h by using the 13C bicarbonate dilution technique, with blood sample collection done either manually in the barn or using an automatic sampling system on pasture. Furthermore, during each collection period physical activity and feeding behaviour of cows were recorded over 3 d using pedometers and behaviour recorders. Milk yield decreased with increasing DIM (P<0.001) but was similar with both treatments. Herbage intake was lower (P<0.01) for grazing cows (16.8 kg dry matter (DM)/d) compared to zero-grazing cows (18.9 kg DM/d). The lowest (P<0.001) intake was observed in S1 and similar intakes were observed in S2 and S3. Within the 6-h measurement period, grazing cows expended 19% more (P<0.001) energy (319 versus 269 kJ/kg metabolic body size (BW0.75)) than zero-grazing cows and differences in EE did not change with increasing DIM. Grazing cows spent proportionally more (P<0.001) time walking and less time standing (P<0.001) and lying (P<0.05) than zero-grazing cows. The proportion of time spent eating was greater (P<0.001) and that of time spent ruminating was lower (P<0.05) for grazing cows compared to zero-grazing cows. In conclusion, lower feed intake along with the unchanged milk production indicates that grazing cows mobilized body reserves to cover additional energy requirements which were at least partly caused by more physical activity. However, changes in cows׳ behaviour between the considered time points during lactation were too small so that differences in EE remained similar between treatments with increasing DIM.

Generation of a recombinant Gag virus-like-particle panel for the evaluation of p24 antigen detection by diagnostic HIV tests.

BACKGROUND Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens. METHODS We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection. RESULTS Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection. CONCLUSIONS The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

The generation of neutrophils in the bone marrow is controlled by autophagy.

Autophagy has been demonstrated to have an essential function in several cellular hematopoietic differentiation processes, for example, the differentiation of reticulocytes. To investigate the role of autophagy in neutrophil granulopoiesis, we studied neutrophils lacking autophagy-related (Atg) 5, a gene encoding a protein essential for autophagosome formation. Using Cre-recombinase mediated gene deletion, Atg5-deficient neutrophils showed no evidence of abnormalities in morphology, granule protein content, apoptosis regulation, migration, or effector functions. In such mice, however, we observed an increased proliferation rate in the neutrophil precursor cells of the bone marrow as well as an accelerated process of neutrophil differentiation, resulting in an accumulation of mature neutrophils in the bone marrow, blood, spleen, and lymph nodes. To directly study the role of autophagy in neutrophils, we employed an in vitro model of differentiating neutrophils that allowed modulating the levels of ATG5 expression, or, alternatively, intervening pharmacologically with autophagy-regulating drugs. We could show that autophagic activity correlated inversely with the rate of neutrophil differentiation. Moreover, pharmacological inhibition of p38 MAPK or mTORC1 induced autophagy in neutrophilic precursor cells and blocked their differentiation, suggesting that autophagy is negatively controlled by the p38 MAPK-mTORC1 signaling pathway. On the other hand, we obtained no evidence for an involvement of the PI3K-AKT or ERK1/2 signaling pathways in the regulation of neutrophil differentiation. Taken together, these findings show that, in contrast to erythropoiesis, autophagy is not essential for neutrophil granulopoiesis, having instead a negative impact on the generation of neutrophils. Thus, autophagy and differentiation exhibit a reciprocal regulation by the p38-mTORC1 axis.

Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification

Rolling Circle Amplification (RCA) is an isothermal enzymatic method generating single-stranded DNA products consisting of concatemers containing multiple copies of the reverse complement of the circular template precursor. Little is known on the compatibility of modified nucleoside triphosphates (dN*TPs) with RCA, which would enable the synthesis of long, fully modified ssDNA sequences. Here, dNTPs modified at any position of the scaffold were shown to be compatible with rolling circle amplification, yielding long (>1 kb), and fully modified single-stranded DNA products. This methodology was applied for the generation of long, cytosine-rich synthetic mimics of telomeric DNA. The resulting modified oligo-nucleotides displayed an improved resistance to fetal bovine serum.

Generation of Aptamers with an Expanded Chemical Repertoire

The enzymatic co-polymerization of modified nucleoside triphosphates (dN*TPs and N*TPs) is a versatile method for the expansion and exploration of expanded chemical space in SELEX and related combinatorial methods of in vitro selection. This strategy can be exploited to generate aptamers with improved or hitherto unknown properties. In this review, we discuss the nature of the functionalities appended to nucleoside triphosphates and their impact on selection experiments. The properties of the resulting modified aptamers will be described, particularly those integrated in the fields of biomolecular diagnostics, therapeutics, and in the expansion of genetic systems (XNAs).

Improving residency training with the help of multisource feedback: Generation of a model of influencing factors

Background: Feedback is considered to be one of the most important drivers of learning. One form of structured feedback used in medical settings is multisource feedback (MSF). This feedback technique provides the opportunity to gain a differentiated view on a doctor’s performance from several perspectives using a questionnaire and a facilitating conversation, in which learning goals are formulated. While many studies have been conducted on the validity, reliability and feasibility of the instrument, little is known about the impact of factors that might influence the effects of MSF on clinical performance. Summary of Work: To study under which circumstances MSF is most effective, we performed a literature review on Google Scholar with focus on MSF and feedback in general. Main key-words were: MSF, multi-source-feedback, multi source feedback, and feedback each combined with influencing/ hindering/ facilitating factors, effective, effectiveness, doctors-intraining, and surgery. Summary of Results: Based on the literature, we developed a preliminary model of facilitating factors. This model includes five main factors influencing MSF: questionnaire, doctor-in-training, group of raters, facilitating supervisor, and facilitating conversation. Discussion and Conclusions: Especially the following points that might influence MSF have not yet been sufficiently studied: facilitating conversation with the supervisor, individual aspects of doctors-in-training, and the causal relations between influencing factors. Overall there are only very few studies focusing on the impact of MSF on actual and long-term performance. We developed a preliminary model of hindering and facilitating factors on MSF. Further studies are needed to better understand under which circumstances MSF is most effective. Take-home messages: The preliminary model might help to guide further studies on how to implement MSF to use it at its full potential.

Generation of an hTERT-restricted, CMV-augmented oncolytic adenovirus capable of tumor-specific transgene expression: Development, characterization, and optimization

Current shortcomings in cancer therapy require the generation of new, broadly applicable, potent, targeted treatments. Here, an adenovirus is engineered to replicate specifically in cells with active human telomerase promotion using a modified hTERT promoter, fused to a CMV promoter element. The virus was also modified to contain a visible reporter transgene, GFP. The virus, Ad/hTC-GFP-E1 was characterized in vitro and demonstrated tumor specific activity both by dose and over time course experiments in a variety of cell lines. In vivo, Ad/hTC-GFP-E1 was affected at suppressing tumor growth and providing a survival benefit without causing any measurable toxicity. To increase the host range of the vector, the fiber region was modified to contain an RGD-motif. The vector, AdRGD/hTC-GFP-E1, was recharacterized in vitro, revealing heightened levels of infectivity and toxicity however maintaining a therapeutic window between cancer and normal cell toxicity. AdRGD/hTC-GFP-E1 was administered in vivo by limb perfusion and was observed to be tumor specific both in expression and replication. To further enhance the efficacy of viral vectors in lung delivery, asthma medications were investigated for their abilities to enhance transgene delivery and expression. A combination of bronchodilators, mast cell inhibitors, and mucolytic agents was devised which demonstrated fold increases in expression in immunocompetent mouse lungs as single agents and more homogenous, intense levels of expression when done in combination of all agents. To characterize the methods in which some cancers are resistant or may become resistant to oncolytic treatments, several small molecule inhibitors of metabolic pathways were applied in combination with oncolytic infection in vitro. SP600125 and PD 98059, respective JNK and ERK inhibitors, successfully suppressed oncolytic toxicity, however did not affect infectivity or transgene expression of Ad/hTC-GFP-E1. JNK and ERK inhibition did significantly suppress viral replication, however, as analyzed by lysate transfer and titration assays. In contrast, SB 203580, an inhibitor for p38, did not demonstrate any protective effects with infected cells. Flow cytometric analysis indicated a possible correlation with G1 arrest and suppressed viral production, however more compounds must be investigated to clarify this observation. ^

Genetic variability and the hypothalamic control of energy balance

Background. Obesity is a major health problem throughout the industrialized world. Despite numerous attempts to curtail the rapid growth of obesity, its incidence continues to rise. Therefore, it is crucial to better understand the etiology of obesity beyond the concept of energy balance.^ Aims. The first aim of this study was to first investigate the relationship between eating behaviors and body size. The second goal was to identify genetic variation associated with eating behaviors. Thirdly, this study aimed to examine the joint relationships between eating behavior, body size and genetic variation.^ Methods. This study utilized baseline data ascertained in young adults from the Training Interventions and Genetics of Exercise (TIGER) Study. Variables assessed included eating behavior (Emotional Eating Scale, Eating Attitudes Test-26, and the Block98 Food Frequency Questionnaire), body size (body mass index, waist and hip circumference, waist/hip ratio, and percent body fat), genetic variation in genes implicated related to the hypothalamic control of energy balance, and appropriate covariates (age, gender, race/ethnicity, smoking status, and physical activity. For the genetic association analyses, genotypes were collapsed by minor allele frequency, and haplotypes were estimated for each gene. Additionally, Bayesian networks were constructed in order to determine the relationships between genetic variation, eating behavior and body size.^ Results. We report that the EAT-26 score, Caloric intake, percent fat, fiber intake, HEAT index, and daily servings of vegetables, meats, grains, and fats were significantly associated with at least one body size measure. Multiple SNPs in 17 genes and haplotypes from 12 genes were tested for their association with body size. Variation within both DRD4 and HTR2A was found to be associated with EAT-26 score. In addition, variation in the ghrelin gene (GHRL) was significantly associated with daily Caloric intake. A significant interaction between daily servings of grains and the HEAT index and variation within the leptin receptor gene (LEPR) was shown to influence body size.^ Conclusion. This study has shown that there is a substantial genetic component to eating behavior and that genetic variation interacts with eating behavior to influence body size.^

Policies and Education in Latin America : From the 'competitive quality' to 'quality for the generation of bicentenary'

En América Latina la injerencia de los organismos internacionales en la política nacional constituye un fenómeno que no se puede soslayar. Se trata de una cuestión candente en el contexto actual y remite a la presencia creciente en los países de la región de una serie de papers, documentos, boletines que, generados en el seno de dichas entidades, señalan desafíos presentes y futuros que deberá atender América Latina. En el campo educativo, marcan las orientaciones de política para la región y representan discursos sobre la educación que es menester analizar críticamente. El objetivo de este trabajo es analizar una serie de ideas, recomendaciones y retóricas político-pedagógicas de los Organismos Internacionales respecto de la denominada calidad de la educación en América Latina con la intención de poner de manifiesto su productividad discursiva en tanto su incidencia está ligada a significantes que operan ordenando las disputas político-discursivas del campo educativo

Policies and Education in Latin America : From the 'competitive quality' to 'quality for the generation of bicentenary'

En América Latina la injerencia de los organismos internacionales en la política nacional constituye un fenómeno que no se puede soslayar. Se trata de una cuestión candente en el contexto actual y remite a la presencia creciente en los países de la región de una serie de papers, documentos, boletines que, generados en el seno de dichas entidades, señalan desafíos presentes y futuros que deberá atender América Latina. En el campo educativo, marcan las orientaciones de política para la región y representan discursos sobre la educación que es menester analizar críticamente. El objetivo de este trabajo es analizar una serie de ideas, recomendaciones y retóricas político-pedagógicas de los Organismos Internacionales respecto de la denominada calidad de la educación en América Latina con la intención de poner de manifiesto su productividad discursiva en tanto su incidencia está ligada a significantes que operan ordenando las disputas político-discursivas del campo educativo

Policies and Education in Latin America : From the 'competitive quality' to 'quality for the generation of bicentenary'

En América Latina la injerencia de los organismos internacionales en la política nacional constituye un fenómeno que no se puede soslayar. Se trata de una cuestión candente en el contexto actual y remite a la presencia creciente en los países de la región de una serie de papers, documentos, boletines que, generados en el seno de dichas entidades, señalan desafíos presentes y futuros que deberá atender América Latina. En el campo educativo, marcan las orientaciones de política para la región y representan discursos sobre la educación que es menester analizar críticamente. El objetivo de este trabajo es analizar una serie de ideas, recomendaciones y retóricas político-pedagógicas de los Organismos Internacionales respecto de la denominada calidad de la educación en América Latina con la intención de poner de manifiesto su productividad discursiva en tanto su incidencia está ligada a significantes que operan ordenando las disputas político-discursivas del campo educativo