Effect of sodium concentration and mucosal pH on apical uptake of acetate and butyrate, and barrier function of the isolated bovine ruminal epithelium.

This study was conducted to investigate the role of Na+ on ruminal short-chain fatty acid (SCFA) absorption and barrier function when isolated ruminal epithelium was exposed to high and low pH ex vivo. Nine Holstein steer calves (322 ± 50.9 kg of body weight) consuming 7.05 ± 1.5 kg dry matter of a total mixed ration were euthanized and ruminal tissue was collected from the caudal-dorsal blind sac. Tissues were mounted between 2 halves of Ussing chambers (3.14 cm2) and exposed to buffers that contained low (10 mM) or high (140 mM) Na+ with low (6.2) or high (7.4) mucosal pH. The same buffer solutions were used on the serosal side except that pH was maintained at 7.4. Buffers used to evaluate SCFA uptake contained bicarbonate to determine total uptake or excluded bicarbonate and included nitrate to determine noninhibitable uptake. Bicarbonate-dependent uptake was calculated as the difference between the total and noninhibitable uptake. Acetate (25 mM) and butyrate (25 mM) were spiked with 2-3H-acetate and 1-14C-butyrate, respectively, and were then added to the mucosal side, incubated for 1 min, and tissues were analyzed to evaluate rates of SCFA uptake. Tissue conductance (Gt) and the mucosal-to-serosal flux of 1-3H-mannitol were used to assess barrier function. There were no Na+ × pH interactions for butyrate or acetate uptake. Decreasing mucosal pH from 7.4 to 6.2 increased total acetate and butyrate uptake, and bicarbonate-dependent acetate uptake. Flux of 1-3H-mannitol was not affected by treatment. However, high Na+ concentration reduced Gt and prevented an increase in Gt from flux period 1 to flux period 2. The results of this study indicate that although providing more Na+ to the ruminal epithelium does not affect SCFA uptake or mannitol flux, it may help stabilize tissue integrity.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation.

A germ-free (GF) chicken model was used to test 2 hypotheses: 1. microbial colonization of the gastrointestinal tract (GIT) influences mucin gene expression and mucin types; and 2. mannan oligosaccharide (MOS) supplementation affects GIT cells directly, without bacteria mediation, compared with bacterial-mediated effect (i.e., indirectly). Gnotobiotic isolators were used: 1) GF, 2) with a single bacteria population, and 3) conventionalized by exposure to cecal bacterial contents. Each was divided to 2 diet groups: with or without MOS (2 kg/t) for 1 wk. Results show that the absence of bacteria in the GIT caused a reduction in neutral and acidic goblet cell (GC) number and density, an increase in sulfated mucin, absence of sialylated GC, and reduced mucin 2 mRNA expression in the small intestine of GF compared with conventional birds. These results indicate a reduced development of mucin production and secretion in the absence of GIT bacteria implying a less mature small intestine mucosa, supporting our first hypothesis. Results from the single bacteria population group were not conclusive and did not support any of the hypotheses. Supplementation of MOS, regardless of microbial presence, caused a reduction in neutral GC number and density but increased neutral GC area. The MOS caused different effects on acidic mucins in conventional and GF birds, causing a reduction in sialylated GC number (conventional) and a reduction in sulfated GC density (GF), all supporting a direct effect of MOS in GF animals, in addition to an indirect effect via gut microflora.

Effect of day length on cause of mortality, leg health, and ocular health in broilers.

An experiment was conducted to study the effect of day length, sex, and genotype (Ross × Ross 308 and 708) on mortality causes, bird mobility, footpad health, and ocular size, with 4 trials within the experiment. Four graded day lengths were chosen to allow the study of relationship between day length and health parameters, including 14L:10D, 17L:7D, 20L:4D, and 23L:1D. The primary statistical tools used to assess the day length relationships were regression analysis (Proc Reg and RSReg of SAS). Data were also analyzed as a 4 (lighting program) × 2 (sex) × 2 (genotype) factorial arrangement. Total mortality, as well as mortality due to metabolic and skeletal disease, decreased linearly with increasing inclusion of darkness (7- to 32-, 7- to 38-, and 7- to 48-d periods). Infectious disorders were quadratically related to day length (7- to 48-d period only), with birds under 20L having the highest level. Day length was linearly or quadratically related to average gait score in a positive fashion, and the incidence of birds falling in painful gait score categories increased linearly with increasing day length. Average footpad lesion scores increased with increasing day length (28 and 35 d). The 23L photoperiod resulted in heavier eye weights than other lighting programs. Males had a higher mortality and morbidity rate and a higher average gait score than females. Average footpad score was lower for males than females (28 and 35 d). Overall mortality was higher for 308 than 708 broilers; hence, levels of specific mortality causes were higher. Average gait scores were lower for 308 than 708 birds in 2 of the 3 time periods measured and footpad lesions were higher. To conclude, many aspects of broiler health improve with decreasing day length.

Dose-dependent effects of dietary zinc oxide on bacterial communities and metabolic profiles in the ileum of weaned pigs.

Pharmacological levels of zinc oxide (ZnO) can improve the health of weaning piglets and influence the intestinal microbiota. This experiment aimed at studying the dose-response effect of five dietary concentrations of ZnO on small intestinal bacteria and metabolite profiles. Fifteen piglets, weaned at 25 ± 1 days of age, were allocated into five groups according to body weight and litter. Diets were formulated to contain 50 (basal diet), 150, 250, 1000 and 2500 mg zinc/kg by adding analytical-grade (>98% purity) ZnO to the basal diet and fed ad libitum for 14 days after a 7-day adaptation period on the basal diet. Ileal bacterial community profiles were analysed by denaturing gradient gel electrophoresis and selected bacterial groups quantified by real-time PCR. Concentrations of ileal volatile fatty acids (VFA), D- and L-lactate and ammonia were determined. Species richness, Shannon diversity and evenness were significantly higher at high ZnO levels. Quantitative PCR revealed lowest total bacterial counts in the 50 mg/kg group. Increasing ZnO levels led to an increase (p = 0.017) in enterobacteria from log 4.0 cfu/g digesta (50 mg/kg) to log 6.7 cfu/g digesta (2500 mg/kg). Lactic acid bacteria were not influenced (p = 0.687) and clostridial cluster XIVa declined (p = 0.035) at highest ZnO level. Concentration of total, D- and L-lactate and propionate was not affected (p = 0.736, p = 0.290 and p = 0.630), but concentrations of ileal total VFA, acetate and butyrate increased markedly from 50 to 150 mg/kg and decreased with further increasing zinc levels and reached low levels again at 2500 mg/kg (p = 0.048, p = 0.048 and p = 0.097). Ammonia decreased (p < 0.006) with increasing dietary ZnO level. In conclusion, increasing levels of dietary ZnO had strong and dose-dependent effects on ileal bacterial community composition and activity, suggesting taxonomic variation in metabolic response to ZnO.

Functional amino acid supplementation postweaning mitigates the response of normal birth weight more than for low birth weight pigs to a subsequent Salmonella challenge.

Previous work has shown that dietary supplementation with key functional amino acids (FAA) improves growth performance and immune status of disease-challenged normal birth weight (NBW) pigs. It is not known whether FAA supplementation attenuates the effects of a subsequent disease challenge or whether this response is similar in low birth weight (LBW) pigs. The objective was to determine the effects of birth weight and FAA supplementation during the postweaning period in Salmonella-challenged pigs. Thirty-two LBW (1.08 ± 0.11 kg) and NBW (1.58 ± 0.11 kg) pigs were assigned to a nursery feeding program at weaning (25 d) for 31 days in a 2 × 2 factorial arrangement. Factors were birth weight category (LBW vs. NBW) and basal (FAA-) or supplemented FAA profile (FAA+; Thr, Met, and Trp at 120% of requirements). At d 31, pigs were placed onto a common grower diet and, after a 7-d adaptation period, were inoculated with Salmonella Typhimurium (ST; 2.2 × 109 colony-forming units/mL) and monitored for 7-d postinoculation. Growth performance, rectal temperature, fecal score, indicators of gut health, ST shedding score in feces, intestinal ST colonization and translocation, and blood parameters of acute-phase response and antioxidant balance were measured pre- and postinoculation. Inoculation with ST increased temperature and fecal score, and the overall rectal temperature was higher in LBW compared to NBW pigs (P < 0.05). Postinoculation (d 7), reduced:oxidized glutathione was increased in NBW compared to LBW pigs (P < 0.05). Salmonella shedding and translocation to spleen were lower in NBW-FAA+ compared to NBW-FAA- pigs (P < 0.05). Postinoculation average daily gain was higher in NBW-FAA+ (P < 0.05) compared to the other groups. Postinoculation haptoglobin, superoxide dismutase, and colonic myeloperoxidase were increased in LBW-FAA- pigs (P < 0.05). Ileal alkaline phosphatase was decreased in LBW compared to NBW (P < 0.05). Overall, FAA supplementation represents a potential strategy to mitigate the effect of enteric disease challenge in NBW, but not LBW pigs.

SNP array analysis in constitutional and cancer genome diagnostics--copy number variants, genotyping and quality control.

Array-based comparative genomic hybridization analysis of genomic DNA was first applied in postnatal diagnosis for patients with intellectual disability (ID) and/or congenital anomalies (CA). Genome-wide single-nucleotide polymorphism (SNP) array analysis was subsequently implemented as the first line diagnostic test for ID/CA patients in our laboratory in 2009, because its diagnostic yield is significantly higher than that of routine cytogenetic analysis. In addition to the detection of copy number variations, the genotype information obtained with SNP array analysis enables the detection of stretches of homozygosity and thereby the possible identification of recessive disease genes, mosaic aneuploidy, or uniparental disomy. Patient-parent (trio) information analysis is used to screen for the presence of any form of uniparental disomy in the patient and can determine the parental origin of a de novo copy number variation. Moreover, the outcome of a genotype analysis is used as a final quality control by ruling out potential sample mismatches due to non-paternity or sample mix-up. SNP array analysis is now also used in our laboratory for patients with disorders for which locus heterogeneity is known (homozygosity pre-screening), in prenatal diagnosis in case of structural ultrasound anomalies, and for patients with leukemia. In this report, we summarize our array findings and experiences in the various diagnostic applications and demonstrate the power of a SNP-based array platform for molecular karyotyping, because it not only significantly improves the diagnostic yield in both constitutional and cancer genome diagnostics, but it also enhances the quality of the diagnostic laboratory workflow.

Intra-individual stability over time of standardized anti-Mullerian hormone in FMR1 premutation carriers.

BACKGROUND: Carriers of a premutation (CGG repeat length 55-200) in the fragile X mental retardation (FMR1) gene are at risk for primary ovarian insufficiency (FXPOI). The anti-Müllerian hormone (AMH) level acts as a useful marker of ovarian follicle reserve and, thus, may serve to predict when this ovarian reserve becomes too low to sustain ovarian function. We investigated the intra-individual variation of AMH levels over time for premutation carriers compared with non-carriers. METHODS: We determined AMH levels in blood samples from 240 women ascertained through fragile X families, of which 127 were premutation carriers and 113 were non-carriers. Linear mixed models were used to assess the effect of age and premutation status on AMH levels and to determine a modeled AMH value. The stability over time of the deviation of observed AMH levels from modeled levels, referred to as standardized AMH values, was assessed through correlation coefficients of 41 longitudinal samples. RESULTS: At all ages, premutation carriers exhibited lower AMH levels. For all women, AMH was found to decrease by 10% per year. The added effect of having a premutation decreased AMH levels by 54%. The deviation of an individual's AMH level from the modeled value showed a reasonable intra-individual correlation. The Pearson correlation coefficient of two samples taken at different ages was 0.36 (P = 0.05) for non-carriers and 0.69 (P = 0.01) for carriers. CONCLUSIONS: We developed a unique standardized AMH value, taking FMR1 premutation status and the subject's age into account, which appears to be stable over time and may serve as a predictor for FXPOI after further longitudinal assessment.

Replacement of corn in the diet of broiler chickens using foxtail millet produced by 2 different cultivation strategies.

Foxtail millet is well-suited to climatic conditions in semi-arid tropic regions where it is cultivated using both agro-ecologic and conventional cultivation practices. This study evaluated the nutritional value, digestibility, and physiological effects of agro-ecologic and conventionally cultivated foxtail millet in comparison with corn. Chemical and TME(n) analysis of foxtail millet cultivated conventionally and agro-ecologically indicated similar nutritional value. In total, 432 eight-day-old Ross 308 broiler chicks, using a 2 × 3 factorial arrangement, were randomly assigned to 4 replicate pens for each of 6 isonitrogenous and isoenergetic diets. Experimental diets were formulated by replacing corn with conventional or agro-ecologic millet at 3 levels (33, 66, or 100% of corn replacement). Body weight at 21 and 42 d of age was higher (P < 0.05) at 100% millet inclusion versus the lower inclusion levels. At 42 d of age, feed intake and feed conversion ratios were also improved (P < 0.05) at the 100% millet inclusion level. Similarly, the apparent ileal digestibility of CP increased (P < 0.05) for 100% millet diets. There were no differences in ileal digestibility of nutrients between millet growth conditions. Millet inclusion level significantly affected small intestinal morphology such that crypt depth was lowest (P < 0.05) in the 100% inclusion group for duodenum, jejunum, and ileum at 28 d of age, and for duodenum and ileum at 42 d of age. The villus crypt ratio was also highest (P < 0.05) in the 100% millet inclusion group for jejunum and ileum at 28 d of age, and duodenum, jejunum, and ileum at 42 d of age. Millet growth condition did not markedly affect small intestinal morphology. Serum antibody responses to Gumboro and Newcastle diseases were not affected by millet inclusion level or growth condition. In conclusion, foxtail millet could be considered as an alternate cereal for inclusion in the diet of broiler chickens. Broiler chicken performance and physiological responses to foxtail millet were similar whether grown conventionally or using agro-ecologic practices.

Development of a Coccidiosis Disease Challenge Model Using a Commercially Available Live Oocyst Vaccine.

With growing cross-disciplinary collaboration among researchers, it is increasingly important to record detailed methodology to prevent the repetition of preliminary experiments. The purpose of this paper is to explain the development of a coccidiosis challenge model for the investigation of dietary interventions to coccidiosis in broiler chickens. The objectives are to select a dose of mixed species coccidial vaccine and evaluate the suitability (ability to produce a consistent, marked change) of selected response variables important to nutritional studies at different times postinfection (PI). Coccivac-B and Coccivac-B52 (Merck Animal Health) were evaluated as the source of coccidia in three trials. Trials 1 and 2 were randomized complete block designs with four doses (0, 10, 20, or 30 times (×) label dose) of Coccivac-B administered to 12 replicate cages of six birds by repeater pipette (Trial 1) or gavaging needle (Trial 2). Trial 3 used a completely randomized design with 0× or 30× label dose of Coccivac-B52 administered by gavaging needle to six replicate cages of six birds. Birds were gavaged at 15 days of age, and response criteria were evaluated 7 days PI in all trials and again at 10 days PI in Trials 1 and 2. All means are reported in order of increasing coccidia dose with significance accepted at P ≤ 0.05. Broiler performance was not affected by coccidia in Trials 1 or 3 but grew poorer with increasing dose from 0 to 7 days PI in Trial 2 (body weight gain, 465, 421, 388, 365 g; feed to gain, 1.37, 1.47, 1.52, 1.58). As coccidia dose increased, nitrogen corrected apparent metabolizable energy decreased (Trial 1, 3387, 3318, 3267, 3170 kcal kg-1; Trial 2, 3358, 2535, 2422, 2309 kcal kg-1; Trial 3, not measured), while relative weight, length, and content for intestinal sections increased (Trials 1through 3). Gross lesion (duodenum, jejunum/ileum, ceca) and oocyst count scores (jejunum/ileum, ceca) increased with dose; however, gross scoring often suggested infection in unchallenged birds, a finding unsupported by oocyst count scores. At 7 days PI there was no correlation between midgut gross lesion score and midgut oocyst count score (r = 0.06, P = 0.705), but cecal scores were weakly correlated (r = 0.55, P < 0.001). Administering coccidia via repeater pipette (Trial 1) resulted in respiratory distress in some birds, while use of the gavaging needle (Trials 2 and 3) successfully induced intestinal damage in chickens without resulting in coccidia related mortality. Thirty times the label dose at 7 days PI resulted in the greatest number of response variables that produced a consistent, marked change. Therefore, consideration should be given to these conditions when designing future coccidiosis challenge models using vaccines as a source of coccidia.

Artículo regular­Desarrollo de un modelo de desafío para coccidiosis utilizando una vacuna de ooquistes vivos disponible comercialmente. Con la creciente colaboración interdisciplinaria entre investigadores, es cada vez más importante registrar la metodología detallada para evitar la repetición de experimentos preliminares. El propósito de este artículo es explicar el desarrollo de un modelo de desafío de coccidiosis para la investigación de intervenciones dietéticas para coccidiosis en pollos de engorde. Los objetivos son seleccionar una dosis de vacuna coccidial de especies mixtas y evaluar la idoneidad (capacidad de producir un cambio marcado y consistente) de las variables de respuesta seleccionadas que son importantes para los estudios nutricionales en diferentes momentos posteriores a la infección (PI). Las vacunas Coccivac-B o Coccivac B-52 (Merck Animal Health) se evaluaron como fuente de coccidias en tres ensayos. Los ensayos 1 y 2 fueron diseños de bloques completamente aleatorios con cuatro dosis (0, 10, 20 o 30 veces (×) la dosis indicada en la etiqueta) de Coccivac-B administradas a 12 jaulas repetidas de seis aves mediante una pipeta repetidora (ensayo 1) o por sonda oral. (Prueba 2). El ensayo 3 utilizó un diseño completamente aleatorio con una dosis de etiqueta de 0 × o 30 × de Coccivac-B52 administrada con una sonda oral en seis jaulas repetidas de seis aves. Las aves fueron inoculadas por sonda a los 15 días de edad y los criterios de respuesta se evaluaron a los 7 días postinoculación en todos los ensayos y nuevamente a los 10 días postinoculación en los ensayos 1 y 2. Todos los promedios se reportan en orden de dosis crecientes de coccidias con significancia aceptada en P ≤ 0.05. El rendimiento de los pollos de engorde no se vio afectado por las coccidias en los Ensayos 1 o 3, pero empeoró al aumentar la dosis de los cero a 7 días después de la inoculación en el Ensayo 2 (aumento de peso corporal, 465, 421, 388, 365 g; alimento para ganar, 1.37, 1.47, 1.52, 1.58). A medida que aumentaba la dosis de coccidia, la energía metabolizable de nitrógeno aparente y corregida disminuyó (Prueba 1, 3387, 3318, 3267, 3170 kcal kg-1; Prueba 2, 3358, 2535, 2422, 2309 kcal kg-1; Prueba 3, no medida), mientras que el peso relativo, la longitud y el contenido de las secciones intestinales aumentaron (ensayos 1 a 3). La lesión macroscópica (duodeno, yeyuno/íleon, ciego) y las puntuaciones del recuento de oocistos (yeyuno/íleon, ciego) aumentaron con la dosis; sin embargo, la puntuación bruta a menudo sugirió infección en aves no desafiadas, un hallazgo que no está respaldado por las puntuaciones del recuento de ooquistes. A los 7 días después de la infección no hubo correlación entre la puntuación de la lesión macroscópica del intestino medio y la puntuación del recuento de oocistos del intestino medio (r= 0,06, P= 0,705), pero las puntuaciones cecales se correlacionaron débilmente (r = 0.55, P <0.001). La administración de coccidias a través de una pipeta repetidora (Ensayo 1) provocó dificultad respiratoria en algunas aves, mientras que el uso de la sonda oral (Ensayos 2 y 3) indujo con éxito el daño intestinal en los pollos sin dar como resultado mortalidad relacionada con los coccidias. Treinta veces la dosis de la etiqueta a los 7 días después de la infección resultó en el mayor número de variables de respuesta que produjeron un cambio marcado y consistente. Por lo tanto, deben tenerse en cuenta estas condiciones al diseñar futuros modelos de exposición a la coccidiosis que utilicen vacunas como fuente de coccidias.

c-sis is translocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia.

By analysis of a series of somatic cell hybrids derived by fusion of either mouse or Chinese hamster cells with leukocytes from different chronic myelocytic leukemia (CML) patients or from normal donors, we have localized the human oncogene, c-sis, on the q11 to qter segment of chromosome 22 and demonstrated its translocation from chromosome 22 to chromosome 9 (q34) in CML.

CNTNAP2 gene dosage variation is associated with schizophrenia and epilepsy.

A homozygous mutation of the CNTNAP2 gene has been associated with a syndrome of focal epilepsy, mental retardation, language regression and other neuropsychiatric problems in children of the Old Order Amish community. Here we report genomic rearrangements resulting in haploinsufficiency of the CNTNAP2 gene in association with epilepsy and schizophrenia. Genomic deletions of varying sizes affecting the CNTNAP2 gene were identified in three non-related Caucasian patients. In contrast, we did not observe any dosage variation for this gene in 512 healthy controls. Moreover, this genomic region has not been identified as showing large-scale copy number variation. Our data thus confirm an association of CNTNAP2 to epilepsy outside the Old Order Amish population and suggest that dosage alteration of this gene may lead to a complex phenotype of schizophrenia, epilepsy and cognitive impairment.

Hu-ets-1 and Hu-ets-2 genes are transposed in acute leukemias with (4;11) and (8;21) translocations.

Human probes identifying the cellular homologs of the v-ets gene, Hu-ets-1 and Hu-ets-2, and two panels of rodent-human cell hybrids were used to study specific translocations occurring in acute leukemias. The human ets-1 gene was found to translocate from chromosome 11 to 4 in the t(4;11)(q21;23), a translocation characteristic of a subtype of leukemia that represents the expansion of a myeloid/lymphoid precursor cell. Similarly, the human ets-2 gene was found to translocate from chromosome 21 to chromosome 8 in the t(8;21)(q22;q22), a nonrandom translocation commonly found in patients with acute myeloid leukemia with morphology M2 (AML-M2). Both translocations are associated with expression different from the expression in normal lymphoid cells of ets genes, raising the possibility that these genes play a role in the pathogenesis of these leukemias.

Intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig.

To study microbial influence on intestinal development pertaining to nutrient digestion, two separate gnotobiotic experiments were performed, each with 16 piglets allocated to four treatment groups: germfree (GF), monoassociation with Escherichia coli, monoassociation with Lactobacillus fermentum or conventionalization with faecal bacteria (CV). Enzyme activity and gene expression of lactase phlorizin hydrolase (LPH) and aminopeptidase N (APN) were measured in isolated enterocytes, harvested on day 14, using specific substrates and quantitative PCR respectively. Enterocytes of CV pigs had reduced APN activity, but had increased gene expression relative to GF, making the specific activity:mRNA (A:G) ratio dramatically lower (p < 0.05). Similarly, LPH A:G ratio was significantly reduced (p < 0.05) in enterocytes of CV pigs as compared with GF. The results of co-incubation of L. fermentum, E. coli and faecal bacteria with APN indicate a direct relationship between enzyme inactivation and specific A:G ratio in enterocytes. We conclude that enterocyte up-regulation of APN expression occurs as either a direct response to microbial colonization or as a feedback mechanism in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.

In vivo digestion characteristics of protein sources fed to broilers.

The rate and extent of protein digestion are relevant to broiler performance and health, but information is lacking on the rate of digestion and the characteristics of the undigested fraction for common protein feed ingredients. Therefore, this study evaluated the digestion kinetics and the distal ileum (DI) digesta protein characteristics of protein meals fed to broiler chickens. Using a completely randomized design, 360 male broilers at 14 D of age were assigned to 60 battery cages and fed semi-purified diets composed of wheat starch (N-free) or wheat starch with either corn distillers dried grains with solubles (CDDGS), corn gluten meal, meat and bone meal, soybean meal, fish meal (FM), porcine meal (PCM), canola meal, blood meal (BM), or feather meal. At day 21, the protein digestion kinetics and total and soluble protein of the DI content were determined. Differences were considered significant when P ≤ 0.05. Protein source affected the extent of amino acid (AA) and CP digestibility at the DI. The results demonstrated differences in digesta mean retention time (MRT) and the rate of digestion of AA and CP among protein sources. FM had the shortest MRT of 46 min, whereas CDDGS had the longest at 142 min. Both FM and PCM had the highest digestion rates for most of the AA evaluated among the protein sources, whereas CDDGS had the lowest. In turn, the total and soluble CP in the distal ileal contents ranged from 54 to 1466 mg and 6 to 347 mg, respectively. In conclusion, dietary protein source influences the amount and solubility of the undigested protein in the DI and the digestion kinetics of AA and CP along the small intestine of broilers. These parameters may contribute to the effects of protein source on muscle deposition and could influence the impact protein sources may have on gut health through protein fermentation.

Predisposition to colorectal cancer: exploiting copy number variation to identify novel predisposing genes and mechanisms.

Although cancer is mostly regarded as an acquired disease, familial predisposition plays a significant role in many cancer types. Thus far, several high penetrant cancer predisposing genes have been identified. As yet, however, these genes explain only a fraction of the familial and/or hereditary cases of cancer. This has led to the exploration of the human genome for novel cancer predisposing genes. The identification of such genes will not only increase our understanding of cancer predisposition and development, but will also have direct implications for genetic counseling and personalized management of the patients and their family members. Here we provide an inventory of currently known molecular mechanisms related to familial colorectal cancer development and an outline of copy number analysis-based strategies to identify new predisposing genes. Finally, we discuss a novel copy number-associated epigenetic mechanism underlying the predisposition to colorectal cancer.

High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression.

Gross cytogenetic anomalies are traditionally being used as diagnostic, prognostic and therapeutic markers in the clinical management of cancer, including childhood acute lymphoblastic leukemia (ALL). Recently, it has become increasingly clear that genetic lesions driving tumorigenesis frequently occur at the submicroscopic level and, consequently, escape standard cytogenetic observations. Therefore, we profiled the genomes of 40 childhood ALLs at high resolution. We detected multiple de novo genetic lesions, including gross aneuploidies and segmental gains and losses, some of which were subtle and affected single genes. Many of these lesions involved recurrent (partially) overlapping deletions and duplications, containing various established leukemia-associated genes, such as ETV6, RUNX1 and MLL. Importantly, the most frequently affected genes were those controlling G1/S cell cycle progression (e.g. CDKN2A, CDKN1B and RB1), followed by genes associated with B-cell development. The latter group includes microdeletions of the B-lineage transcription factors PAX5, EBF, E2-2 and IKZF1 (Ikaros), as well as genes with other established roles in B-cell development, that is RAG1 and RAG2, FYN, PBEF1 or CBP/PAG. The fact that we frequently encountered multiple lesions affecting genes involved in cell cycle regulation and B-cell differentiation strongly suggests that both these processes need to be targeted independently and simultaneously to trigger ALL development.

Effects of peripartum propylene glycol supplementation on nitrogen metabolism, body composition, and gene expression for the major protein degradation pathways in skeletal muscle in dairy cows.

Early-lactating dairy cows mobilize body protein to provide amino acids that are directed toward gluconeogenesis and milk protein synthesis. Propylene glycol (PG) is a precursor of ruminal propionate, and feeding PG has been reported to improve energy supply by increasing blood glucose. Our hypothesis was that feeding PG could spare body protein by providing an alternative source of carbon for gluconeogenesis. The major objectives of this study were 1) to delineate the effects of pre- and postpartum PG supplementation in transition dairy cows on whole-body nitrogen balance, urinary 3-methylhistidine (3-MH) excretion, body composition, and gene expression profiles for the major protein degradation pathways in skeletal muscle; and 2) to characterize the changes in body protein metabolism during the periparturient period. Sixteen pregnant cows (7 primiparous and 9 multiparous) were paired based on expected calving dates and then randomly assigned within each pair to either a basal diet (control) or basal diet plus 600 mL/d of PG. Diets were fed twice daily for ad libitum intake, and PG was fed in equal amounts as a top dress from d -7 to d 45. All measurements were conducted at 3 time intervals starting at d -14 +/- 5, d 15, and d 38 relative to calving. Propylene glycol had no effect on whole-body N balance, urinary 3-MH excretion, or body composition. However, N balance was lower at d 15 and 38, compared with d -14. Urinary excretion of 3-MH was lower at d -14 than at d 15 and 38. Supplemental PG had no effect on body weight (BW) and all components of empty BW. On average, cows fed both diets mobilized 19 kg of body fat and 14 kg of body protein between d -14 and d 38. Supplemental PG had no effect on mRNA abundance in skeletal muscle for m-calpain, and the 14-kDa ubiquitin-carrier protein E2 (14-kDa E2) and proteasome 26S subunit-ATPase components of the ubiquitin-mediated proteolytic pathway; however, PG supplementation downregulated mRNA expression for mu-calpain at d 15, and tended to downregulate mRNA expression for ubiquitin at d 15 and 38. Relative to calving, mRNA abundance for m- and mu-calpain, ubiquitin, and 14-kDa E2 were greater at d 15 compared with d -14 and d 38. In summary, these results indicate that transitional effects on whole-body metabolism and gene expression for the Ca(2+)-dependent and ubiquitin-mediated proteolytic pathways in skeletal muscle were more pronounced than those elicited by PG supplementation.

Effect of live yeast Saccharomyces cerevisiae (Actisaf Sc 47) supplementation on the performance and hindgut microbiota composition of weanling pigs.

As an alternative to antibiotic growth promoters, live yeast supplementation has proven useful in reducing weaning stress and improving performance parameters of piglets. Here, we compared the performance and hindgut microbiota of weanling piglets subjected to different pre- and post-weaning yeast supplementation regimens using a live strain of Saccharomyces cerevisiae (Actisaf Sc 47). Average feed intake and average daily weight gain of piglets within Yeast-Control and Yeast-Yeast groups were higher than those in the Control-Control group. Yeast supplementation resulted in development of microbial communities that were phylogenetically more homogenous and less dispersed compared to the microbiota of control piglets. Key bacterial taxa overrepresented in the microbiota of yeast supplemented piglets included phylum Actinobacteria, specifically family Coriobacteriaceae, as well as Firmicutes families Ruminococcaceae, Clostridiaceae, Peptostreptococcaceae, and Peptococcaceae. Correlation network analysis revealed that yeast supplementation was associated with enrichment of positive correlations among proportions of different bacterial genera within the hindgut ecosystem. In particular, within the cecal microbiota of supplemented piglets, higher numbers of positive correlations were observed among potentially beneficial genera of the phyla Actinobacteria and Firmicutes, suggesting a mechanism by which yeast supplementation may contribute to regulation of intestinal homeostasis and improved performance of piglets.

Molecular mechanisms underlying the MiT translocation subgroup of renal cell carcinomas.

Renal cell carcinomas (RCCs) represent a heterogeneous group of neoplasms, which differ in histological, pathologic and clinical characteristics. The tumors originate from different locations within the nephron and are accompanied by different recurrent (cyto)genetic anomalies. Recently, a novel subgroup of RCCs has been defined, i.e., the MiT translocation subgroup of RCCs. These tumors originate from the proximal tubule of the nephron, exhibit pleomorphic histological features including clear cell morphologies and papillary structures, and are found predominantly in children and young adults. In addition, these tumors are characterized by the occurrence of recurrent chromosomal translocations, which result in disruption and fusion of either the TFE3 or TFEB genes, both members of the MiT family of basic helix-loop-helix/leucine-zipper transcription factor genes. Hence the name MiT translocation subgroup of RCCs. In this review several features of this RCC subgroup will be discussed, including the molecular mechanisms that may underlie their development.