Maternal nutrient restriction in mid-to-late gestation influences fetal mRNA expression in muscle tissues in beef cattle.

BACKGROUND: Manipulating maternal nutrition during specific periods of gestation can result in re-programming of fetal and post-natal development. In this experiment we investigated how a feed restriction of 85% compared with 140% of total metabolizable energy requirements, fed to cows during mid-to-late gestation, influences phenotypic development of fetuses and mRNA expression of growth (Insulin-Like Growth Factor family and Insulin Receptor (INSR)), myogenic (Myogenic Differentiation 1 (MYOD1), Myogenin (MYOG), Myocyte Enhancer Factor 2A (MEF2A), Serum Response Factor (SRF)) and adipogenic (Peroxisome Proliferator Activated Receptor Gamma (PPARG)) genes in fetal longissimus dorsi (LD) and semitendinosus (ST) muscle. DNA methylation of imprinted genes, Insulin Like Growth Factor 2 (IGF2) and Insulin Like Growth Factor 2 Receptor (IGF2R), and micro RNA (miRNA) expression, were also examined as potential consequences of poor maternal nutrition, but also potential regulators of altered gene expression patterns. RESULTS: While the nutrient restriction impacted dam body weight, no differences were observed in phenotypic fetal measurements (weight, crown-rump length, or thorax circumference). Interestingly, LD and ST muscles responded differently to the differential pre-natal nutrient levels. While LD muscle of restricted fetal calves had greater mRNA abundances for Insulin Like Growth Factor 1 and its receptor (IGF1 and IGF1R), IGF2R, INSR, MYOD1, MYOG, and PPARG, no significant differences were observed for gene expression in ST muscle. Similarly, feed restriction had a greater impact on the methylation level of IGF2 Differentially Methylated Region 2 (DMR2) in LD muscle as compared to ST muscle between treatment groups. A negative correlation existed between IGF2 mRNA expression and IGF2 DMR2 methylation level in both LD and ST muscles. Differential expression of miRNAs 1 and 133a were also detected in LD muscle. CONCLUSIONS: Our data suggests that a nutrient restriction of 85% as compared to 140% of total metabolizable energy requirements during the 2nd half of gestation can alter the expression of growth, myogenic and adipogenic genes in fetal muscle without apparent differences in fetal phenotype. It also appears that the impact of feed restriction varies between muscles suggesting a priority for nutrient partitioning depending on muscle function and/or fiber composition. Differences in the methylation level in IGF2, a well-known imprinted gene, as well as differences in miRNA expression, may be functional mechanisms that precede the differences in gene expression observed, and could lead to trans-generational epigenetic programming.

Community Pharmacist Training-and-Communication Network and Drug-Related Problems in Patients With CKD: A Multicenter, Cluster-Randomized, Controlled Trial.

BACKGROUND: Appropriate training for community pharmacists may improve the quality of medication use. Few studies have reported the impact of such programs on medication management for patients with chronic kidney disease (CKD). STUDY DESIGN: Multicenter, cluster-randomized, controlled trial. SETTING & PARTICIPANTS: Patients with CKD stage 3a, 3b, or 4 from 6 CKD clinics (Quebec, Canada) and their community pharmacies. INTERVENTION: Each cluster (a pharmacy and its patients) was randomly assigned to either ProFiL, a training-and-communication network program, or the control group. ProFiL pharmacists completed a 90-minute interactive web-based training program on use of medications in CKD and received a clinical guide, patients' clinical summaries, and facilitated access to the CKD clinic. OUTCOMES: Drug-related problems (primary outcome), pharmacists' knowledge and clinical skills, and patients' clinical attributes (eg, blood pressure and glycated hemoglobin concentration). MEASUREMENTS: Drug-related problems were evaluated the year before and after the recruitment of patients using a validated set of significant drug-related problems, the Pharmacotherapy Assessment in Chronic Renal Disease (PAIR) criteria. Pharmacists' questionnaires were completed at baseline and after 1 year. Clinical attributes were documented at baseline and after 1 year using available information in medical charts. RESULTS: 207 community pharmacies, 494 pharmacists, and 442 patients with CKD participated. After 1 year, the mean number of drug-related problems per patient decreased from 2.16 to 1.60 and from 1.70 to 1.62 in the ProFiL and control groups, respectively. The difference in reduction of drug-related problems per patient between the ProFiL and control groups was -0.32 (95% CI, -0.63 to -0.01). Improvements in knowledge (difference, 4.5%; 95% CI, 1.6%-7.4%) and clinical competencies (difference, 7.4%; 95% CI, 3.5%-11.3%) were observed among ProFiL pharmacists. No significant differences in clinical attributes were observed across the groups. LIMITATIONS: High proportion of missing data on knowledge and clinical skills questionnaire (34.6%) and clinical attributes (11.1%). CONCLUSIONS: Providing community pharmacists with essential clinical data, appropriate training, and support from hospital pharmacists with expertise in nephrology increases pharmacists' knowledge and reduces drug-related problems in patients with CKD who are followed up in clinics incorporating a multidisciplinary health care team.

The imitation switch ATPase Snf2l is required for superovulation and regulates Fgl2 in differentiating mouse granulosa cells.

Imitation switch (ISWI) proteins are catalytic subunits of chromatin remodeling complexes that alter nucleosome positioning by hydrolyzing ATP to regulate access to DNA. In mice, there are two paralogs, SNF2-homolog (SNF2H) and SNF2-like (SNF2L), which participate in different complexes and have contrasting patterns of expression. Here we investigate the role of SNF2L in ovaries by characterizing a mouse bearing an inactivating deletion of exon 6 that disrupts the ATPase domain. Snf2l mutant mice produce significantly fewer eggs than control mice when superovulated. Gonadotropin stimulation leads to a significant deficit in secondary follicles and an increase in abnormal antral follicles. Mutant females also failed to induce fibrinogen-like 2 (Fgl2) in response to human chorionic gonadotropin (hCG) stimulation, while overexpression of SNF2L was sufficient to drive its expression in granulosa cells. SNF2L was also shown to directly interact with the nuclear receptor co-activator flightless I (FLI-I) as shown by immunoprecipitation. These results begin to establish a role for SNF2L in the precise coordination of gene expression in granulosa cells during folliculogenesis and its broader implications in fertility.

The oncolytic poxvirus JX-594 selectively replicates in and destroys cancer cells driven by genetic pathways commonly activated in cancers.

Oncolytic viruses are generally designed to be cancer selective on the basis of a single genetic mutation. JX-594 is a thymidine kinase (TK) gene-inactivated oncolytic vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and lac-Z transgenes that is designed to destroy cancer cells through replication-dependent cell lysis and stimulation of antitumoral immunity. JX-594 has demonstrated a favorable safety profile and reproducible tumor necrosis in a variety of solid cancer types in clinical trials. However, the mechanism(s) responsible for its cancer-selectivity have not yet been well described. We analyzed the replication of JX-594 in three model systems: primary normal and cancer cells, surgical explants, and murine tumor models. JX-594 replication, transgene expression, and cytopathic effects were highly cancer-selective, and broad spectrum activity was demonstrated. JX-594 cancer-selectivity was multi-mechanistic; replication was activated by epidermal growth factor receptor (EGFR)/Ras pathway signaling, cellular TK levels, and cancer cell resistance to type-I interferons (IFNs). These findings confirm a large therapeutic index for JX-594 that is driven by common genetic abnormalities in human solid tumors. This appears to be the first description of multiple selectivity mechanisms, both inherent and engineered, for an oncolytic virus. These findings have implications for oncolytic viruses in general, and suggest that their cancer targeting is a complex and multifactorial process.

Development and validation of the PAIR (Pharmacotherapy Assessment in Chronic Renal Disease) criteria to assess medication safety and use issues in patients with CKD.

BACKGROUND: Explicit criteria for judging medication safety and use issues in patients with chronic kidney disease (CKD) are lacking. STUDY DESIGN: Quality improvement report. SETTING & PARTICIPANTS: Nephrologists (n = 4), primary care physicians (n = 2), hospital pharmacists with expertise in nephrology (n = 4), and community pharmacists (n = 2). The PAIR (Pharmacotherapy Assessment in Chronic Renal Disease) criteria were applied retrospectively to 90 patients with CKD in a randomized study. QUALITY IMPROVEMENT PLAN: Development of an explicit set of criteria to enable rapid and systematic detection of drug-related problems (DRPs). Using a RAND method, experts judged the clinical significance of DRPs and the appropriateness of a community pharmacist intervention. The PAIR criteria include 50 DRPs grouped into 6 categories. OUTCOMES: DRPs detected using the PAIR criteria compared with implicit clinical judgment by nephrology pharmacists. MEASUREMENTS: Prevalence of DRPs and reliability, validity, and responsiveness of the PAIR criteria. RESULTS: A mean of 2.5 DRPs/patient (95% CI, 2.0-3.1) was identified based on the PAIR criteria compared with 3.9 DRPs/patient (95% CI, 3.4-4.5) based on clinical judgment of nephrology pharmacists. Inter-rater reliability coefficients (κ) by PAIR category varied from 0.80-1.00, with an intraclass correlation coefficient (ICC) of 0.93 (95% CI, 0.89-0.95) for total DRPs per patient. Test-retest reliability coefficients by category varied from 0.74-1.00, with an ICC of 0.91 (95% CI, 0.82-0.96) for total DRPs per patient. During the study, the mean number of DRPs per patient did not change significantly when assessed using the PAIR criteria and clinical judgment. LIMITATION: The prevalence of PAIR DRPs may be underestimated due to the retrospective nature of the validation. CONCLUSION: The prevalence of DRPs requiring the intervention of community pharmacists is high in patients with CKD. The PAIR criteria are reliable, but their responsiveness remains to be shown.

Genetic potential for residual feed intake and diet fed during early- to mid-gestation influences post-natal DNA methylation of imprinted genes in muscle and liver tissues in beef cattle.

Discovery of epigenetic modifications associated with feed efficiency or other economically important traits would increase our understanding of the molecular mechanisms underlying these traits. In combination with known genetic markers, this would provide opportunity to improve genomic selection accuracy in cattle breeding programs. It would also allow cattle to be managed to improve favorable gene expression. The objective of this study was to identify variation in DNA methylation between beef cattle of differential pre-natal nutrition and divergent genetic potential for residual feed intake (RFI). Purebred Angus offspring with the genetic potential for either high (HRFI) or low (LRFI) RFI were prenatally exposed to either a restricted maternal diet of 0.5 kg/d average daily gain (ADG) or a moderate maternal diet of 0.7 kg/d ADG from 30 to 150 d of gestation. We performed DNA methylation analysis of differentially methylated regions (DMR) of imprinted genes (Insulin-like growth factor 2 (IGF2) DMR2, IGF2/H19 imprinting control region (ICR) and IGF2 receptor (IGF2R) DMR2) using post-natal samples of longissimus dorsi (LD) muscle taken from male and female calves at birth and weaning, and of LD muscle, semimembranosus (SM) muscle, and liver samples collected from steers at slaughter (17 months of age). Interestingly, for all three DMR investigated in liver, LRFI steers had higher levels of methylation than HRFI steers. In LD muscle, IGF2/H19 ICR methylation differences for heifers at birth were due to pre-natal diet, while for steers at birth they were mostly the result of genetic potential for RFI with LRFI steers again having higher levels of methylation than HRFI steers. While results from repeated measures analysis of DNA methylation in steers grouped by RFI revealed few differences, in steers grouped by diet, we found higher methylation levels of IGF2 DMR2 and IGF2R DMR2 in LD muscle of restricted diet steers at weaning and slaughter than at birth, as well as increased methylation in LD muscle of restricted diet steers compared with moderate diet steers at weaning and/or slaughter. Our results suggest that differential pre-natal nutrition, and divergent genetic potential for RFI, induces tissue- and sex-specific alterations in post-natal IGF2 and IGF2R methylation patterns and that these patterns can vary with age in Angus beef cattle.

Quantitative assay for the detection of the V617F variant in the Janus kinase 2 (JAK2) gene using the Luminex xMAP technology.

BACKGROUND: The availability of clinically valid biomarkers contribute to improve the diagnosis and clinical management of diseases. A valine-to-phenylalanine substitution at position 617 (V617F) in the Janus kinase 2 (JAK2) gene has been recently associated with key signaling abnormalities in the transduction of haemopoietic growth-factor receptors and is now considered as a useful clinical marker of myeloproliferative neoplasms. Several methods have recently been reported to detect the JAK2 V617F point mutation and show variable sensitivity. METHODS: Using the Luminex xMAP technology, we developed a quantitative assay to detect the JAK2V617F variant. The method was based on polymerase chain reaction (PCR) followed by hybridization to specific probes coupled with internally dyed microspheres. The assay comprises 3 steps: genomic DNA extraction, end point PCR reaction, direct hybridization of PCR fragments and quantification. It has been tested with different sources of nucleic acid. RESULTS: Applied to whole blood samples, this quantitative assay showed a limit of detection of 2%. A highly sensitive allele-specific primer extension reaction performed in parallel allowed to validate the results and to identify the specimens with values below 2%. CONCLUSION: Direct hybridization assay using the Luminex xMAP technology allows sensitive quantification of JAK2V617F from blood spots. It is simple and can be easily performed in a clinical setting.

Temporal regulation of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA expression in the oocyte, granulosa and theca cells of developing preovulatory follicles in the pig.

This study aimed to describe the abundance and localization of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA during pig preovulatory follicular development and to evaluate their implication in improving follicular maturity in the preovulatory period preceding the second versus first post-weaning oestrus. Oocytes, granulosa (GC) and theca cells (TC) were recovered from antral follicles of primiparous sows at day 1, 2 and 4 after weaning and at day 14, 16 and 20 of their subsequent oestrous cycle. Real-time PCR analysis revealed that with the exception of BMP6 mRNA, which was absent in GC, all genes were expressed in every cell type. Although BMP6, BMP15 and GDF9 mRNA were most abundant in the oocyte, their expression remained relatively constant during follicular development. By contrast, receptor BMPR1B and TGFBR1 expressions in the GC and TC were temporally regulated. BMPR1B mRNA abundance was positively correlated with plasma oestradiol (E2) suggesting that its regulation by oestrogen may be implicated in normal folliculogenesis. Interestingly, the increase in BMPR1B mRNA and protein abundance during the periovulatory period in GC and TC suggests a role for bone morphogenetic protein (BMP) 15 in the ovulatory process. Finally, expression of these ligands and receptors was not associated with potential differences in follicle maturity observed during the second versus first post-weaning preovulatory follicular wave. In conclusion, our results clearly demonstrate the presence of a complex signalling system within the pig follicle involving the transforming growth factor-beta superfamily and their receptors, and provide evidence to support a role for BMP15 and BMPR1B during ovulation.

Proteomics of dark cutting longissimus thoracis muscle from heifer and steer carcasses.

Studies have suggested that the phenomenon of dark cutting (Canada B4) beef may also be related to muscle glycolytic proteins. The objective of this study, therefore, was to analyze longissimus thoracis (LT; n=23), from Canada AA (n=8), atypical (AB4; pH<5.9, n=8) and typical (TB4; pH>5.9, n=7) B4 heifer and steer carcasses, for sarcoplasmic and myofibrillar proteins using 2-D gel electrophoresis and LC-MS/MS mass spectrometry. Results indicated that AB4 LT had intramuscular pH and lactate concentration similar to Canada AA but lower (P<0.05) L* and b*. Moreover, AB4 LT were tougher than Canada AA even at 21days post-mortem, unlike TB4. Canada AB4 LT had reduced (P<0.05) levels of creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and glycerol-3-phosphate dehydrogenase [NAD(+)], indicating a compromised glycolytic capacity in AB4. Canada AB4 LT had increased (P<0.05) abundances of phosphatidylethanolamine-binding protein 1 and small heat shock proteins.

Pharmacist Web-based training program on medication use in chronic kidney disease patients: impact on knowledge, skills, and satisfaction.

INTRODUCTION: Chronic kidney disease (CKD) patients are multimorbid elderly at high risk of drug-related problems. A Web-based training program was developed based on a list of significant drug-related problems in CKD patients requiring a pharmaceutical intervention. The objectives were to evaluate the impact of the program on community pharmacists' knowledge and skills and their satisfaction with the training. METHODS: Pharmacists were randomized to the training program or the control group. Training comprised a 60-minute Web-based interactive session supported by a clinical guide. Pharmacists completed a questionnaire on knowledge (10 multiple-choice questions) and skills (2 clinical vignettes) at baseline and a second time within 1 month. Trained pharmacists completed a written satisfaction questionnaire. Semidirected telephone interviews were conducted with 8 trained pharmacists. Changes in knowledge and skills scores were compared between the groups. RESULTS: Seventy pharmacists (training: 52; control: 18) were recruited; the majority were women with <15 years' experience. Compared with the control group, an adjusted incremental increase in the knowledge score (22%; 95% confidence interval [CI]: 16%-27%) and skills score (24%; 95% CI: 16%-33%) was observed in the training group. Most pharmacists (87%-100%) rated each aspect of the program "excellent'' or "very good." Additional training and adding a discussion forum were suggested to complement the program. DISCUSSION: Pharmacists like the Web-based continuing education program. Over a short time span, the program improved their knowledge and skills. Its impact on their clinical practices and quality of medication use in CKD patients remains to be assessed.

Pig preovulatory oocytes modulate cumulus cell protein and gene expression in vitro.

This study investigated the changes in protein and gene expression in oocytectomized cumulus cells (OOX) of medium-sized follicles from gilts, cultured with or without denuded oocytes isolated from large oestrogenic sow follicles. Proteomic analysis identified 14 proteins that were differentially expressed in OOX, of which the protein 14-3-3 eta, a signal transduction pathway modulator, was down-regulated in the presence of oocytes. Oocyte co-culture also down-regulated FSHR mRNA expression in OOX, as measured by real-time PCR, and FSHR and 14-3-3 eta mRNA abundance were positively correlated. The oocyte also up-regulated HSD3B mRNA, suggesting an effect on cumulus cell progesterone synthesis. Together with data on gene expression in granulosa cells during the follicular phase of the sow oestrous cycle, this study suggests that modulation of the expression of steroidogenesis related proteins and genes in cumulus cells by the porcine preovulatory oocyte reflects the specific physiological requirements of the preovulatory follicle.

RNA interference as a tool to study gene function in bovine oocytes.

RNA interference (RNAi) has become a well-established technique to study gene function in several species. Our objective was to develop a RNAi approach to study gene function in bovine oocytes. In the first experiment, three different treatments including a 20 min exposure to cytochalasin B, a 6 hr maturation in cycloheximide, and a combination of these two treatments were tested to improve oocyte survival following microinjection. The cycloheximide/cytochalasin B treatment greatly increased (P<0.02) the survival rate of the microinjected oocytes. In the second experiment, we assessed the effect of both cyclin B1 and GFP dsRNA on cyclin B1 mRNA and protein expression. The injection of cyclin B1 dsRNA resulted in a decrease in cyclin B1 mRNA and protein, while the cyclin B2 mRNA remained unaffected. Furthermore, the injection of GFP dsRNA did not interfere with cyclin B1 mRNA or protein nor with the ability of the oocyte to mature properly. In addition, the lack of cyclin B1 in the oocyte led to activation in 10% of the oocytes as evidenced by the presence of a pronucleus. However, the use of an additional 10 hr of maturation in the presence of 6-dimethylaminopurine (6-DMAP) prevented germinal vesicle breakdown and allowed a longer exposure to dsRNA. This procedure increased the percentage of activated oocytes to 33% and is likely to result from an increased length of time for dsRNA processing and for degradation of the cyclin B1 mRNA to occur. In conclusion, RNAi represents a useful technique to study gene function in the bovine oocyte.

Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation.

Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.

Pitx1 in vivo promoter activity and mechanisms of positive autoregulation.

During early mouse embryogenesis, Pitx1 (pituitary homeobox 1), a member of the bicoid subgroup of PAIRED homeobox-containing transcription factors, marks the stomodeum, oral ectoderm, pituitary and first branchial arch in the anterior part of the embryo and lateral plate mesoderm only in the posterior half of the embryo. We have now defined PITX1 promoter fragments that mimic the anterior but not posterior expression of PITX1 in transgenic mice. In addition, we show positive regulation of this promoter in transfection studies by three members of the Pitx1 family (Pitx1, Pitx1b, Pitx2), as well as by a related factor, Otx1. PITX1 autoregulation depends on DNA-binding and trans-activation domains of Pitx1 and it may be responsible for establishment and/or maintenance of the Pitx1 expression domain.