Touchscreen cognitive deficits, hyperexcitability and hyperactivity in males and females using two models of Cdkl5 deficiency.

Many neurodevelopmental disorders (NDDs) are the result of mutations on the X chromosome. One severe NDD resulting from mutations on the X chromosome is CDKL5 deficiency disorder (CDD). CDD is an epigenetic, X-linked NDD characterized by intellectual disability (ID), pervasive seizures and severe sleep disruption, including recurring hospitalizations. CDD occurs at a 4:1 ratio, with a female bias. CDD is driven by the loss of cyclin-dependent kinase-like 5 (CDKL5), a serine/threonine kinase that is essential for typical brain development, synapse formation and signal transmission. Previous studies focused on male subjects from animal models, likely to avoid the complexity of X mosaicism. For the first time, we report translationally relevant behavioral phenotypes in young adult (8-20 weeks) females and males with robust signal size, including impairments in learning and memory, substantial hyperactivity and increased susceptibility to seizures/reduced seizure thresholds, in both sexes, and in two models of CDD preclinical mice, one with a general loss-of-function mutation and one that is a patient-derived mutation.

Conserved Proline Residues of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB Are Important for Substrate Interaction and Cleavage.

Intramembrane metalloproteases (IMMPs) regulate diverse biological processes by cleaving membrane-associated substrates within the membrane or near its surface. SpoIVFB is an intramembrane metalloprotease of Bacillus subtilis that cleaves Pro-σK during endosporulation. Intramembrane metalloproteases have a broadly conserved NPDG motif, which in the structure of an archaeal enzyme is located in a short loop that interrupts a transmembrane segment facing the active site. The aspartate residue of the NPDG motif acts as a ligand of the zinc ion involved in catalysis. The functions of other residues in the short loop are less well understood. We found that the predicted short loop of SpoIVFB contains two highly conserved proline residues, P132 of the NPDG motif and P135. Mutational analysis revealed that both proline residues are important for Pro-σK cleavage in Escherichia coli engineered to synthesize the proteins. Substitutions for either residue also impaired the Pro-σK interaction with SpoIVFB in copurification assays. Disulfide cross-linking experiments showed that the predicted short loop of SpoIVFB is in proximity to the N-terminal pro-sequence region (Proregion) of Pro-σK. Alanine substitutions for N129 and P132 of the SpoIVFB NPDG motif reduced cross-linking between its predicted short loop and the Proregion more than a P135A substitution. Conversely, the SpoIVFB P135A substitution reduced Pro-σK cleavage more than the N129A and P132A substitutions during sporulation of B. subtilis. We conclude that all three conserved residues of SpoIVFB are important for substrate interaction and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. IMPORTANCE Intramembrane metalloproteases (IMMPs) function in numerous signaling pathways. Bacterial IMMPs govern stress responses, including the sporulation of some species, thus enhancing the virulence and persistence of pathogens. Knowledge of IMMP-substrate interactions could aid therapeutic design, but structures of IMMP·substrate complexes are unknown. We examined the interaction of the IMMP SpoIVFB with its substrate Pro-σK, whose cleavage is required for Bacillus subtilis endosporulation. We found that conserved proline residues in a short loop predicted to interrupt a SpoIVFB transmembrane segment are important for Pro-σK binding and cleavage. The corresponding residues of the Escherichia coli IMMP RseP have also been shown to be important for substrate interaction and cleavage, suggesting that this is a broadly conserved feature of IMMPs, potentially suitable as a therapeutic target.

An in vivo Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective in vivo delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection. Secreted ZF activated the imprinted paternal Ube3a in a transgenic reporter mouse and ameliorated motor deficits in a Ube3a deletion Angelman Syndrome (AS) mouse. Intrathecally administered autologous rhesus MSCs were well-tolerated for 3 weeks following administration and secreted ZF protein was detectable within the cerebrospinal fluid (CSF), midbrain, and spinal cord. This approach is less invasive when compared to direct intracranial injection which requires a surgical procedure.

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene.

A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.

Maternal pregnancy intention and its association with low birthweight and pregnancy complications in Bangladesh: findings from a hospital-based study.

BACKGROUND: The investigation of the potential impact of unintended pregnancy on maternal and child health is important to design effective interventions. This study explored the associations between unintended pregnancy and low birthweight (LBW) and pregnancy complications. METHODS: A cross-sectional survey was conducted among 400 randomly selected women in the postnatal wards of Rajshahi Medical College Hospital, Bangladesh. Multivariate logistic regression analyses were used to identify associations. RESULTS: Results of this study indicate that 30.5% of all pregnancies were unintended and 29.3% of babies were born with LBW. Additionally, 79.3% of women experienced any pregnancy complication (AC), 69.5% experienced medical complications and 44.3% experienced obstetric complications (OCs) during their last pregnancy. Unintended pregnancy was significantly associated with LBW (adjusted odds ratio [AOR]: 3.18, 95% CI: 1.79 to 5.54), maternal experience of OCs (AOR: 1.83, 95% CI: 1.03 to 3.28) and AC (AOR: 2.93, 95%: 1.14 to 7.58). Women with unintended pregnancies were at higher risk of developing high blood pressure and anemia during pregnancy. CONCLUSIONS: Women with unintended pregnancies are at increased risk of producing LBW babies and experiencing complications during pregnancy. Therefore, maternal pregnancy intention should be addressed in interventions aimed to reduce maternal and child morbidity and mortality.

Yellow Mealworm Larvae (Tenebrio molitor) Fed Mycotoxin-Contaminated Wheat-A Possible Safe, Sustainable Protein Source for Animal Feed?

The aim of this study was to determine the potential for accumulation of deoxynivalenol (DON) in yellow mealworm larvae (Tenebrio molitor) reared on high DON Fusarium-infected wheat and investigate the effects on production, survival and nutritional traits. Wheat containing 200 µg/kg DON was used as the control diet. A different source of wheat was sorted into six fractions and mixed to obtain low (2000 µg/kg), medium (10,000 µg/kg) and high (12,000 µg/kg) levels of DON. Each diet was replicated five times with 300 or 200 mealworms per replicate for the feeding and breeding trials, respectively. Trial termination occurred when the first two pupae were observed (32-34 days). There was no difference in the concentrations of DON detected in the larvae between diets that ranged from 122 ± 19.3 to 136 ± 40.5 µg/kg (p = 0.88). Excretion of DON was 131, 324, 230 and 742 µg/kg for control, low, medium and high, respectively. Nutritional analysis of larvae showed maximum crude protein of 52% and crude fat of 36%. Ash, fiber, chitin, fatty-acids and amino-acid content were consistent across diets. Survival was greater than 96% for all life stages and average daily gain ranged from 1.9 ± 0.1 to 2.1 ± 0.1 mg/day per mealworm. Larvae accumulated low levels of DON from Fusarium-infected wheat diets suggesting contaminated wheat could be used to produce a sustainable, safe protein source.

Congenital nuclear cataracts in a Holstein dairy herd.

This report describes congenital nuclear cataracts and posterior lenticonus in a closed purebred Holstein dairy herd in Canada. Ophthalmic examinations were completed on 30 male and 249 female cattle aged newborn to 10 years old. Nutritional, infectious, and toxic etiologies were investigated. Necropsies of 3 affected calves were performed and eyes of 2 additional affected calves were examined with light microscopy. Bilateral nuclear cataracts were identified in 53/279 (19%) animals. Additional congenital anomalies observed included posterior lenticonus, iris to lens persistent pupillary membranes (n = 7), and lenticular colobomata (n = 1). Heifers did not give birth to calves with congenital nuclear cataracts (0/105), whereas the incidence of affected calves born to multiparous cows was 31% (53/171). The animals with nuclear cataracts ranged from newborn to 8 years old. The cataracts appeared to be non- or minimally progressive. Light microscopic examination of 10 affected globes confirmed nuclear cataract with posterior lenticonus (n = 10). Pedigree analysis was inconclusive. Polymerase chain reaction and sequence analysis for the NID1 gene deletion were negative. The etiology of the congenital lenticular anomalies was not determined.

Cataractes nucléaires congénitales dans un troupeau laitier Holstein. Cette étude rapporte les résultats d'une enquête sur des cataractes nucléaires congénitales et des lenticônes postérieurs dans un troupeau laitier fermé Holstein pur-sang au Canada. Des examens ophtalmiques ont été réalisés sur 30 bovins mâles et 249 bovins femelles qui étaient âgés de nouveau-né à 10 ans. Les étiologies nutritionnelles, infectieuses et toxiques ont été étudiées. Des nécropsies ont été effectuées pour trois veaux touchés et les yeux de deux veaux affectés additionnels ont été examinés à l'aide de microscopie photonique. Des cataractes nucléaires bilatérales ont été identifiées chez 53/279 (19 %) des animaux. Les anomalies congénitales additionnelles observées incluaient le lenticône postérieur, des membranes pupillaires persistantes de l'iris à la lentille (n = 7) et le colobome lenticulaire (n = 1). Les taures n'ont pas donné naissance à des veaux avec des cataractes nucléaires congénitales (0/105), tandis que l'incidence des veaux affectés nés de vaches multipares était de 31 % (53/171). Les animaux avec des cataractes nucléaires étaient âgés de nouveau-né à 8 ans. Les cataractes semblaient être non progressives ou minimalement progressives. Un examen à l'aide d'un microscope photonique de 10 globes affectés a confirmé la cataracte nucléaire avec un lenticône postérieur (n = 10). L'analyse du pedigree a été non-concluante. Une réaction d'amplification en chaîne par la polymérase et une analyse de séquence pour la suppression du gène NID1 ont été négatives. L'étiologie des anomalies lenticulaires congénitales n'a pas été déterminée.(Traduit par Isabelle Vallières).

Domestic violence and the place of fear in mother/baby relationships: "what was I afraid of ? Of making it worse.".

It should come as no surprise that when women who have raised babies in domestic violence come together to discuss the formation of relationships with their babies they raise issues of fear. Yet in current attachment studies about the formation of relationships between women and their babies, knowledge of fear based in lived experiences is undervalued. This article draws on a qualitative study of such experiences to explore ways in which fear impacted on 16 women and their babies. From this study it is discerned that fear impacts in diverse ways on women, babies, and their relationships with each other. Women's experiences show that fear is a complex emotion that cannot be understood outside of context, relations, and subjectivity. Furthermore, fear can be the motivation for protection, whereby actions by women are in the interests of safety of their babies. These insights look beyond attachment theory to the manifestations of and responses to fear identified by women who have raised babies while enduring domestic violence.

Characterization of two pro-opiomelanocortin gene variants and their effects on carcass traits in beef cattle.

BACKGROUND: Carcass quantity (lean meat yield) and quality (degree of marbling) in beef cattle determines much of their economic value. Consequently, it is important to study genes that are part of the appetite pathway and that may ultimately affect carcass composition. Pro-opiomelanocortin is a prohormone that codes for many different peptides, several of which are involved in the appetite pathway. A single nucleotide polymorphism (SNP) c.288C>T in pro-opiomelanocortin (POMC) has previously been associated with hot carcass weight (HCW) and shipping weight (Ship wt) in beef cattle. RESULTS: While developing a commercial real time PCR test for POMC c.288C>T a 12 bp deletion (POMC c.293_304delTTGGGGGCGCGG) was identified. The deletion results in the removal of four amino acids (a valine, two glycines, and an alanine). Both the POMC c.288C>T and the deletion were genotyped in 386 crossbred steers and evaluated for associations with carcass traits. The animals with one copy of the deletion had a significantly smaller carcass rib-eye area (7.91 cm²; P = 0.02) in comparison to homozygous normal animals. Significant associations were observed between POMC c.288C>T with start-of-finishing weight (SOF WT; P = 0.04), hot carcass weight (HCW; P = 0.02), average fat and grade fat (both P = 0.05), carcass rib-eye area (REA; P = 0.03) and marbling (P = 0.02). CONCLUSIONS: These results suggest that it could be beneficial for beef producers to know both the deletion and POMC c.288C>T genotypes when making marketing and culling decisions.

Hypothalamic-pituitary-adrenal axis activation in obstructive sleep apnea: the effect of continuous positive airway pressure therapy.

CONTEXT: Obstructive sleep apnea (OSA) is a common condition with significant cardiovascular and metabolic comorbidity. We hypothesized that these may result from OSA-induced perturbations of endogenous ultradian hypothalamic-pituitary-adrenal axis activity. OBJECTIVE: The aim of the study was to investigate ACTH and cortisol ultradian patterns using an automated, repetitive blood sampling technique. DESIGN: Samples for ACTH and cortisol were collected from 10 patients with moderate to severe OSA under basal conditions, at 10-min intervals over 24 h, at diagnosis and 3 months after compliant continuous positive airway pressure (CPAP) therapy. Multiple-parameter deconvolution estimated specific measures of ACTH and cortisol pulsatile secretion from blood hormone concentrations. RESULTS: Mean total ACTH and cortisol production were elevated pre-CPAP compared to post-CPAP (ACTH, 1459.8 +/- 123.0 vs. 808.1 +/- 97.9 pg/ml, P < 0.001; cortisol, 5748.9 +/- 364.9 vs. 3817.7 +/- 351.7 nmol/liter, P < 0.001) as were mean total pulsatile production (ACTH, 764.1 +/- 86.3 vs. 383.5 +/- 50.0 pg/ml, P = 0.002; cortisol, 4715.9 +/- 253.3 vs. 3227.7 +/- 258.8 nmol/liter, P < 0.001). ACTH and cortisol secretory burst mean half-duration were higher at diagnosis (12.3 +/- 0.7 and 13.5 +/- 0.7 vs. 7.8 +/- 0.4 and 8.4 +/- 0.6 min, respectively, P < 0.001); thus, 95% of each ACTH secretion occurred in 21.0 +/- 1.2 vs. 12.9 +/- 0.8 min post-CPAP (P < 0.001) and for cortisol in 23.0 +/- 1.2 vs. 14.2 +/- 1.1 min post-CPAP (P < 0.001). Approximate entropy (ApEn) revealed greater disorderliness in both ACTH (P = 0.03) and cortisol (P = 0.001) time series pre-CPAP. Forward and reverse cross-ApEn suggested nodal disruption at central and adrenal levels pre-CPAP (P = 0.01). Significantly elevated cortisol responses to a single breath of 35% CO(2) occurred pre-CPAP (P = 0.006). CONCLUSIONS: Untreated compared to treated OSA is associated with marked disturbances in ACTH and cortisol secretory dynamics, resulting in prolonged tissue exposure to disordered, elevated hormone levels.

Plasma apelin levels in obstructive sleep apnea and the effect of continuous positive airway pressure therapy.

Apelin is a peptide hormone with cardiovascular and glucose homeostasis properties, and obstructive sleep apnea (OSA) is complicated by cardiovascular and metabolic comorbidities. Plasma apelin has not been previously assessed in OSA. We investigated the response of plasma apelin to a 2-h 75 g oral glucose tolerance test (OGTT) and the effect of 3 months compliant continuous positive airway pressure (CPAP) therapy in 15 obese males with newly diagnosed OSA. Plasma apelin and serum cortisol were recorded 10 minutely, while serum insulin and glucose were measured 30 minutely. Ten subjects had plasma apelin measured at intervals across a 24-h period to investigate for circadian variation in apelin levels, and this was repeated following 3 months compliant CPAP therapy. Fasting (0.342+/-0.038 vs 0.288+/-0.024 ng/ml, P=0.04), 30 min (0.399+/-0.035 vs 0.312+/-0.036 ng/ml, P=0.007) and 120 min (0.402+/-0.030 vs 0.259+/-0.024 ng/ml, P<0.001) apelin levels were reduced following CPAP. The area under curve for apelin OGTT response was lower post-CPAP (44.1+/-3.3 vs 35.8+/-2.3 ng/ml per min, P<0.001). Mean OGTT apelin levels showed a significant treatment effect (P=0.006) and a time effect (P<0.001), and the effect of time was different pre- versus post-CPAP (P=0.005). No significant variability in apelin levels existed across the 24-h period at diagnosis. Lower levels were evident overnight following treatment (P=0.004). Improvements in insulin and glucose parameters and reduced cortisol levels were found post-CPAP. In summary, untreated OSA was associated with elevated plasma apelin levels, altered apelin secretory dynamics in response to oral glucose and lack of an apparent circadian variability, which was restored following CPAP.

Central hypoventilation with PHOX2B expansion mutation presenting in adulthood.

The case history is described of a 41 year old male who presented with acute-on-chronic respiratory failure due to PHOX2B mutation-associated central hypoventilation with a quantified impaired response to hypercapnia. He was successfully treated with non-invasive ventilation, which has been continued nocturnally at home.

Genetic diversity in a feral horse population from Sable Island, Canada.

The present-day Sable Island horse population, inhabiting an island off the eastern coast of Canada, is believed to have originated mainly from horses confiscated from the early French settlers in Nova Scotia in the latter half of the 18th century. In 1960, the Sable Island horses were given legal protected status and no human interference has since been allowed. The objective of this study was to characterize the current genetic diversity in Sable Island horses in comparison to 15 other horse breeds commonly found in Canada and 5 Spanish breeds. A total of 145 alleles from 12 microsatellite loci were detected in 1093 horses and 40 donkeys. The average number of alleles per locus ranged from 4.67 in the Sable Island horse population to 8.25 in Appaloosas, whereas the mean observed heterozygosity ranged from 0.626 in the Sable Island population to 0.787 in Asturcons. Various genetic distance estimates and clustering methods did not permit to support that the Sable Island horses originated from shipwrecked Spanish horses, according to a popular anecdote, but closely resemble light draft and multipurpose breeds commonly found in eastern Canada. Based on the Weitzman approach, the loss of the Sable Island horse population to the overall diversity in Canada is comparable or higher than any other horse breed. The Sable Island horse population has diverged enough from other breeds to deserve special attention by conservation interest groups.

Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels.

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.