Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.     

Exenatide does not evoke pancreatitis and attenuates chemically induced pancreatitis in normal and diabetic rodents

The risk of developing pancreatitis is elevated in type 2 diabetes and obesity. Cases of pancreatitis have been reported in type 2 diabetes patients treated with GLP-1 (GLP-1R) receptor agonists. To examine whether the GLP-1R agonist exenatide potentially induces or modulates pancreatitis, the effect of exenatide was evaluated in normal or diabetic rodents. Normal and diabetic rats received a single exenatide dose (0.072, 0.24, and 0.72 nmol/kg) or vehicle. Diabetic ob/ob or HF-STZ mice were infused with exenatide (1.2 and 7.2 nmol·kg(-1)·day(-1)) or vehicle for 4 wk. Post-exenatide treatment, pancreatitis was induced with caerulein (CRN) or sodium taurocholate (ST), and changes in plasma amylase and lipase were measured. In ob/ob mice, plasma cytokines (IL-1β, IL-2, IL-6, MCP-1, IFNγ, and TNFα) and pancreatitis-associated genes were assessed. Pancreata were weighed and examined histologically. Exenatide treatment alone did not modify plasma amylase or lipase in any models tested. Exenatide attenuated CRN-induced release of amylase and lipase in normal rats and ob/ob mice but did not modify the response to ST infusion. Plasma cytokines and pancreatic weight were unaffected by exenatide. Exenatide upregulated Reg3b but not Il6, Ccl2, Nfkb1, or Vamp8 expression. Histological analysis revealed that the highest doses of exenatide decreased CRN- or ST-induced acute inflammation, vacuolation, and acinar single cell necrosis in mice and rats, respectively. Ductal cell proliferation rates were low and similar across all groups of ob/ob mice. In conclusion, exenatide did not modify plasma amylase and lipase concentrations in rodents without pancreatitis and improved chemically induced pancreatitis in normal and diabetic rodents.     

CART peptide: central mediator of leptin-induced adipose tissue apoptosis?

Because of connections between CART peptide containing neurons and the sympathetic nervous system (SNS) and the possible role of the SNS in leptin-induced adipose apoptosis, CART may act as a downstream effector of leptin-induced adipose apoptosis. Male Sprague-Dawley rats received continuous intracerebroventricular (i.c.v.) infusion for 4 days of either artificial cerebrospinal fluid (aCSF, 12 microl/day), leptin (15 microg/day), or CART55-102 at 2.4 microg/day (CART2.4) or 9.6 microg/day (CART9.6). Food intake (FI) was decreased 10.8% for CART2.4, 41.9% for CART9.6 and 33.4% for leptin (p<0.05). CART9.6 and leptin reduced meal size and meal number. Body weight (BW) was reduced by CART9.6 (14.6%) and leptin (11.6%) (p<0.05), but not by CART2.4. CART9.6 and CART2.4, but not leptin, caused hypothermia, and CART9.6 inhibited physical activity (p<0.05). Epididymal, inguinal and retroperitoneal fat pad weights were reduced (p<0.05) by both CART treatments and leptin; CART9.6 also reduced gastrocnemius muscle weight (18.1%, p<0.05). Leptin, but not CART, increased serum free fatty acid concentrations by 31.1% (p<0.05) and increased adipose apoptosis by 48% (p<0.05). These data show that although leptin and CART55-102 have some similar actions, CART55-102 is probably not a mediator for leptin-induced adipose apoptosis in the brain.     

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic β-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP’s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable β-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.     

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane‐rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast‐targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici‐populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora‐specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N‐terminal signal‐dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant‐specific sorting signals to traffic within plant cells.     

Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance

Background

Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial ‘set-point’ may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the ‘reference range of normality’ remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport.

Methodology/Principal Findings

Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm²), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 μA/cm²; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport.

Conclusions/Significance

Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine.     

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

Age and sex differentially affect regional changes in one repetition maximum strength

To assess the influences of age and sex on regional changes in 1 repetition maximum (1RM) strength, 10 young men (20-30 years), 8 young women (20-30 years), 11 older men (65- 75 years), and 10 older women (65-75 years) were studied before and after a 24-week whole-body strength training program. Changes in 1RM strength were analyzed for each individual exercise, as well as by calculating a total body score (TBS), an upper body score (UBS), and a lower body score (LBS). The effect of age and sex on changes in 1RM strength was analyzed using a repeated measures analysis of variance. When changes in strength for individual exercises were analyzed, the chest press, lat pulldown, shoulder press, and triceps pushdown were affected by both age (p < 0.05) and sex (p < 0.05), while the biceps curls were only influenced by age (p < 0.05). For the lower body, the leg press changes in 1RM strength were influenced by age (p < 0.0001), while leg extension was influenced by sex (p < 0.05). Total body score, UBS, and LBS showed significant increases with 24 weeks of ST (p < 0.001, all). Changes in TBS and UBS were affected by age (p < 0.001, both) and sex (p < 0.05 and p < 0.001, respectively). Younger subjects showed a greater increase in strength than older subjects, and men showed a greater increase in strength compared with women. Changes in LBS were affected by age (p < 0.001), with younger subjects showing a greater increase in strength compared with the older subjects, but not by sex (p = 0.464). These data indicate that regional increases in strength are differentially affected by age and sex.