Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes.

Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We find that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin. In iPSC-derived human neurons, knockin of the strongly hyperactive LRRK2-p.R1441H mutation causes striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knockout of the opposing protein phosphatase 1H (PPM1H) phenocopies the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuates transport defects in both p.R1441H knockin and PPM1H knockout neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive "tug-of-war" between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy.

The potential use of skin and liver as biomarkers to estimate mercury in the brain, kidney, and muscle of bottlenose dolphins (Tursiops truncatus).

For marine cetaceans, Hg biomagnification can negatively affect neurological, hepatic, renal, and immune functions. To evaluate the use of biomarkers for Hg in dolphins, multiple tissues were analyzed from 127 stranded common bottlenose dolphins (Tursiops truncatus) from the estuarine and oceanic waters of Virginia, USA. Twenty-two percent of liver Hg concentrations exceeded the published observed effect level for liver abnormalities, and 26 % of cerebrum samples exceeded the published threshold for neurochemical changes, suggesting that Hg may have impacted dolphin health. Mercury tissue levels were similar to or lower than those reported from other locations (liver range: 1.4-943 µg/g-dw). Significant correlations were found between tissue types, indicating that skin or liver can be used as a biomarker to estimate the total Hg concentrations in the other tissue types (kidney, liver, cerebrum, cerebellum, pons). This is the first study to measure Hg concentrations in multiple brain regions of T. truncatus.

Exploring and Mapping Screening Tools for Cognitive Impairment and Traumatic Brain Injury in the Homelessness Context: A Scoping Review.

Cognitive impairment is common amongst people experiencing homelessness, yet cognitive screening and the collection of history of brain injury rarely features in homelessness service delivery practice. The purpose of this research was to scope and map strategies for screening for the potential presence of cognitive impairment or brain injury amongst people experiencing homelessness and identify instruments that could be administered by homelessness service staff to facilitate referral for formal diagnosis and appropriate support. A search was conducted across five databases, followed by a hand search from relevant systematic reviews. A total of 108 publications were included for analysis. Described in the literature were 151 instruments for measuring cognitive function and 8 instruments screening for history of brain injury. Tools that were described in more than two publications, screening for the potential presence of cognitive impairment or history of brain injury, were included for analysis. Of those regularly described, only three instruments measuring cognitive function and three measuring history of brain injury (all of which focused on traumatic brain injury (TBI)) may be administered by non-specialist assessors. The Trail Making Test (TMT) and the Ohio State University Traumatic Brain Injury Identification Method (OSU TBI-ID) are both potentially viable tools for supporting the identification of a likely cognitive impairment or TBI history in the homelessness service context. Further population-specific research and implementation science research is required to maximise the potential for practice application success.

A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function.

At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 caused levamisole hypersensitivity and had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. We also examined other genes that resulted in a levamisole-hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR-dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

Screening for Geriatric Syndromes: Falls, Urinary/Fecal Incontinence, and Osteoporosis.

The geriatric syndromes of falls, incontinence, and osteoporosis are concerns in older adults because of their potential impact on quality of life. Asking about history of falls or a fear of falling should prompt a multifactorial assessment of fall risk and targeted interventions to reduce falls. Urinary and fecal incontinence should be screened because they are common conditions that are underreported due to embarrassment and general perception that incontinence is a normal part of aging. Women over age 65, men over age 70, and younger patients with high-risk characteristics should be screened with bone mineral density testing with dual-energy x-ray absorptiometry.

Incubation of food craving is independent of macronutrient composition.

Cues previously paired with rewarding stimuli induce a time-dependent increase in the motivational craving state (incubation of craving). Whether there is an increase in craving for high-fat (HF) food over time, which may contribute to overeating and obesity, has not been determined. We hypothesized that cues paired with HF pellets would elicit a greater incubation of craving effect than those paired with standard chow (SC) pellets. Rats exposed to cues associated with either HF or SC pellets demonstrated equivalent levels of craving over an abstinence period of 30 days. Diet preference tests between SC pellets and LabDiet revealed that SC pellets were preferred over LabDiet. Rats reared on SC pellets exclusively, did not display incubation of craving for SC pellets, suggesting that prior history with the food plays an important role in cue-induced seeking behavior. Results identified cues previously associated with food undergo a comparable magnitude of incubation of craving. When ingestive behavior was measured after 30 days of abstinence, rats significantly increased their consumption of HF pellets. Our results indicate that food cues gain importance over time, trigger increased approach behaviors, and increased consumption of HF food following abstinence. This may contribute to overeating and the development of obesity.

The Quality of School Physical Activity Policies Within Maryland and Virginia.

BACKGROUND: Since the adoption of the Healthy, Hunger-Free Kids Act of 2010, many researchers have examined changes in the school nutrition environment; however, far less research has focused on the evaluation of physical activity (PA) policies within public schools. METHODS: School district wellness policies (n = 144) of Virginia and Maryland were coded using a previously validated audit tool with a scale of 0 (weakest, least comprehensive) to 1 (strongest, most comprehensive). RESULTS: Mean policy strength was weak (.20 ± .15), and, on average, policies were moderately comprehensive (.40 ± .22). The strongest (.73 ± .44) and most comprehensive (.79 ± .40) policy subgroup addressed daily recess in elementary schools. Virginia had significantly higher scores in 9 policy groups, while Maryland had higher significant policy scores in the 2 following groups: (1) the strength and comprehensiveness of a written physical education (PE) curriculum for each grade level (Ps < .05) and (2) the strength and comprehensiveness of addressing the use of PE waivers (Ps < .05). CONCLUSIONS: PA wellness policies in Maryland and Virginia are extremely weak and only moderately comprehensive; it is unlikely that these policies will significantly influence school-based PA.

Babesia microti infection, eastern Pennsylvania, USA.

Infection with Babesia microti has not been well-described in eastern Pennsylvania, USA, despite the vector of this organism being prevalent. We report 3 cases of babesiosis in eastern Pennsylvania in persons without recent travel outside the region or history of blood transfusions, suggesting emergence of this infection.

School wellness policies: effects of using standard templates.

BACKGROUND: Public school policies related to physical activity and nutrition recently have become the focal point for policymakers to evaluate the effect of regulations on the childhood obesity epidemic. State school board associations have begun to provide school districts templates for wellness policies, and little research exists that evaluates the effect of a template on the strength and comprehensiveness of these policies. PURPOSE: To determine the strength and comprehensiveness of school wellness policies developed using a standard template when compared to those that do not. METHODS: In 2011, a random sample of wellness policies from school districts in Virginia (ten locally developed wellness policies and ten template-based policies) was coded using a previously validated audit tool for strength and comprehensiveness. Data were reduced to a scale ranging from 0 to 1, with higher scores representing stronger and more-comprehensive policies, and compared using t-tests. RESULTS: Overall, only 17% of school wellness policies met all federal requirements. On average, locally developed policies met five of six federal requirements, whereas VSBA policies met four of six, t(2, 21)=2.161, p<0.05. Both types of policies were ranked on a scale from 0 (weakest) to 1 (strongest); both types were weak (M=0.16±0.13) and only mildly comprehensive (M=0.37±0.16). There was a difference in policy comprehensiveness and strength between locally developed policies and template-based policies. Locally developed policies were stronger, t(2, 21)= -1.82, p<0.05, and more comprehensive, t(2, 21)= -2.5, p<0.05, than template-based policies. CONCLUSIONS: In this sample, locally developed policies were stronger than template-based policies. If replicated in large studies, these findings suggest that further research is needed about how best to support schools that wish to develop school wellness policies.

SHP-2/PTPN11 mediates gliomagenesis driven by PDGFRA and INK4A/ARF aberrations in mice and humans.

Recent collaborative efforts have subclassified malignant glioblastomas into 4 clinical relevant subtypes based on their signature genetic lesions. Platelet-derived growth factor receptor α (PDGFRA) overexpression is concomitant with a loss of cyclin-dependent kinase inhibitor 2A (CDKN2A) locus (encoding P16INK4A and P14ARF) in a large number of tumors within one subtype of glioblastomas. Here we report that activation of PDGFRα conferred tumorigenicity to Ink4a/Arf-deficient mouse astrocytes and human glioma cells in the brain. Restoration of p16INK4a but not p19ARF suppressed PDGFRα-promoted glioma formation. Mechanistically, abrogation of signaling modules in PDGFRα that lost capacity to bind to SHP-2 or PI3K significantly diminished PDGFRα-promoted tumorigenesis. Furthermore, inhibition of SHP-2 by shRNAs or pharmacological inhibitors disrupted the interaction of PI3K with PDGFRα, suppressed downstream AKT/mTOR activation, and impaired tumorigenesis of Ink4a/Arf-null cells, whereas expression of an activated PI3K mutant rescued the effect of SHP-2 inhibition on tumorigenicity. PDGFRα and PDGF-A are coexpressed in clinical glioblastoma specimens, and such co-expression is linked with activation of SHP-2/AKT/mTOR signaling. Together, our data suggest that in glioblastomas with Ink4a/Arf deficiency, overexpressed PDGFRα promotes tumorigenesis through the PI3K/AKT/mTOR-mediated pathway regulated by SHP-2 activity. These findings functionally validate the genomic analysis of glioblastomas and identify SHP-2 as a potential target for treatment of glioblastomas.

Sweet cues: How heparan sulfate modification of fibronectin enables growth factor guided migration of embryonic cells.

Growth factors regulate a diverse array of cellular functions including proliferation, survival, movement, and the ability to do this often involves interactions with the extracellular matrix (ECM) and particularly heparan sulfate proteoglycans (HSPGs). HSPGs have been shown to sequester growth factors, and to act as growth factor co-receptors or receptors themselves. Recent studies, however, have revealed a new role for HSPGs in mediating the interactions of growth factors with the ECM. Specifically, heparan sulfate has been shown to modulate fibronectin structure to reveal previously masked growth factor binding sites. In vivo, this mechanism appears to control the guidance of migrating cells during embryonic development as HSPG-modification of fibronectin enables direct platelet derived growth factor-fibronectin interactions necessary for this process. A model based on this observation is discussed here as well as the possibility that other growth factors/morphogens utilize similar mechanisms involving fibronectin or additional ECM proteins.

PDGF-A interactions with fibronectin reveal a critical role for heparan sulfate in directed cell migration during Xenopus gastrulation.

Platelet-derived growth factor (PDGF) signaling is essential for processes involving cell motility and differentiation during embryonic development in a wide variety of organisms including the mouse, frog, zebrafish, and sea urchin. In early Xenopus laevis embryos, PDGF-AA provides guidance cues for the migration of anterior mesendoderm cells as they move across a fibronectin-rich extracellular matrix. The long form of PDGF-A includes a positively charged carboxyl-terminal retention motif that can interact with the extracellular matrix and heparan sulfate proteoglycans (HSPGs). In this study we demonstrate that PDGF-AA binds directly to fibronectin and that this association is greatly enhanced by heparin. The PDGF-AA-fibronectin binding occurs across a broad range of pHs (5.5-9), which is significant because the PDGF-guided migration of Xenopus mesendoderm cells occurs under basic extracellular conditions (pH 8.4). We further demonstrate that endogenous HSPG's are required for the PDGF-AA-guided mesendoderm movement, suggesting an in vivo role for HSPGs in mediating the interaction between PDGF-AA and fibronectin.

Feeding Drosophila a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns.

Energy restriction increases stress resistance and lifespan in Drosophila melanogaster and other species. The roles of individual nutrients in stress resistance and longevity are largely unknown. The vitamin biotin is a potential candidate for mediating these effects, given its known roles in stress signaling and gene regulation by epigenetic mechanisms, i.e. biotinylation of histones. Here, we tested the hypothesis that prolonged culture of Drosophila on biotin-deficient (BD) medium increases stress resistance and lifespan. Flies were fed a BD diet for multiple generations; controls were fed a biotin-normal diet. In some experiments, a third group of flies was fed a BD diet for 12 generations and then switched to control diets for 2 generations to eliminate potential effects of short-term biotin deficiency. Flies fed a BD diet exhibited a 30% increase in lifespan. This increase was associated with enhanced resistance to the DNA-damaging agent hydroxyurea and heat stress. Also, fertility increased significantly compared with biotin-normal controls. Biotinylation of histones was barely detectable in biotin-deprived flies, suggesting that epigenetic events might have contributed to effects of biotin deprivation.

Mutation of smooth muscle myosin causes epithelial invasion and cystic expansion of the zebrafish intestine.

Zebrafish meltdown (mlt) mutants develop cystic expansion of the posterior intestine as a result of stromal invasion of nontransformed epithelial cells. Positional cloning identified zebrafish smooth muscle myosin heavy chain (myh11) as the responsible gene. The mlt mutation constitutively activates the Myh11 ATPase, which disrupts smooth muscle cells surrounding the posterior intestine. Adjacent epithelial cells ectopically express metalloproteinases, integrins, and other genes implicated in human cancer cell invasion. Knockdown and pharmacological inhibition of these genes restores intestinal structure in mlt mutants despite persistent smooth muscle defects. These data identify an essential role for smooth muscle signaling in the maintenance of epithelial architecture and support gene expression analyses and other studies that identify a role for stromal genes in cancer cell invasion. Furthermore, they suggest that high-throughput screens to identify regulators of cancer cell invasion may be feasible in zebrafish.

Intestinal growth and differentiation in zebrafish.

Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.

DNA hypermethylation near the transcription start site of collagen alpha2(I) gene occurs in both cancer cell lines and primary colorectal cancers.

Collagen production plays a significant role in tumor development, especially in breast cancer, hepatocarcinomas, and colorectal carcinoma. However, collagen production is decreased during oncogenic transformation of cells in culture. This study demonstrates that methylation of the collagen alpha2(I) gene transcription start site occurs frequently in human cancer cell lines (9 of 10), including breast cancer cell lines (MCF-7 and Hs578T), hepatocellular carcinoma cell lines (SNU387, SNU449, SNU398, and PLC/PRF/5), a fibrosarcoma cell line (HT1080), and colorectal carcinoma cell lines (HCT116, SW480, and SW620). In addition, the collagen gene is more methylated in colorectal cancer tissues compared with normal mucosa. The increased DNA methylation of the collagen gene in cell lines is inversely correlated with collagen mRNA steady-state levels. Most importantly, treatment of fibrosarcoma or breast carcinoma cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, resulted in lower methylation and reactivation of the collagen gene in a dose-responsive manner. This is the first demonstration that the collagen alpha2(I) gene is methylated in multiple cancer cell lines correlating with loss of collagen expression and also methylated in primary cancer tissues. These data also suggest that methylation-induced repression of collagen transcription may be a frequent occurrence in cancer.