The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.     

‘None of my ancestors ever discussed this disease before!’ How disease information shapes adaptive capacity of marginalised rural populations in India

Smallholder farmer and tribal communities are often characterised as marginalised and highly vulnerable to emerging zoonotic diseases due to their relatively poor access to healthcare, worse-off health outcomes, proximity to sources of disease risks, and their social and livelihood organisation. Yet, access to relevant and timely disease information that could strengthen their adaptive capacity remain challenging and poorly characterised in the empirical literature. This paper addresses this gap by exploring the role of disease information in shaping the adaptive capacity of smallholder farmer and tribal groups to Kyasanur Forest Disease (KFD), a tick-borne viral haemorrhagic fever. We carried out household surveys (n = 229) and in-depth interviews (n = 25) in two affected districts–Shimoga and Wayanad–in the Western Ghats region.Our findings suggest that, despite the generally limited awareness about KFD, access to disease information improved households’ propensity to implement adaptation strategies relative to households that had no access to it. Of the variety of adaptation strategies implemented, vaccination, avoiding forest visits, wearing of protective clothing and footwear, application of dimethyl phthalate (DMP) oil and income diversification were identified by respondents as important adaptive measures during the outbreak seasons. Even so, we identified significant differences between individuals in exposure to disease information and its contribution to substantive adaptive action. Households reported several barriers to implement adaptation strategies including, lack of disease information, low efficacy of existing vaccine, distrust, religio-cultural sentiments, and livelihood concerns. We also found that informal information sharing presented a promising avenue from a health extension perspective albeit with trade-offs with potential distortion of the messages through misinformation and/or reporting bias. Altogether, our findings stress the importance of contextualising disease information and implementing interventions in a participatory way that sufficiently addresses the social determinants of health in order to bolster households’ adaptive capacity to KFD and other neglected endemic zoonoses.     

Liquid-based vs. conventional smears in fine needle aspiration of bone and soft tissue tumors

OBJECTIVE: To compare the accuracy of fine needle aspiration cytology of bone and soft tissue tumors utilizing ThinPrep (TP) (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) vs. conventional smears (CS). STUDY DESIGN: Fine needle aspiration cytology from bone and soft tissue tumors was processed and assessed for cellularity, nuclear and cytoplasmic preservation, cellular architecture and stromal background with both the TP liquid-based smear technique and conventional methods. RESULTS: An accurate diagnosis was made in 13% of TP cases as compared to 64% in CS cases. CONCLUSION: CS of fine needle aspiration sample is far superior to TP in diagnosing tumors of bone and soft tissues. Preservation of cytoplasmic features and cellular architecture was superior in conventionally prepared smears.     

Bioluminescence imaging of period1 gene expression in utero

The use of real-time reporters has accelerated our understanding of gene expression in vivo. This study examined the feasibility of a luciferase-based reporter to image spatiotemporal changes in fetal gene expression in utero. We chose to monitor Period1 (Per1) because it is expressed broadly in the body and plays a role in circadian rhythmicity. Using rats carrying a Per1::luc transgene, we repetitively imaged fetuses in utero throughout gestation. We found that bioluminescence was specific to transgenic pups, increased dramatically on embryonic day 10 (10 days after successful mating), and continued to increase logarithmically until birth. Diurnal fluctuations in Per1 expression were apparent several days prior to birth. These results demonstrate the feasibility of in utero imaging of mammalian gene expression, tracking of fetal gene expression from the same litter, and early detection of mammalian clock gene expression. We conclude that luciferase-based reporters can provide a sensitive, noninvasive measure of in utero gene expression.     

Preparation of photoluminescent nano-biocomposite nacre from graphene oxide and polylactic acid

Nano-biocomposites of inorganic and organic components wereprepared to produce long-persistent phosphorescent artificial nacre-like materials. Biodegradable polylactic acid (PLA), graphene oxide (GO), and nanoparticles (13–20 nm) of lanthanide-doped aluminate pigment (NLAP) were used in a simple production procedure of an organic/inorganic hybrid nano-biocomposite. Both polylactic acid and GO nanosheets were chemically modified to form covalent and hydrogen bonding. The high toughness, good tensile strength, and great endurance of those bonds were achieved by their interactions at the interfaces. Long-persistent and reversible photoluminescence was shown by the prepared nacre substrates. Upon excitation at 365 nm, the nacre substrates generated an emission peak at 517 nm. When ultraviolet light was shone on luminescent nacres, they displayed a bright green colour. The high superhydrophobicity of the generated nacres was obtained without altering their mechanical characteristics.     

Production of islet-like structures from neonatal porcine pancreatic tissue in suspension bioreactors

The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i.e., aggregates or islet-like structures) in suspension bioreactors. Key issues addressed included (i) serum-free media, (ii) cell inoculation density, (iii) medium pH, and (iv) aggregate dissociation. Suspension bioreactors were inoculated with pancreatic neonatal tissue and operated under controlled conditions for a 9-day period. Medium studies showed that a new serum-free medium developed in our laboratory was capable of supporting endocrine cell expansion. An inoculation density of 127,000 cells/mL resulted in more than a 7.5-fold increase in the number of insulin-positive cells after 9 days. The resulting population consisted of single cells and many islet-like aggregates that contained all of the endocrine cell types (including insulin-positive, glucagon-positive, somatostatin-positive, and pancreatic polypeptide-positive cells). Furthermore, the cell aggregates exhibited a glucose-responsive behavior. This study represents a significant milestone on the path to the effective expansion of human islet-like tissue in bioreactors that may be used for cell therapy to treat Type 1 diabetes.     

Pharmacokinetics of theanine enantiomers in rats

Theanine, first discovered in tea, is a chiral nonproteinic amino acid that has been reported to have cardiovascular, neurological, and oncological effects. It is being considered as a therapeutic/medicinal agent and additive in consumer products. The present study evaluated the pharmacokinetics of D-theanine, L-theanine, and D,L-theanine in plasma and urine using LC-ESI/MS in rats after oral (p.o.) and intraperitoneal (i.p.) administration. Oral administration data indicated that gut absorption of d-theanine was far less than that of L-theanine. However, after i.p. administration, plasma theanine concentrations of L- and D-theanine were similar. This indicated that D- and L-theanine may exhibit a competitive effect with respect to intestinal absorption. Regardless of the route of administration, p.o. or i.p., the presence of the other enantiomer always decreased theanine plasma concentrations, indicating D,L-theanine competition with respect to urinary reabsorption. Data on urinary concentrations of D-theanine suggested that the D-isomer may be eliminated with minimal metabolism. L-Theanine appeared to be preferentially reabsorbed and metabolized by the kidney while D-theanine was preferentially excreted. Clearly, the bioequivalencies of D,L-theanine and its enantiomers were found to be quite different from one another. Consequently, the efficacy of commercial theanine products containing D-theanine, L-theanine, or D,L-theanine may be quite different.     

Sugar and alcohol molecules provide a therapeutic strategy for the serpinopathies that cause dementia and cirrhosis

Mutations in neuroserpin and alpha1-antitrypsin cause these proteins to form ordered polymers that are retained within the endoplasmic reticulum of neurones and hepatocytes, respectively. The resulting inclusions underlie the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) and Z alpha1-antitrypsin-associated cirrhosis. Polymers form by a sequential linkage between the reactive centre loop of one molecule and beta-sheet A of another, and strategies that block polymer formation are likely to be successful in treating the associated disease. We show here that glycerol, the sugar alcohol erythritol, the disaccharide trehalose and its breakdown product glucose reduce the rate of polymerization of wild-type neuroserpin and the Ser49Pro mutant that causes dementia. They also attenuate the polymerization of the Z variant of alpha1-antitrypsin. The effect on polymerization was apparent even when these agents had been removed from the buffer. None of these agents had any detectable effect on the structure or inhibitory activity of neuroserpin or alpha1-antitrypsin. These data demonstrate that sugar and alcohol molecules can reduce the polymerization of serpin mutants that cause disease, possibly by binding to and stabilizing beta-sheet A.     

Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7

Expression of an oncogene in a primary cell can, paradoxically, block proliferation by inducing senescence or apoptosis through pathways that remain to be elucidated. Here we perform genome-wide RNA-interference screening to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of human primary fibroblasts and melanocytes. Surprisingly, we find a secreted protein, IGFBP7, has a central role in BRAFV600E-mediated senescence and apoptosis. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAFV600E-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses growth of BRAFV600E-positive tumors in xenografted mice. Immunohistochemical analysis of human skin, nevi, and melanoma samples implicates loss of IGFBP7 expression as a critical step in melanoma genesis.