International Health Security: A Summative Assessment by ACAIM Consensus Group

International health security (IHS) encompasses any natural or anthropogenic occurrence that can threaten the safety of human health and well-being. The American College of Academic International Medicine IHS Consensus Group (ACAIM-CG) developed a summative assessment highlighting the main issues that can impact IHS including emerging infectious diseases; chronic health conditions; bioterrorism; planetary changes (volcanic eruptions, earthquakes, wildfires, and climate change); nuclear incidents; information and cyber health; industrialization; globalization; pharmaceutical production; and communication platforms (social media). These concerns can directly and indirectly impact IHS both in the long and short term. When considering IHS, we aim to emphasize the utility of applying a predefined framework to effectively approach health security threats. This framework comprises of prevention, detection, assessment, reporting, response, addressing needs, and the perpetual repetition of the above cycle (inclusive of appropriate mitigation measures). It is hoped that this collective work will provide a foundation for further research within the redefined, expanded scope of IHS

Regulation of excitation and inhibition balance in C. elegans

Proper neurological function requires a regulated balance of excitation and inhibition in the nervous system. An imbalance of these two forms of neuronal transmission is a hallmark of the neurological disorder, epilepsy. In this dissertation I describe my work on a C. elegans model mimicking the neurological condition of epilepsy. A gain of function mutation in the acetylcholine receptor subunit gene, acr-2, causes increased cholinergic excitation and decreased GABAergic inhibition, resulting in whole body convulsions. Through screening for genetic suppressors of this mutant the composition of the ACR-2 receptor was determined. Further studies of a unique genetic suppressor identified a key role for TRPM channels and systemic ion homeostasis in the control of excitation and inhibition imbalance. Lastly, I found that the cholinergic motor neurons use neuropeptides to alleviate the severity of convulsions caused by the acr-2(gf) mutation. My thesis work identifies a new model for studying excitation and inhibition balance in vivo. While traditional work on neuronal network balance has focused on fast excitatory or inhibitory transmitter systems my work highlights the role of nonneuronal and neuromodulatory regulation of this balanc

alms1 mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants.

Multiple cilia-associated genes have been shown to affect hair cells in zebrafish (Danio rerio), including the human deafness gene dcdc2, the radial spoke gene rsph9, and multiple intraflagellar transport (IFT) and transition zone genes. Recently a zebrafish alms1 mutant was generated. The ALMS1 gene is the gene mutated in the ciliopathy Alström Syndrome a disease that causes hearing loss among other symptoms. The hearing loss seen in Alström Syndrome may be due in part to hair cell defects as Alms1 mutant mice show stereocilia polarity defects and a loss of hair cells. Hair cell loss is also seen in postmortem analysis of Alström patients. The zebrafish alms1 mutant has metabolic defects similar to those seen in Alström syndrome and Alms1 mutant mice. We wished to investigate if it also had hair cell defects. We, however, failed to find any hair cell related phenotypes in alms1 mutant zebrafish. They had normal lateral line hair cell numbers as both larvae and adults and normal kinocilia formation. They also showed grossly normal swimming behavior, response to vibrational stimuli, and FM1-43 loading. Mutants also showed a normal degree of sensitivity to both short-term neomycin and long-term gentamicin treatment. These results indicate that cilia-associated genes differentially affect different hair cell types

Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics.

The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca(2+) entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca(2+) entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone. DOI: http://dx.doi.org/10.7554/eLife.01180.001

Neuropeptides function in a homeostatic manner to modulate excitation-inhibition imbalance in C. elegans.

Neuropeptides play crucial roles in modulating neuronal networks, including changing intrinsic properties of neurons and synaptic efficacy. We previously reported a Caenorhabditis elegans mutant, acr-2(gf), that displays spontaneous convulsions as the result of a gain-of-function mutation in a neuronal nicotinic acetylcholine receptor subunit. The ACR-2 channel is expressed in the cholinergic motor neurons, and acr-2(gf) causes cholinergic overexcitation accompanied by reduced GABAergic inhibition in the locomotor circuit. Here we show that neuropeptides play a homeostatic role that compensates for this excitation-inhibition imbalance in the locomotor circuit. Loss of function in genes required for neuropeptide processing or release of dense core vesicles specifically modulate the convulsion frequency of acr-2(gf). The proprotein convertase EGL-3 is required in the cholinergic motor neurons to restrain convulsions. Electrophysiological recordings of neuromuscular junctions show that loss of egl-3 in acr-2(gf) causes a further reduction of GABAergic inhibition. We identify two neuropeptide encoding genes, flp-1 and flp-18, that together counteract the excitation-inhibition imbalance in acr-2(gf) mutants. We further find that acr-2(gf) causes an increased expression of flp-18 in the ventral cord cholinergic motor neurons and that overexpression of flp-18 reduces the convulsion of acr-2(gf) mutants. The effects of these peptides are in part mediated by two G-protein coupled receptors, NPR-1 and NPR-5. Our data suggest that the chronic overexcitation of the cholinergic motor neurons imposed by acr-2(gf) leads to an increased production of FMRFamide neuropeptides, which act to decrease the activity level of the locomotor circuit, thereby homeostatically modulating the excitation and inhibition imbalance

Neuropeptide modulation primarily affects GABAergic neuromuscular transmission.

<p>(A) Shown are the electrophysiology recording data on the neuromuscular junctions. Top panels are representative traces of genotypes indicated. Middle panels are mean rates of endogenous EPSCs and IPSCs; and bottom panels are cumulative fractions of the animal number with endogenous EPSC rate or IPSC rate less than indicated values in X-axis of genotype indicated. (B) Mean amplitudes of endogenous EPSCs and IPSCs from genotypes shown in A. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Statistics, two-tailed t-test, <i>***</i>, <i>p</i><0.001; <i>*</i>, <i>p</i><0.05.</p

Loss of both <i>flp-1</i> and <i>flp-18</i> enhances <i>acr-2(gf)</i> convulsions.

<p>Null mutants of candidate neuropeptide genes were tested for effects on <i>acr-2(gf)</i> convulsions. <i>flp-18(lf)</i> indicates <i>flp-18(tm2179)</i>; the allele number for other genes are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003472#s4" target="_blank">materials and methods</a>. No significant effects were observed for selected FMRF-amide (<i>flp</i>) (A), neuropeptide like proteins (<i>nls</i>) (B), or insulins (<i>ins</i>) (C). (D) Double mutants of candidate peptide genes with <i>flp-18</i>. Loss of both <i>flp-1</i> and <i>flp-18</i> leads to a significant enhancement of acr-2(gf) convulsions. Numbers in the graph indicate sample sizes. Mean convulsion frequencies are shown. Error bars indicate SEM. Statistics: *: <i>p</i><0.05 by ANOVA and Dunnett's post hoc test.</p

<i>flp-1</i> and <i>flp-18</i> act as inhibitory neuropeptides in the <i>acr-2(gf)</i> background.

<p>(A) Convulsion frequency of <i>acr-2(gf)</i> in combination with loss of function (lf) mutations in <i>flp-1(yn4)</i>, <i>flp-18(tm2179)</i>, or <i>flp-18(db99)</i>. The enhanced convulsion frequency of <i>flp-1(lf); flp-18(lf) acr-2(gf)</i> animals is rescued with transgenic expression of <i>flp-1</i> under the pan-neuronal promoter <i>Prgef-1</i>. Two independent transgenic lines were tested as indicated by line 1 and 2. Mean convulsion frequencies are shown. Error bars indicate SEM. Numbers in the graph indicate sample sizes. Statistics: ***: <i>p</i><0.001, **: <i>p</i><0.01 by ANOVA and Dunnett's post-hoc test. (B, C) Rate of paralysis on 150 µM aldicarb plates in <i>acr-2(gf)</i> background. <i>flp-1(lf); flp-18(lf)</i> mutants in the <i>acr-2(gf)</i> background showed enhanced aldicarb sensitivity compared to <i>acr-2(gf)</i> (B). Pan-neuronal expression of <i>flp-1</i> rescues the increased aldicarb sensitivity of the <i>flp-1(lf); flp-18(lf) acr-2(gf)</i> mutants (C). n = 10 for one group per trial; and results of three to five independent trials are shown. Mean rate of paralysis are shown for each time point. Error bars indicate SEM. Two independent transgenic lines were tested, only one is shown in the graph. Statistics in B, C: **: <i>p</i><0.01, *: <i>p</i><0.05 by two-way ANOVA and Bonferroni post-hoc test.</p

NPR-1, NPR-4, and NPR-5 act together to mediate the effects of neuropeptides on convulsions.

<p>(A–B). Convulsion frequencies of <i>acr-2(gf)</i> combined with loss of function mutations in <i>npr-1(ok1447)</i>, <i>npr-4(tm1782)</i>, <i>npr-5(ok1583)</i> (A) and with <i>flp-1(yn4)</i> (B). (C) Convulsion frequency of animals with cell type-specific expression of <i>npr-1</i> and <i>npr-5</i>. <i>npr-5</i> expression in the muscle rescued the increased convulsion frequency of <i>npr-5(lf); npr-1(lf) acr-2(gf)</i> triple mutant; two independent lines were tested. <i>npr-1</i> expression in GABAergic motor neurons did not significantly rescue the increased convulsion frequency; three lines were tested. All strains contain <i>acr-2(gf)</i>. Mean convulsion frequencies are shown. Error bars indicate SEM. Statistics: ***: <i>p</i><0.001, *: <i>p</i><0.05 by ANOVA and Bonferroni post hoc test. <i>(+/+)</i> indicates strain with no mutations in any of the neuropeptide receptor genes.</p

Neuropeptide processing and release pathway regulate <i>acr-2(gf)</i> convulsions.

<p>All mutations are loss of function alleles, except for <i>acr-2(gf)</i>, which designates <i>acr-2(n2420)</i>. Mean convulsion frequencies are shown. Error bars indicate SEM. Numbers in the graph indicate sample sizes. Statistics: ***: <i>p</i><0.001, **: <i>p</i><0.01, *: <i>p</i><0.05 by ANOVA and Bonferroni post hoc test. (A) Loss of function in <i>egl-3</i> and <i>sbt-1</i> significantly enhances <i>acr-2(gf)</i> convulsions; and the increased convulsion caused by egl-3(lf) is dependent on <i>unc-31</i>. (B) <i>egl-3</i> functions in the cholinergic motor neurons to suppress <i>acr-2(gf)</i> convulsions. The number of independent transgenic lines tested are the following: <i>Prgef-1::egl-3</i>; 4 lines, <i>Punc-17β::egl-3</i>; 3 lines, <i>Pglr-1::egl-</i>3; 3 lines, <i>Pacr-2</i>; 2 lines. Quantification data is shown for one representative line.</p