Peroxisomes take shape.

Peroxisomes carry out various oxidative reactions that are tightly regulated to adapt to the changing needs of the cell and varying external environments. Accordingly, they are remarkably fluid and can change dramatically in abundance, size, shape and content in response to numerous cues. These dynamics are controlled by multiple aspects of peroxisome biogenesis that are coordinately regulated with each other and with other cellular processes. Ongoing studies are deciphering the diverse molecular mechanisms that underlie biogenesis and how they cooperate to dynamically control peroxisome utility. These important challenges should lead to an understanding of peroxisome dynamics that can be capitalized upon for bioengineering and the development of therapies to improve human health.

Revealing molecular determinants governing mambalgin-3 pharmacology at acid-sensing ion channel 1 variants.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

A Peer-Based Strategy to Overcome HPV Vaccination Inequities in Rural Communities: A Physical Distancing-Compliant Approach.

The human papilloma virus (HPV) vaccine is the world's first proven and effective vaccine to prevent cancers in males and females when administered pre-exposure. Like most of the US, barely half of Vermont teens are up-to-date with the vaccination, with comparable deficits in New Hampshire and Maine. The rates for HPV vaccine initiation and completion are as low as 33% in rural New England. Consequently, there is a compelling responsibility to communicate its importance to unvaccinated teenagers before their risk for infection increases. Messaging in rural areas promoting HPV vaccination is compromised by community-based characteristics that include access to appropriate medical care, poor media coverage, parental and peer influence, and skepticism of science and medicine. Current strategies are predominantly passive access to literature and Internet-based information. Evidence indicates that performance-based messaging can clarify the importance of HPV vaccination to teenagers and their parents in rural areas. Increased HPV vaccination will significantly contribute to the prevention of a broadening spectrum of cancers. Reducing rurality-based inequities is a public health priority. Development of a performance-based peer-communication intervention can capture a window of opportunity to provide increasingly effective and sustained HPV protection. An effective approach can be partnering rural schools and regional health teams with a program that is nimble and scalable to respond to public health policies and practices compliant with COVID-19 pandemic-related modifications on physical distancing and interacting in the foreseeable future.

GluClR-mediated inhibitory postsynaptic currents reveal targets for ivermectin and potential mechanisms of ivermectin resistance.

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold.

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric ß-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.

Combining inferred regulatory and reconstructed metabolic networks enhances phenotype prediction in yeast.

Gene regulatory and metabolic network models have been used successfully in many organisms, but inherent differences between them make networks difficult to integrate. Probabilistic Regulation Of Metabolism (PROM) provides a partial solution, but it does not incorporate network inference and underperforms in eukaryotes. We present an Integrated Deduced And Metabolism (IDREAM) method that combines statistically inferred Environment and Gene Regulatory Influence Network (EGRIN) models with the PROM framework to create enhanced metabolic-regulatory network models. We used IDREAM to predict phenotypes and genetic interactions between transcription factors and genes encoding metabolic activities in the eukaryote, Saccharomyces cerevisiae. IDREAM models contain many fewer interactions than PROM and yet produce significantly more accurate growth predictions. IDREAM consistently outperformed PROM using any of three popular yeast metabolic models and across three experimental growth conditions. Importantly, IDREAM's enhanced accuracy makes it possible to identify subtle synthetic growth defects. With experimental validation, these novel genetic interactions involving the pyruvate dehydrogenase complex suggested a new role for fatty acid-responsive factor Oaf1 in regulating acetyl-CoA production in glucose grown cells.

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating.

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (µ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Evidence that auxin is required for normal seed size and starch synthesis in pea.

In recent years the biosynthesis of auxin has been clarified with the aid of mutations in auxin biosynthesis genes. However, we know little about the effects of these mutations on the seed-filling stage of seed development. Here we investigate a key auxin biosynthesis mutation of the garden pea, which results in auxin deficiency in developing seeds. We exploit the large seed size of this model species, which facilitates the measurement of compounds in individual seeds. The mutation results in small seeds with reduced starch content and a wrinkled phenotype at the dry stage. The phenotypic effects of the mutation were fully reversed by introduction of the wild-type gene as a transgene, and partially reversed by auxin application. The results indicate that auxin is required for normal seed size and starch accumulation in pea, an important grain legume crop.

Body Temperature after EMS Transport: Association with Traumatic Brain Injury Outcomes.

INTRODUCTION: Low body temperatures following prehospital transport are associated with poor outcomes in patients with traumatic brain injury (TBI). However, a minimal amount is known about potential associations across a range of temperatures obtained immediately after prehospital transport. Furthermore, a minimal amount is known about the influence of body temperature on non-mortality outcomes. The purpose of this study was to assess the correlation between temperatures obtained immediately following prehospital transport and TBI outcomes across the entire range of temperatures. METHODS: This retrospective observational study included all moderate/severe TBI cases (CDC Barell Matrix Type 1) in the pre-implementation cohort of the Excellence in Prehospital Injury Care (EPIC) TBI Study (NIH/NINDS: 1R01NS071049). Cases were compared across four cohorts of initial trauma center temperature (ITCT): <35.0°C [Very Low Temperature (VLT)]; 35.0-35.9°C [Low Temperature (LT)]; 36.0-37.9°C [Normal Temperature (NT)]; and ≥38.0°C [Elevated Temperature (ET)]. Multivariable analysis was performed adjusting for injury severity score, age, sex, race, ethnicity, blunt/penetrating trauma, and payment source. Adjusted odds ratios (aORs) with 95% confidence intervals (CI) for mortality were calculated. To evaluate non-mortality outcomes, deaths were excluded and the adjusted median increase in hospital length of stay (LOS), ICU LOS and total hospital charges were calculated for each ITCT group and compared to the NT group. RESULTS: 22,925 cases were identified and cases with interfacility transfer (7361, 32%), no EMS transport (1213, 5%), missing ITCT (2083, 9%), or missing demographic data (391, 2%) were excluded. Within this study cohort the aORs for death (compared to the NT group) were 2.41 (CI: 1.83-3.17) for VLT, 1.62 (CI: 1.37-1.93) for LT, and 1.86 (CI: 1.52-3.00) for ET. Similarly, trauma center (TC) LOS, ICU LOS, and total TC charges increased in all temperature groups when compared to NT. CONCLUSION: In this large, statewide study of major TBI, both ETs and LTs immediately following prehospital transport were independently associated with higher mortality and with increased TC LOS, ICU LOS, and total TC charges. Further study is needed to identify the causes of abnormal body temperature during the prehospital interval and if in-field measures to prevent temperature variations might improve outcomes.

Molecular mechanisms of system responses to novel stimuli are predictable from public data.

Systems scale models provide the foundation for an effective iterative cycle between hypothesis generation, experiment and model refinement. Such models also enable predictions facilitating the understanding of biological complexity and the control of biological systems. Here, we demonstrate the reconstruction of a globally predictive gene regulatory model from public data: a model that can drive rational experiment design and reveal new regulatory mechanisms underlying responses to novel environments. Specifically, using ∼ 1500 publically available genome-wide transcriptome data sets from Saccharomyces cerevisiae, we have reconstructed an environment and gene regulatory influence network that accurately predicts regulatory mechanisms and gene expression changes on exposure of cells to completely novel environments. Focusing on transcriptional networks that induce peroxisomes biogenesis, the model-guided experiments allow us to expand a core regulatory network to include novel transcriptional influences and linkage across signaling and transcription. Thus, the approach and model provides a multi-scalar picture of gene dynamics and are powerful resources for exploiting extant data to rationally guide experimentation. The techniques outlined here are generally applicable to any biological system, which is especially important when experimental systems are challenging and samples are difficult and expensive to obtain-a common problem in laboratory animal and human studies.

Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of ϕ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. ϕ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.

δ-Conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails.

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition.

Global analysis of condition-specific subcellular protein distribution and abundance.

Cellular control of protein activities by modulation of their abundance or compartmentalization is not easily measured on a large scale. We developed and applied a method to globally interrogate these processes that is widely useful for systems-level analyses of dynamic cellular responses in many cell types. The approach involves subcellular fractionation followed by comprehensive proteomic analysis of the fractions, which is enabled by a data-independent acquisition mass spectrometry approach that samples every available mass to charge channel systematically to maximize sensitivity. Next, various fraction-enrichment ratios are measured for all detected proteins across different environmental conditions and used to group proteins into clusters reflecting changes in compartmentalization and relative conditional abundance. Application of the approach to characterize the response of yeast proteins to fatty acid exposure revealed dynamics of peroxisomes and novel dynamics of MCC/eisosomes, specialized plasma membrane domains comprised of membrane compartment occupied by Can1 (MCC) and eisosome subdomains. It also led to the identification of Fat3, a fatty acid transport protein of the plasma membrane, previously annotated as Ykl187.

Dual feedback loops in the GAL regulon suppress cellular heterogeneity in yeast.

Transcriptional noise is known to be an important cause of cellular heterogeneity and phenotypic variation. The extent to which molecular interaction networks may have evolved to either filter or exploit transcriptional noise is a much debated question. The yeast genetic network regulating galactose metabolism involves two proteins, Gal3p and Gal80p, that feed back positively and negatively, respectively, on GAL gene expression. Using kinetic modeling and experimental validation, we demonstrate that these feedback interactions together are important for (i) controlling the cell-to-cell variability of GAL gene expression and (ii) ensuring that cells rapidly switch to an induced state for galactose uptake.

The insecticidal potential of venom peptides.

Pest insect species are a burden to humans as they destroy crops and serve as vectors for a wide range of diseases including malaria and dengue. Chemical insecticides are currently the dominant approach for combating these pests. However, the de-registration of key classes of chemical insecticides due to their perceived ecological and human health risks in combination with the development of insecticide resistance in many pest insect populations has created an urgent need for improved methods of insect pest control. The venoms of arthropod predators such as spiders and scorpions are a promising source of novel insecticidal peptides that often have different modes of action to extant chemical insecticides. These peptides have been optimized via a prey-predator arms race spanning hundreds of millions of years to target specific types of insect ion channels and receptors. Here we review the current literature on insecticidal venom peptides, with a particular focus on their structural and pharmacological diversity, and discuss their potential for deployment as insecticides.

Unique scorpion toxin with a putative ancestral fold provides insight into evolution of the inhibitor cystine knot motif.

The three-disulfide inhibitor cystine knot (ICK) motif is a fold common to venom peptides from spiders, scorpions, and aquatic cone snails. Over a decade ago it was proposed that the ICK motif is an elaboration of an ancestral two-disulfide fold coined the disulfide-directed ß-hairpin (DDH). Here we report the isolation, characterization, and structure of a novel toxin [U(1)-liotoxin-Lw1a (U(1)-LITX-Lw1a)] from the venom of the scorpion Liocheles waigiensis that is the first example of a native peptide that adopts the DDH fold. U(1)-LITX-Lw1a not only represents the discovery of a missing link in venom protein evolution, it is the first member of a fourth structural fold to be adopted by scorpion-venom peptides. Additionally, we show that U(1)-LITX-Lw1a has potent insecticidal activity across a broad range of insect pest species, thereby providing a unique structural scaffold for bioinsecticide development.

A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

Environment-responsive transcription factors bind subtelomeric elements and regulate gene silencing.

Subtelomeric chromatin is subject to evolutionarily conserved complex epigenetic regulation and is implicated in numerous aspects of cellular function including formation of heterochromatin, regulation of stress response pathways and control of lifespan. Subtelomeric DNA is characterized by the presence of specific repeated segments that serve to propagate silencing or to protect chromosomal regions from spreading epigenetic control. In this study, analysis of genome-wide chromatin immunoprecipitation and expression data, suggests that several yeast transcription factors regulate subtelomeric silencing in response to various environmental stimuli through conditional association with proto-silencing regions called X elements. In this context, Oaf1p, Rox1p, Gzf1p and Phd1p control the propagation of silencing toward centromeres in response to stimuli affecting stress responses and metabolism, whereas others, including Adr1p, Yap5p and Msn4p, appear to influence boundaries of silencing, regulating telomere-proximal genes in Y' elements. The factors implicated here are known to control adjacent genes at intrachromosomal positions, suggesting their dual functionality. This study reveals a path for the coordination of subtelomeric silencing with cellular environment, and with activities of other cellular processes.

Histone chaperone Chz1p regulates H2B ubiquitination and subtelomeric anti-silencing.

Chz1p is a histone chaperone that interacts physically and functionally with the histone variant Htz1p, which has been implicated in establishing and maintaining boundaries between transcriptionally inactive heterochromatin and active euchromatin. To investigate the role of Chz1p in chromatin organization, we performed genome-wide expression arrays and chromatin immunoprecipitations of SIR complex components and modified histones in a CHZ1 deletion strain. Deletion of CHZ1 led to reduced ubiquitination of subtelomere-associated H2B, reduced subtelomeric H3K79 di-methylation, and increased binding of Sir3p, and Sir4p at telomere-distal euchromatin regions, correlating with decreased gene expression in subtelomeric regions. This anti-silencing defect appears to be mediated by enhanced association of de-ubiquitinase Ubp10p with subtelomeric DNA, as detected by chromatin immunoprecipitation analysis. In support of this, we show that deletion of UBP10 can antagonize the subtelomeric silencing phenotype of Deltachz1. Taken together, the results demonstrate a novel role for Chz1p in epigenetic regulation, through H2B de-ubiquitination by Ubp10p.

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.