Substrate dynamics contribute to enzymatic specificity in human and bacterial methionine adenosyltransferases Protein conformational change can facilitate the binding of non-cognate substrates and underlie promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here we analyse human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, non-cognate substrates bind in a stable conformation to allow catalysis. In contrast, non-cognate substrates rarely sample stable productive binding modes in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrate likely drove the evolutionary divergence of substrate specificity in these orthologs. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated level in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.
Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23-complex) imports proteins with cleavable pre-sequences, and comes in two flavours: the TIM23SORT complex mediates inner mitochondrial membrane (IMM) protein insertion and the TIM23MOTOR complex delivers proteins to the matrix. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how {Delta}{psi} and ATP hydrolysis, the two main driving forces for import through the TIM23MOTOR complex, contribute to the import of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the OMM and initiation of IMM transport by the pre-sequence. The rates of these steps are influenced by PCP properties such as size and net charge, but not total amount of PCP imported - emphasising the importance of collecting rapid kinetic data, achieved here, to elucidate mechanistic detail. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintaining mitochondrial fitness.
MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1. Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1.This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo HM kinase of S6K1. We show that rapamycin resistant S6K1 truncation mutant {Delta}NH{Delta}CT, continues to display HM phosphorylation with selective sensitivity toward torin-1. We also show that HM phosphorylation of wildtype S6K1and {Delta}NH{Delta}CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis analysis highlights the involvement of a conserved 19 amino acid stretch of S6K1 in mediating mTORC2 dependent HM phosphorylation. We finally show that deletion of this amino acid stretch leads to complete loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data identifies mTORC2 as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complex 1 and 2 act in tandem to activate the enzyme. Dug: A Semantic Search Engine Leveraging Peer-Reviewed Knowledge to Span Biomedical Data Repositories MotivationAs the number of public data resources continues to proliferate, identifying relevant datasets across heterogenous repositories is becoming critical to answering scientific questions. To help researchers navigate this data landscape, we developed Dug: a semantic search tool for biomedical datasets utilizing evidence-based relationships from curated knowledge graphs to find relevant datasets and explain why those results are returned.

ResultsDeveloped through the National Heart, Lung, and Blood Institutes (NHLBI) BioData Catalyst ecosystem, Dug has indexed more than 15,911 study variables from public datasets. On a manually curated search dataset, Dugs total recall (total relevant results/total results) of 0.79 outperformed default Elasticsearchs total recall of 0.76. When using synonyms or related concepts as search queries, Dug (0.36) far outperformed Elasticsearch (0.14) in terms of total recall with no significant loss in the precision of its top results.

Availability and ImplementationDug is freely available at https://github.com/helxplatform/dug. An example Dug deployment is also available for use at https://search.biodatacatalyst.renci.org/.

Contactawaldrop@rti.org or scox@renci.org Multivariate genome-wide association study on tissue-sensitive diffusion metrics identifies key molecular pathways for axonal growth, synaptogenesis, and astrocyte-mediated neuroinflammation The molecular determinants of tissue composition of the human brain remain largely unknown. Recent genome-wide association studies (GWAS) on this topic have had limited success due to methodological constraints. Here, we apply advanced whole-brain analyses on multi-shell diffusion imaging data and multivariate GWAS to two large scale imaging genetic datasets (UK Biobank and the Adolescent Brain Cognitive Development study) to identify and validate genetic association signals. We discovered 503 unique genetic loci that have impact on multiple regions of human brain. Among them, more than 79% were validated in either of two large-scale independent imaging datasets. Key molecular pathways involved in axonal growth, astrocyte-mediated neuroinflammation, and synaptogenesis during development were found to significantly impact the measured variations in tissue-specific imaging features. Our results shed new light on the biological determinants of brain tissue composition and their potential overlap with the genetic basis of neuropsychiatric disorders. Membrane remodeling due to mixture of multiple curvature proteins We present an extension of the Monte Carlo based mesoscopic membrane model, where the membrane is represented as a dynamically triangulated surface and the proteins are modeled as anisotropic inclusions formulated as in-plane nematic field variables adhering to the deformable elastic sheet. In the extended model, we have augmented the Hamiltonian to study membrane deformation due to a mixture of multiple types of curvature generating proteins. This feature opens the door for understanding how multiple kinds of curvature-generating proteins may be working in a coordinated manner to induce desired membrane morphologies. For example, among other things, we study membrane deformations and tubulation due to a mixture of positive and negative curvature proteins as mimics of various proteins from BAR domain family working together for curvature formation and stabilization. We also study the effect of membrane anisotropy, which manifests as membrane localization and differential binding affinity of a given curvature protein, leading to insights into the tightly regulated cargo sorting and transport processes. Our simulation results show different morphology of deformed vesicles that depend on the curvatures and densities of the participating proteins as well as on the protein-protein and membrane-proteins interactions.

SignificanceMembrane remodeling requires highly orchestrated interactions between several types of lipids and curvature proteins. Experimentally probing the membrane deformation intermediates is non-trivial. For example, it is not known exactly how two or more different kinds of BAR domain proteins work in concert to induce and stabilize certain membrane curvatures. In this work, we use mesoscopic continuum modeling and explore the factors that induce and stabilize a range of membrane deformation due to a mixture of positive and negative curvature proteins. Neurological mechanism of sensory deficits after developmental exposure to non-dioxin-like polychlorinated biphenyls (PCBs) in zebrafish larvae The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to chemicals can result in neurobehavioral disorders. To uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits, we investigated the effects of the non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response using zebrafish as a vertebrate model. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. Morphological and biochemical data demonstrate that while exposure to PCB153 induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling is the dominant mechanism associated with motor movement. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and, with the startle response being critical for the survival of fish, to evolutionary adaptation in wildlife. Analysis of Drosophila cardiac hypertrophy by micro-computerized tomography for genetic dissection of heart growth mechanisms Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

NEW AND NOTEWORTHYGenetic analysis of cardiac hypertrophy in Drosophila holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. However, this potential has been hindered by a lack of rapid and effective methods for analysis of heart size in flies. Here we demonstrate that analysis of the Drosophila heart with micro-computerized tomography yields accurate and highly reproducible heart size measurements that can be used to efficiently analyze heart growth and cardiac hypertrophy in Drosophila. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. A Multi-omics Method Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem Cell-derived Cardiomyocytes Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show immense promise for patient-specific disease modeling, cardiotoxicity screening, and regenerative therapy development. However, hPSC-CMs in culture have not recapitulated the structural or functional properties of adult CMs in vivo thus far. To gain global insight into hPSC-CM biology, we established a multi-omics method for analyzing the hPSC-CM metabolome and proteome from the same cell culture, creating multi-dimensional profiles of hPSC-CMs. Specifically, we developed a sequential extraction to capture metabolites and proteins from the same hPSC-CM monolayer cultures, and analyzed these extracts using high-resolution mass spectrometry (MS). Using this method, we annotated 205 metabolites/lipids and 4,008 proteins from 106 cells with high reproducibility. We further integrated the proteome and metabolome measurements to create network profiles of molecular phenotypes for hPSC-CMs. Out of 310 pathways identified using metabolomics and proteomics, 40 pathways were considered significantly overrepresented (FDR-corrected p [&le;] 0.05). Highly populated pathways included those involved in protein synthesis (ribosome, spliceosome), ATP generation (oxidative phosphorylation), and cardiac muscle contraction. This multi-omics method achieves deep coverage of metabolites and proteins, creating a multidimensional view of the hPSC-CM phenotype, which provides a strong technological foundation to advance the understanding of hPSC-CM biology. Thermal modulation of Zebrafish exploratory statistics reveals constraints on individual behavioral variability BackgroundVariability is a hallmark of animal behavior. It contributes to survival by endowing individuals and populations with the capacity to adapt to ever-changing environmental conditions. Intra-individual variability is thought to reflect both endogenous and exogenous modulations of the neural dynamics of the central nervous system. However, how variability is internally regulated and modulated by external cues remains elusive. Here we address this question by analyzing the statistics of spontaneous exploration of freely swimming zebrafish larvae, and by probing how these locomotor patterns are impacted when changing the water temperatures within an ethologically relevant range.

ResultsWe show that, for this simple animal model, five short-term kinematic parameters - interbout interval, turn amplitude, travelled distance, turn probability and orientational flipping rate - together control the long-term exploratory dynamics. We establish that the bath temperature consistently impacts the means of these parameters, but leave their pairwise covariance unchanged. These results indicate that the temperature merely controls the sampling statistics within a well-defined kinematic space delineated by this robust statistical structure. At a given temperature, individual animals explore the behavioral space over a timescale of tens of minutes, suggestive of a slow internal state modulation that could be externally biased through the bath temperature. By combining these various observations into a minimal stochastic model of navigation, we show that this thermal modulation of locomotor kinematics results in a thermophobic behavior, complementing direct gradient-sensing mechanisms.

ConclusionsThis study establishes the existence of a well-defined locomotor space accessible to zebrafish larvae during spontaneous exploration, and quantifies self-generated modulation of locomotor patterns. Intra-individual variability reflects a slow diffusive-like probing of this space by the animal. The bath temperature in turn restricts the sampling statistics to sub-regions, endowing the animal with basic thermophobicity. This study suggests that in Zebrafish, as well as in other ectothermic animals, ambient temperature could be used to efficiently manipulate internal states in a simple and ethological way. Climate warming changes synchrony of plants and pollinators Climate warming changes the phenology of many species. When interacting organisms respond differently, climate change may disrupt their interactions and affect the stability of ecosystems. Here, we used GBIF occurrence records to examine phenology trends in plants and their associated insect pollinators in Germany since the 1980s. We found strong phenological advances in plants, but differences in the extent of shifts among pollinator groups. The temporal trends in plant and insect phenologies were generally associated with interannual temperature variation, and thus likely driven by climate change. When examining the synchrony of species-level plant-pollinator interactions, their temporal trends differed among pollinator groups. Overall, plant-pollinator interactions become more synchronized, mainly since the phenology of plants responded more strongly to climate change than that of the pollinators. However, if the observed trends continue, many interactions may become more asynchronous again in the future. Our study suggests that climate change affects the phenologies of both plants and insects, and that it also influences the synchrony of plant-pollinator interactions. mTOR driven gene transcription is required for cholesterol production in neurons of the developing cerebral cortex Dysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood.

Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the sterol/cholesterol biosynthesis pathway to be transcriptionally regulated by mTOR complex 1 (mTORC1) signaling in vitro in primary neurons and in vivo in the developing cerebral cortex of the mouse. We find that these genes are shared targets of the transcription factors SREBP, SP1 and NF-Y. Prenatal as well as postnatal mTORC1 inhibition downregulated expression of these genes which directly translated into reduced cholesterol levels pointing towards a substantial metabolic function of the mTORC1 signaling cascade. Altogether, our results indicate that mTORC1 is an essential transcriptional regulator of the expression of sterol/cholesterol biosynthesis genes in the developing brain. Altered expression of these genes may be an important factor contributing to the pathogenesis of neurodevelopmental disorders associated with dysregulated mTOR signaling. Nelfinavir induces cytotoxicity towards high-grade serous ovarian cancer cells, involving induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction of HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction of clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho- eIF2, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile. On Rank Deficiency in Phenotypic Covariance This paper is concerned with rank deficiency in phenotypic covariance matrices: first to establish it is a problem by measuring it, and then proposing methods to treat for it. Significant rank deficiency can mislead current measures of whole-shape phenotypic integration, because they rely on eigenvalues of the covariance matrix, and highly rank deficient matrices will have a large percentage of meaningless eigenvalues. This paper has three goals. The first is to examine a typical geometric morphometric data set and establish that its covariance matrix is rank deficient. We employ the concept of information, or Shannon, entropy to demonstrate that a sample of dire wolf jaws is highly rank deficient. The different sources of rank deficiency are identified, and include the Generalized Procrustes analysis itself, use of the correlation matrix, insufficient sample size, and phenotypic covariance. Only the last of these is of biological interest.

Our second goal is to examine a test case where a change in integration is known, allowing us to document how rank deficiency affects two measures of whole shape integration (eigenvalue standard deviation and standardized generalized variance). This test case utilizes the dire wolf data set from Part 1, and introduces another population that is 5000 years older. Modularity models are generated and tested for both populations, showing that one population is more integrated than the other. We demonstrate that eigenvalue variance characterizes the integration change incorrectly, while the standardized generalized variance lacks sensitivity. Both metrics are impacted by the inclusion of many small eigenvalues arising from rank deficiency of the covariance matrix. We propose a modification of the standardized generalized variance, again based on information entropy, that considers only the eigenvalues carrying non-redundant information. We demonstrate that this metric is successful in identifying the integration change in the test case.

The third goal of this paper is to generalize the new metric to the case of arbitrary sample size. This is done by normalizing the new metric to the amount of information present in a permuted covariance matrix. We term the resulting metric the  relative dispersion, and it is sample size corrected. As a proof of concept we us the new metric to compare the dire wolf data set from the first part of this paper to a third data set comprising jaws of Smilodon fatalis. We demonstrate that the Smilodon jaw is much more integrated than the dire wolf jaw. Finally, this information entropy-based measures of integration allows comparison of whole shape integration in dense semilandmark environments, allowing characterization of the information content of any given shape, a quantity we term  latent dispersion. Engineered bacteria detect tumor DNA Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to diseases such as gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can easily access the entire gastrointestinal tract via oral administration8, and they can produce outputs that can be noninvasively measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangement11, has been used to allow bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells and organoids. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered orally or rectally into mice injected with orthotopic donor CRC organoids. We observe horizontal gene transfer from the tumor to the sensor bacteria in vivo, allowing their detection in stool. The sensor bacteria achieved 100% discrimination between mice with and without CRC using both delivery methods. Our findings establish a framework for biosensing applications that require the detection of mutations or organisms within environments that are difficult to sample. In addition, the platform can be readily expanded to include in situ production and delivery of therapeutic payloads at the detection site. In search of a core cellular network with single cell transcriptome data in the fruit fly Along with specialized functions, cells of multicellular organisms also perform essential functions. Whether diverse cells do this by using the same set of genes, interacting in a fixed coordinated fashion, remains a central question in biology. Single-cell RNA-sequencing (scRNA-seq) measures gene expression of individual cells, enabling researchers to discover gene expression patterns that contribute to the diversity of cell functions. Current efforts focus primarily on identifying differentially expressed genes. However, patterns of co-expression between genes are more indicative of biological processes than are the expression of individual genes. Here, we constructed cell type-specific gene co-expression networks using the fly brain transcriptome atlas to search for a core cellular network. We detected a set of highly coordinated genes preserved across cell types and defined this set as the core network. This network was further validated through two additional datasets profiling fly brains or heads. This core has a small size and shows dense connectivity. Modules within this core are enriched for basic cellular functions and gene members of these modules have distinct evolutionary signatures. Overall, we demonstrated that a core cellular network exists in diverse cell types of fly brains and this core exhibits unique topological, structural, functional and evolutionary properties. FGF8-mediated signaling regulates tooth developmental pace during odontogenesis The developing human and mouse teeth constitute an ideal model system to study the regulatory mechanism underlying organ growth control due to the fact that their teeth share highly conserved and well-characterized developmental processes and their developmental tempo vary notably. In the current study, we manipulated heterogenous recombination between human and mouse dental tissues and demonstrate that the dental mesenchyme dominates the tooth developmental tempo and FGF8 could be a critical player during this developmental process. Forced activation of FGF8 signaling in the dental mesenchyme of mice promoted cell proliferation, prevented cell apoptosis via p38 and perhaps PI3K-Akt intracellular signaling, and impelled the transition of the cell cycle from G1- to S-phase in the tooth germ, resulting in the slowdown of the tooth developmental pace. Our results provide compelling evidence that extrinsic signals can profoundly affect tooth developmental tempo and the dental mesenchymal FGF8 could be a pivotal factor in controlling developmental pace in a non-cell-autonomous manner during mammalian odontogenesis. An energy-dissipative state of the major antenna complex of plants Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as non-photochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants survival under abiotic stress. Here, we tested whether we could isolate photosynthetic subcomplexes (from plants in which qH was induced) that would remain in an energy-dissipative state. Interestingly chlorophyll fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein or lipid content of antenna with active or inactive qH, and no evident differences were detected. Finally, we investigated whether specific antenna subunits of the major antenna were required for qH but found it insensitive to trimer composition. Because qH can occur in the absence of specific xanthophylls, and no changes in pigments were detected, we propose that the energy-dissipative state reported here may stem from chlorophyll-chlorophyll excitonic interaction. Carbon Dot-based Fluorescent Antibody Nanoprobes as Brain Tumour Glioblastoma Diagnostics. The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters.

Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems. The TPLATE subunit is essential for structural assembly of the endocytic TSET complex All eukaryotic cells rely on endocytosis to regulate the plasma membrane proteome and lipidome. Most eukaryotic groups, with the exception of fungi and animals, have retained the evolutionary ancient TSET complex as a regulator of endocytosis. Despite the presence of similar building blocks in TSET, compared to other coatomer complexes, structural insight into this adaptor complex is lacking. Here, we elucidate the molecular architecture of the octameric plant TSET complex (TPLATE complex/TPC) using an integrative structural approach. This allowed us to describe a plant-specific connection between the TML subunit and the AtEH/Pan1 proteins and show a direct interaction between the complex and the plasma membrane without the need for any additional protein factors. Furthermore, we identify the appendage of TPLATE as crucial for complex assembly. Structural elucidation of this ancient adaptor complex vastly advances our functional as well as evolutionary insight into the process of endocytosis.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Royal knifefish generate powerful suction feeding through large neurocranial elevation and high epaxial muscle power Suction feeding in ray-finned fishes involves powerful buccal cavity expansion to accelerate water and food into the mouth. Previous XROMM studies in largemouth bass (Micropterus salmoides), bluegill sunfish (Lepomis macrochirus), and channel catfish (Ictalurus punctatus) have shown that more than 90% of suction power in high performance strikes comes from the axial musculature. Thus, the shape of the axial muscles and skeleton may impact suction feeding mechanics. Royal knifefish (Chitala blanci) have an unusual postcranial morphology, with a ventrally flexed vertebral column and relatively large mass of epaxial muscle. Based on their body shape, we hypothesized that royal knifefish would generate high power strikes by utilizing large neurocranial elevation, vertebral column extension, and epaxial shortening. As predicted, C. blanci generated high suction expansion power compared to the other three species studied to date (up to 160 W), which was achieved by increasing both the rate of volume change and the intraoral subambient pressure. The large epaxial muscle (25% of body mass) shortened at high velocities to produce large neurocranial elevation and vertebral extension (up to 41 deg, combined), as well as high muscle mass-specific power (up to 800 W kg-1). For the highest power strikes, axial muscles generated 95% of the power, and 64% of the axial muscle mass consisted of the epaxial muscles. The epaxial-dominated suction expansion of royal knifefish supports our hypothesis that postcranial morphology may be a strong predictor of suction feeding biomechanics.

SUMMARY STATEMENTRoyal knifefish rely on their distinct postcranial morphology--with a curved vertebral column and large dorsal body muscles--to produce large neurocranial elevation and powerful suction feeding. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like Escherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during E. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine via the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, via the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms. The perceptual relevance of the speech envelope-following response in normal hearing and age-related hearing impaired listeners The aim of this work was to investigate the perceptual relevance of the frequency following response to the syllable /da/ for speech intelligibility in noise based on age and hearing deficits. Recordings of the auditory evoked potential from young normal hearing (NH) and older individuals with both normal hearing and high-frequency (HF) hearing loss were analyzed. EFR metrics obtained in quiet and noise condition were calculated and correlated with speech reception. The envelope following responses were analyzed in terms of amplitude, latency and noise robustness. The response was first simulated to form predictions on the effect of cochlear synaptopathy and outer hair cell loss on the EFR. The experimental findings were in line with the computational predictions in the found observation that the EFR was reduced as a consequence of ageing and HF hearing loss. Both the audiogram and the speech EFR magnitude fell short in the individual prediction of SRT in stationary noise, but they accounted well for group performance. We also obtained within-group EFR latency with a cross covariance matrix. Validation of the method confirmed that speech EFR latency was predictive of click ABR Wave V peak latency. Moreover, statistical analysis not only showed that the robustness of the EFR obtained in the noise condition was dependent on the degree of high-frequency hearing loss in the older NH adults, but also dependent on the EFR magnitude in the NH younger adults. These findings provide evidence towards the important role of the EFR in speech-in-noise perception. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals.