Differential regulation of alternative promoters emerges from unified kinetics of enhancer-promoter interaction Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. Here, we apply single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, we find that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, we show that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the kinetic mechanisms of complex eukaryotic gene regulation.
Designer Scaffolds for Interfacial Bioengineering In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for studying the complex microenvironment of this interface remain lacking. Here, we study these complex living interfaces by manufacturing modular "blocks" of naturally porous decellularized plant-derived scaffolds of varying shapes and sizes with a computer numerical controlled mill. We demonstrate how each scaffold can be seeded with different cell types and easily assembled in a manner akin to LEGO bricks to create an engineered tissue interface (ETI). Cells were observed to migrate across the interface formed between an empty scaffold and a scaffold pre-seeded with cells. However, when both scaffolds contained cells, only a shallow cross-over zone of cell infiltration formed at the interface. As a proof-of-concept study, we utilized ETIs to investigate the interaction between lab grown bone and connective tissues. Consistent with the above, a cross-over zone of the two distinct cell types formed at the interface between scaffolds, otherwise the populations remained distinct. Finally, we demonstrate how ETIs are biocompatible in vivo, becoming vascularized and integrated into surrounding tissue after implantation. This work creates new tissue design avenues for understanding biological processes or the development of synthetic artificial tissues.
G protein-coupled estrogen receptor activation by Bisphenol-A disrupts protection from apoptosis conferred by estrogen receptors ERα and ERβ in pancreatic beta cells 17{beta}-estradiol protects pancreatic {beta}-cells from apoptosis via the estrogen receptors ER, ER{beta} and GPER. Conversely, the endocrine disruptor Bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ER and ER{beta}, or in dispersed islet cells from ER{beta} knockout (BERKO) mice. Yet, the ER and ER{beta} agonists, PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ER and ER{beta} form homodimers and heterodimers. Molecular dynamic simulations together with proximity ligand assay and coimmunoprecipitation experiments indicated that the interaction of BPA with ER and ER{beta} as well as the GPER activation by G1 decreased ER{beta} heterodimers. We propose that ER{beta} heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decrease in ER{beta} heterodimers leads to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

HighlightsO_LIPharmacological blockade and gene silencing of estrogen receptors ER, ER{beta} and GPER indicate that they are antiapoptotic in basal conditions.
C_LIO_LIGPER activation by G1 and BPA triggered apoptosis via a crosstalk with ER and ER{beta}.
C_LIO_LIBPA interaction with ER and ER{beta} as well as GPER activation decreased ER{beta} heterodimers, which was associated to increased apoptosis.
C_LIO_LIThis pathway represents a novel molecular initiating event underlying the pro-apoptotic effect of BPA
C_LIO_LIThe EndoC-{beta}H1 cell line may be a valid model of human {beta}-cells for identifying diabetogenic pollutants.
C_LI Land use change through the lens of macroecology: insights from Azorean arthropods and the Maximum Entropy Theory of Ecology Human activity and land management practices, in particular land use change, have resulted in the global loss of biodiversity. These types of disturbance affect the shape of macroecological patterns, and therefore analyzing these patterns can provide insights into how ecosystems are affected by land use change. We here use arthropod census data from 96 sites at Terceira Island in the Azores archipelago across four different land uses of increasing management intensity: native forest, exotic forest, semi-natural pasture, and intensive pasture, to examine the effects of land use type on three macroecological patterns: the species abundance distribution, the metabolic rate distribution of individuals, and the species-area relationship. The Maximum Entropy Theory of Ecology (METE) has successfully predicted these patterns across habitats and taxa in undisturbed ecosystems, and thus provides a null expectation for their shapes. Across these patterns, we find that the forest habitats are the best fit by METE predictions, while the semi-natural pasture is consistently the worst fit, and the intensive pasture is intermediately well fit. We show that the direction of failure of the METE predictions at the pasture sites is likely due to the hyper-dominance of introduced spider species present there. We hypothesize that the particularly poor fit for the semi-natural pasture is due to the mix of arthropod communities out of equilibrium, leading to greater heterogeneity in composition and complex dynamics that violate METEs assumption of static state variables. The comparative better fit for the intensive pasture plausibly results from more homogeneous arthropod communities that are well adapted to intensive management, and thus whose state variables are less in flux. Analyzing deviations from theoretical predictions across land use type provides useful information about how land use and disturbance affect ecosystems, and such comparisons could be useful across other habitats and taxa. A MALDI-TOF assay identifies nilotinib as an inhibitor of inflammation in acute myeloid leukaemia Inflammatory responses are important in cancer, particularly in the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs in human monocytes derived from acute myeloid leukaemia (AML), by tracking several biological features ionizing from only 2,500 cells using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that the BCR-ABL inhibitor nilotinib, but not the structurally similar imatinib, blocks inflammatory responses. In order to identify the cellular (off-)targets of nilotinib, we performed thermal proteome profiling (TPP). Unlike imatinib, nilotinib and other later generation BCR-ABL inhibitors inhibit the p38-MK2/3 signalling axis which suppressed the expression of inflammatory cytokines, cell adhesion and innate immunity markers in activated human monocytes derived from AML. Thus, our study provides a tool for the discovery of new anti-inflammatory drugs, which could contribute to the treatment of inflammation in myeloid neoplasms and other diseases.

Key PointsLabel-free cell-based assay identifies new anti-inflammatory drugs using MALDI-TOF MS. Nilotinib reduces inflammation by inhibition of MAPK14-MK2/3 signalling axis in AML. The genome of the cereal pest Sitophilus oryzae: a transposable element haven BackgroundThe rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions.

ResultsWe sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 Kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino-acids required for insect development and cuticle biosynthesis.

ConclusionsHere we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes. White matter connections of high-level visual areas predict cytoarchitecture better than category-selectivity in childhood, but not adulthood Ventral temporal cortex (VTC) consists of high-level visual regions that are arranged in consistent anatomical locations across individuals. This consistency has led to several hypotheses about the factors that constrain the functional organization of VTC. A prevailing theory is that white matter connections influence the organization of VTC, however, the nature of this constraint is unclear. Here, we test two hypotheses: (1) white matter tracts are specific for each category or (2) white matter tracts are specific to cytoarchitectonic areas of VTC. To test these hypotheses, we used diffusion magnetic resonance imaging (dMRI) to identify white matter tracts and functional magnetic resonance imaging (fMRI) to identify category-selective regions in VTC in children and adults. We find that in childhood, white matter connections are linked to cytoarchitecture rather than category-selectivity. In adulthood, however, white matter connections are linked to both cytoarchitecture and category-selectivity. These results suggest a rethinking of the view that category-selective regions in VTC have category-specific white matter connections early in development. Instead, these findings suggest that the neural hardware underlying the processing of categorical stimuli may be more domain-general than previously thought, particularly in childhood. Multi-omic integrated curvature study on pan-cancer genomic data In this work, we introduce a new mathematical framework based on network curvature to extract significant cancer subtypes from multi-omics data. This extends our previous work that was based on analyzing a fixed single-omics data class (e,g, CNA, gene expression, etc.). Notably, we are able to show that this new methodology provided us with significant survival differences on Kaplan-Meier curves across almost every cancer that we considered. Moreover, the variances in Ollivier-Ricci curvature was explored to investigate its usefulness in network topology analysis as this curvature may be capturing subtle functional changes between various cancer subtypes. Revealing the inherent susceptibility of pathogenic bacteria to magnetic hyperthermia The growing number of multiple drug resistant (MDR) bacteria and the dwindling pipeline of new antibiotics are driving us towards a  post-antibiotic era in which even common infections would become difficult to treat. To address this, an antibiotic-free strategy that can combat multiple bacteria is recommended. Most of the proposed approaches nevertheless have several limitations, including bacterial targeting. To overcome such limitations, the proposed strategy employs the bacterial machinery to self-destruct. Herein, the biosynthesis of magnetic nanoparticle (MNP) is reported for the first time in multiple pathogenic bacteria, including MDR bacteria. The intracellular MNPs composed of superparamagnetic zinc ferrites were formed in presence of iron and zinc precursors. Exposure of the treated bacteria/biofilms to an alternating magnetic field (AMF) exhibited hyperthermia (5-6{degrees}C) and a dramatic decrease in bacterial viability, suggesting the MNPs therapeutic potential. Likewise, the bacteria existing in vivo biosynthesize the MNPs by mining these elements from the host. To determine its therapeutic efficacy, the infected tissues were exposed directly to AMF. A 3-4 log reduction in bacterial burden, as compared to antibiotics treatment, confirmed the significance of using naturally existing MNPs to combat bacterial infections. The proposed broad-spectrum approach can therefore aid in overcoming the challenges facing anti-bacterial therapies. Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats Venom proteome profiling of Naja naja from the Western Ghats region in Kerala was achieved through SDS-PAGE and RP-HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 115 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified through conventional and 39 peptides through homology-driven proteomics approaches. Fourteen peptides derived through de novo complements the Mascot data indicating the importance of homology-driven approaches in improving protein sequence information. Among the protein families identified, glutathione peroxidase and endonuclease were reported for the first time in the Indian cobra venom. Immunological cross-reactivity assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 g/mL) value than that of PSAV (6.04 g/mL) and Virchow (6.03 g/mL) antivenoms. Western blotting experiments indicated that all the antivenoms elicited poor binding specificities, especially towards low molecular mass proteins. Second-generation antivenomics studies revealed that VINS antivenom was less efficient to detect many low molecular mass proteins such as three-finger toxins and Kunitz-type serine protease Inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja from the Western Ghats and emphasized the need for developing more efficient antivenoms.

HighlightsO_LIProteomics of cobra venom resulted in the identification of 115 proteins representing 17 snake venom protein families.
C_LIO_LIDe novo approaches exclusively yielded 39 peptides harbouring multiple amino acid mutations.
C_LIO_LIGlutathione peroxidase and endonuclease were identified for the first time in Indian cobra venom.
C_LIO_LIIndian polyvalent antivenoms showed varying cross-reactivity towards cobra venom.
C_LIO_LIVINS antivenom was less efficient to detect many low molecular mass proteins (< 20 kDa).
C_LI A nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an olfactory chemoattractant for the grass puffer Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this unknown function TTX analog as an olfactory chemoattractant. Plasma proteome analyses in individuals of European and African ancestry identify cis-pQTLs and models for proteome-wide association studies Improved understanding of genetic regulation of proteome can facilitate the identification of causal mechanisms for complex traits. We analyzed data on 4,657 plasma proteins from 7,213 European American (EA) and 1,871 African American (AA) individuals from the ARIC study, and further replicated findings on 467 AA individuals from the AASK study. Here we identified 2,004 proteins in EA and 1,618 in AA, with majority overlapping, which showed associations with common variants in cis-regions. Availability of AA samples led to smaller credible sets and significant number of population-specific cis-pQTLs. Elastic-net produced powerful models for protein prediction in both populations. An application of proteome-wide association studies (PWAS) to serum urate and gout, implicated several proteins, including IL1RN, revealing the promise of the drug anakinra to treat acute gout flares. Our study demonstrates the value of large and diverse ancestry study for genetic mechanisms of molecular phenotypes and their relationship with complex traits. Neuroinvasion and neurotropism by SARS-CoV-2 variants in the K18-hACE2 mouse Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, also after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally orientated regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates, and broadly support investigation of agents with adequate penetration into relevant regions of the CNS. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. 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. Flexible reprogramming of Pristionchus pacificus motivation for attacking Caenorhabditis elegans in predator-prey competition Animals with diverse diets must adapt their food priorities to a wide variety of environmental conditions. This diet optimization problem is especially complex for predators that compete with prey for food. Although predator-prey competition is widespread and ecologically critical, it remains difficult to disentangle predatory and competitive motivations for attacking competing prey. Here, we dissect the foraging decisions of the omnivorous nematode Pristionchus pacificus to reveal that its seemingly failed predatory attempts against Caenorhabditis elegans are actually motivated acts of efficacious territorial aggression. While P. pacificus easily kills and eats larval C. elegans with a single bite, adult C. elegans typically survives and escapes from bites. However, nonfatal biting can provide competitive benefits by reducing access of adult C. elegans and its progeny to bacterial food that P. pacificus also eats. We show that P. pacificus considers costs and benefits of both predatory and territorial outcomes to decide which food goal, prey or bacteria, should guide its motivation for biting. These predatory and territorial motivations impose different sets of rules for adjusting willingness to bite in response to changes in bacterial abundance. In addition to biting, predatory and territorial motivations also influence which search tactic P. pacificus uses to increase encounters with C. elegans. When treated with an octopamine receptor antagonist, P. pacificus switches from territorial to predatory motivation for both biting and search. Overall, we demonstrate that P. pacificus assesses alternate outcomes of attacking C. elegans and flexibly reprograms its foraging strategy to prioritize either prey or bacterial food. Mechanisms Underlying WNT-mediated Priming of Human Embryonic Stem Cells Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs) called gastruloids. Our previous work demonstrated that during early self-organization of gastruloids, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent Activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening, but rather, acts by inducing the expression of the SMAD2 co-factor, EOMES. Expression of EOMES is sufficient to replace WNT upstream of Activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. An Essential Role of UBXN3B in B Lymphopoiesis Hematopoiesis is finely regulated to enable timely production of the right numbers and types of mature immune cells to maintain tissue homeostasis. Dysregulated hematopoiesis may compromise antiviral immunity and/or exacerbate immunopathogenesis. Herein, we report an essential role of UBXN3B in maintenance of hematopoietic homeostasis and restriction of immunopathogenesis during respiratory viral infection. Ubxn3b deficient (Ubxn3b-/-) mice are highly vulnerable to SARS-CoV-2 and influenza A infection, characterized by more severe lung immunopathology, lower virus-specific IgG, significantly fewer B cells, but more myeloid cells than Ubxn3b+/+ littermates. This aberrant immune compartmentalization is recapitulated in uninfected Ubxn3b-/- mice. Mechanistically, UBXN3B controls precursor B-I (pre-BI) transition to pre-BII and subsequent proliferation in a cell-intrinsic manner, by maintaining BLNK protein stability and pre-BCR signaling. These results reveal an essential role of UBXN3B for the early stage of B cell development. Common Dynamic Determinants Govern Quorum Quenching Activity in N-terminal Serine Hydrolases Growing concerns about microbial antibiotic resistance have motivated extensive research into ways of overcoming antibiotic resistance. Quorum quenching (QQ) processes disrupt bacterial communication via quorum sensing, which enables bacteria to sense the surrounding bacterial cell density and markedly affects their virulence. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and may thus avoid inducing resistance. Although many enzymes display QQ activity against various bacterial signaling molecules, their mechanisms of action are poorly understood, limiting their potential optimization as QQ agents. Here we evaluate the capacity of three N-terminal serine hydrolases to degrade N-acyl homoserine lactones that serve as signaling compounds for Gram-negative bacteria. Using molecular dynamics simulations of the free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulations of their initial catalytic steps, we clarify the molecular processes underpinning their QQ activity. We conclude that all three enzymes degrade bacterial signaling molecules via similar reaction mechanisms. Moreover, we experimentally confirmed the activity of two penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), adding these biotechnologically well-optimized enzymes to the QQ toolbox. We also observed enzyme- and substrate-dependent differences in the catalytic actions of these enzymes, arising primarily from the distinct structures of their acyl-binding cavities and the dynamics of their molecular gates. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate had an elevated energy barrier because its shallow acyl binding site could not accommodate a productive substrate-binding configuration. Conversely, aPGA in complex with both substrates exhibited unfavorable energetics in both reaction steps due to the dynamics of the residues gating the acyl binding cavity entrance. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to the unique positioning of Arg297{beta} in this enzyme. The discovery of these dynamic determinants will guide future efforts to design robust QQ agents capable of selectively controlling virulence in resistant bacterial species. Supervised Learning Model Predicts Protein Adsorption to Carbon Nanotubes Engineered nanoparticles are advantageous for numerous biotechnology applications, including biomolecular sensing and delivery. However, testing the compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where unpredictable biofouling via protein corona formation often prevents effective implementation. Moreover, rational design of biomolecule-nanoparticle conjugates requires prior knowledge of such interactions or extensive experimental testing. Toward better applying engineered nanoparticles in biological systems, herein, we develop a random forest classifier (RFC) trained with proteomic mass spectrometry data that identifies proteins that adsorb to nanoparticles, based solely on the proteins amino acid sequence. We model proteins that populate the corona of a single-walled carbon nanotube (SWCNT)-based optical nanosensor and study whether there is a relationship between the proteins amino acid-based properties and the proteins adsorption to SWCNTs. We optimize the classifier and characterize the classifier performance against other models. To evaluate the predictive power of our model, we apply the classifier to rapidly identify proteins with high binding affinity to SWCNTs, followed by experimental validation. We further determine protein features associated with increased likelihood of SWCNT binding: high content of solvent-exposed glycine residues and non-secondary structure-associated amino acids. Conversely, proteins with high content of leucine residues and beta-sheet-associated amino acids are less likely to form the SWCNT protein corona. The classifier presented herein provides a step toward undertaking the otherwise intractable problem of predicting protein-nanoparticle interactions, which is needed for more rapid and effective translation of nanobiotechnologies from in vitro synthesis to in vivo use.



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org.highwire.dtl.DTLVardef@bbc175org.highwire.dtl.DTLVardef@9a0b94org.highwire.dtl.DTLVardef@16e22e1org.highwire.dtl.DTLVardef@1b40a84_HPS_FORMAT_FIGEXP  M_FIG C_FIG Local fitness and epistatic effects lead to distinct patterns of linkagedisequilibrium in protein-coding genes Selected mutations interfere and interact with evolutionary processes at nearby loci, distorting allele frequency trajectories and creating correlations between pairs of mutations. A number of recent studies have used patterns of linkage disequilibrium (LD) between selected variants to test for selective interference and epistatic interactions, with some disagreement over interpreting observations from data. Interpretation is hindered by a lack of analytic or even numerical expectations for patterns of variation between pairs of loci under the combined effects of selection, dominance, epistasis, and demography. Here, I develop a numerical approach to compute the expected two-locus sampling distribution under diploid selection with arbitrary epistasis and dominance, recombination, and variable population size. I use this to explore how epistasis and dominance affect expected signed LD, including for non-steadystate demography relevant to human populations. Using whole-genome sequencing data from humans, I explore genome-wide patterns of LD within protein-coding genes. I show that positive LD between missense mutations within genes is driven by strong positive allele-frequency correlations between pairs of mutations that fall within the same annotated conserved domain, pointing to compensatory mutations or antagonistic epistasis as the prevailing mode of interaction within conserved genic elements. LD between missense mutations is reduced outside of conserved domains, as would expected under Hill-Robertson interference. This variation in both mutational fitness effects and selective interactions within proteincoding genes calls for more refined inferences of the joint distribution of fitness and interactive effects, and the methods presented here should prove useful in that pursuit. FACT regulates pluripotency through distal regulation of gene expression in murine embryonic stem cells The FACT complex is a conserved histone chaperone with essential roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear. To determine this mechanism, we used inducible depletion of FACT subunit SPT16 in murine embryonic stem cells paired with genomic factor localization, nascent transcription, and chromatin accessibility analyses. Over a timecourse of SPT16 depletion, nucleosomes invade loci bound by master pluripotency factors and gene-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (OCT4), Sox2, Nanog, and enhancer RNAs produced at the genes' associated enhancers are downregulated, suggesting that FACT regulates expression of the pluripotency factors themselves. We find that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency. An alignment free approach confirms semantic properties of species proteomes Alignment-based methods dominate molecular biology. However, by primarily allowing one-to-one comparisons, these methods are focused on a gene-centered viewpoint and lack the broad context essential to analyze how complex biological systems function and evolve. In actuality, a gene is part of genome where more than one sequence contributes to the functional network and evolutionary trajectory of the cell. The need for conservation of established interactions, is arguably more important to the evolutionary success of species than conservation of individual function. To test whether such contextual information exists, a distributional semantics method - Latent Semantic Analysis (LSA), was applied to thousands of species proteomes. Using natural language processing, Latent Taxonomic Signatures (LTSs) were identified that outperformed existing alignment-based BLAST methods when random protein sequences were being mapped to annotated taxonomy according to GenBank. LTSs are a novel proteome distributed feature, suggesting the existence of evolutionary constraints imposed on individual proteins by their proteome context. Even orphan proteins are exhibiting LTSs, which makes their uniqueness linked to a specific taxonomic level questionable. Unlike more simple bias, LTSs represent a self-similarity pattern, where random sets of species proteins show the same statistical properties of a complete proteome at many scales. Natural language processing and machine learning provide insights not easily discernable using alignment based methods suggestive there is more to species related differences than just translational optimization. Single-Cell Multiomics Reveals Distinct Cell States at the Top of the Human Hematopoietic Hierarchy The advent of single cell (Sc) genomics has challenged the dogma of haematopoiesis as a tree-like structure of stepwise lineage commitment through distinct and increasingly restricted progenitor populations. Instead, analysis of ScRNA-seq has proposed that the earliest events in human hematopoietic stem cell (HSC) differentiation are characterized by only subtle molecular changes, with hematopoietic stem and progenitor cells (HSPCs) existing as a continuum of low-primed cell-states that gradually transition into a specific lineage (CLOUD-HSPCs). Here, we combine ScRNA-seq, ScATAC-seq and cell surface proteomics to dissect the heterogeneity of CLOUD-HSPCs at different stages of human life. Within CLOUD-HSPCs, pseudotime ordering of both mRNA and chromatin data revealed a bifurcation of megakaryocyte/erythroid and lympho/myeloid trajectories immediately downstream a subpopulation with an HSC-specific enhancer signature. Importantly, both HSCs and lineage-restricted progenitor populations could be prospectively isolated based on correlation of their molecular signatures with CD35 and CD11A expression, respectively. Moreover, we describe the changes that occur in this heterogeneity as hematopoiesis develops from neonatal to aged bone marrow, including an increase of HSCs and depletion of lympho-myeloid biased MPPs. Thus, this study dissects the heterogeneity of human CLOUD-HSPCs revealing distinct HSPC-states of relevance in homeostatic settings such as ageing. Algal photosystem I dimer and high resolution model of PSI:plastocyanin complex Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here, we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Our cryo-EM structure identifies that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-LHCI monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH, and thus partially overlaps with the surface area that would bind one of the LHCII complexes in state transitions. We also define the most accurate available PSI-LHCI model at 2.3 [A] resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations of all the pigments, as well as 621 water molecules that affect energy transfer pathways.