title: Quantitative stable-isotope probing (qSIP) with metagenomics links microbial physiology and activity to soil moisture in Mediterranean-climate grassland ecosystems

abstract: The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are likely dependent on current and prior soil moisture levels. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) to an H218O labeling experiment to determine which microbial community members, and with what capacities, are growing under in situ conditions. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve an active subset of soil community members and identify the ecophysiological traits that characterize them. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to bacteria that were heavily isotopically labeled, we identified abundant isotope-labeled phages suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capabilities to decompose the diverse complex carbohydrates common in soil necromass or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation.


title: ATP induced conformational change of axonemal outer dynein arms studied by cryo-electron tomography

abstract: Axonemal dyneins in the outer dynein arm (ODA) generate force for ciliary beating. We analyzed three states of ODA during the power stroke cycle using in situ cryo-electron tomography, subtomogram averaging and classification. These states of force generation depict the pre-power stroke, post-power stroke conformations and an intermediate state. Comparison of these conformations to published in vitro atomic structures of cytoplasmic dynein, ODA and Shulin-ODA complex showed that the orientation and position of the dynein head and linker differs. Our analysis shows that all dynein linkers in the in the absence of ATP interact with AAA3/AAA4, indicating interaction to the adjacent B-tubule direct dynein orientation. For the pre-power stroke conformation, we found changes of the tail anchored on the A-tubule. We built pseudo-atomic models from high-resolution structures to generate a best fitting atomic model to the in-situ pre- and post-power stroke ODA, thereby showing that the Shulin-ODA display similar conformation as the active pre-power stroke ODA conformation in the axoneme.

category: molecular biology


title: Extreme tolerable winds for seabirds are determined by morphology

abstract: Flying seabirds are adapted for windy environments1,2. Despite this, storms can cause widespread strandings and wrecks, demonstrating that these seabirds are not always able to avoid or compensate for extreme conditions3,4,5,6,7. The maximum wind speeds that birds can operate in should vary with morphology and flight style8, but this has been hard to quantify due to the challenges of collecting data during infrequent events9. Yet this information is crucial for predicting how seabirds are im-pacted by and respond to extreme events, which are expected to increase in intensity and frequency under climate change10,11. We analyzed > 300,000 hours of tracking data from 18 seabird species, representing all major seabird guilds in terms of flight style. We quantified the range of wind speeds that seabirds use during their foraging trips in relation to the wind speeds available, and assessed evidence for avoidance of particular wind conditions. The maximum wind speeds that birds flew in increased with wing loading, in line with general aeronautical predictions. Two species of albatross flew in extreme winds > 23 m s-1. Within the 18 species studied, we found no general preference or avoidance of specific wind speeds. Nonetheless, in a very small number of instances, albatrosses avoided speeds below their operable maxima, demonstrating that even the most wind-adapted birds avoid extreme speeds in particular scenarios. The Atlantic yellow-nosed albatross and the wandering albatross avoided the maximum wind speeds by flying towards and tracking the eye of the storm. Extreme winds therefore might pose context-dependent risks to seabirds, and there is a need for more information on the factors that determine the hierarchy of risk, given the impact of global change on storm intensity.

category: animal behavior and cognition


title: Scalable sequence-informed embedding of single-cell ATAC-seq data with CellSpace

abstract: Standard scATAC-seq analysis pipelines represent cells as sparse numeric vectors relative to an atlas of peaks or genomic tiles and consequently ignore genomic sequence information at accessible loci. We present CellSpace, an efficient and scalable sequence-informed embedding algorithm for scATAC-seq that learns a mapping of DNA k-mers and cells to the same space. CellSpace captures meaningful latent structure in scATAC-seq datasets, including cell subpopulations and developmental hierarchies, and scores the activity of transcription factors in single cells based on proximity to binding motifs embedded in the same space. Importantly, CellSpace implicitly mitigates batch effects arising from multiple samples, donors, or assays, even when individual datasets are processed relative to different peak atlases. Thus, CellSpace provides a powerful tool for integrating and interpreting large-scale scATAC-seq compendia.

category: bioinformatics


title: A new species of deepwater Lethrinops (Cichlidae) from Lake Malawi

abstract: A new species of cichlid fish, Lethrinops atrilabris is described from specimens collected by trawling at a depth of around 90m off Monkey Bay, southern Lake Malawi. It is assigned to the genus Lethrinops on the basis of its vertical flank barring, lack of enlarged cephalic lateral line canal pores and the form of the lower jaw dental arcade. It can be distinguished from congeneric species by its male breeding dress of contrasting flank barring and dark ventral surface, most strikingly on the lips, throat and chest, its relatively small maximum size (<75mm SL), large eyes (38-41% head length), laterally compressed body (depth 2.5-2.7 times max head width) and lower gillraker count (13-14).

category: zoology


title: EGR2 promoter antisense RNA coordinates chromatin remodeling and spatial genome organization in Schwann cells.

abstract:
The EGR2 promoter antisense RNA (AS-RNA) recruits chromatin remodeling complexes to inhibit EGR2 transcription following peripheral nerve injury. Here we show that the EGR2-AS-RNA modulates chromatin accessibility and serves as scaffold for two distinct histone modification complexes. It binds to EZH2 and WDR5 and enables coordinate targeting of H3K27me3 and H3K4me3 to promoters of EGR2and C-JUN. Expression of the AS-RNA results in reorganization of the global chromatin landscape and quantitative changes in loop formation and in contact frequency at domain boundaries exhibiting enrichment for AP-1 genes. In addition, the EGR2-AS-RNA induces changes in hierarchical TADs and increases transcriptional activity on an inter-TAD loop between a super-enhancer regulatory hub and the promoter of mTOR. Our results show that the EGR2-AS-RNA may serve as regulator of coordinate chromatin remodeling and spatial genome organization to regulate cellular plasticity-associated transcription in Schwann cells.

Highlights- Overexpression of the EGR2-AS-RNA regulates gene expression in Schwann Cells
- The EGR2-AS-RNA binds and recruits repressive and activating chromatin modifiers on the EGR2 and C-JUN promoters, repsectively
- ATAC-seq data showed an increase in the AP-1 network accessibility/TF activity
- The EGR2-AS-RNA promotes reorganization of the global chromatin landscape, quantitative changes in loop formation and in contact frequency at domain boundaries exhibiting enrichment for AP-1 genes

category: neuroscience


title: Perchlorate-Specific Proteomic Stress Responses of Debaryomyces hansenii Could Enable Microbial Survival in Martian Brines

abstract: If life exists on Mars, it would face several challenges including the presence of perchlorates, which destabilize biomacromolecules by inducing chaotropic stress. However, little is known about perchlorate toxicity for microorganism on the cellular level. Here we present the first proteomic investigation on the perchlorate-specific stress responses of the halotolerant yeast Debaryomyces hansenii and compare these to generally known salt stress adaptations. We found that the responses to NaCl and NaClO4-induced stresses share many common metabolic features, e.g., signaling pathways, elevated energy metabolism, or osmolyte biosynthesis. However, several new perchlorate-specific stress responses could be identified, such as protein glycosylation and cell wall remodulations, presumably in order to stabilize protein structures and the cell envelope. These stress responses would also be relevant for life on Mars, which - given the environmental conditions - likely developed chaotropic defense strategies such as stabilized confirmations of biomacromolecules and the formation of cell clusters.

category: cell biology


title: Insight into RNA-DNA primer length counting by human primosome

abstract: The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Pol), synthesizes chimeric RNA-DNA primers for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA-DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (~35-mer): 1) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58C) with the primer 5-end, and 2) flexible tethering of p58C and the DNA polymerase alpha catalytic core domain (p180core) to the primosome platform domain by extended linkers. The obtained data allows us to conclude that p58C is a key regulator of all steps of RNA-DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.

category: biochemistry


title: Highly dense and scalable protein arrays for single-molecule studies

abstract: Single-molecule protein studies are challenging because both single-molecule sensitivity and a high dynamic range are needed to comprehensively analyze the complex mixture of proteins present in biologically relevant samples. One way to achieve both high sensitivity and dynamic range would be to create a protein array that arranges single molecules with uniform spacing to achieve billions of well-separated molecules on a patterned surface. So far, no approach has combined both a uniformly patterned surface and the possibility to deposit a single protein at each spot. In addition, current patterning approaches cannot be scaled to the size needed to interrogate billions of molecules. Here, we present a scalable method for fabricating dense protein arrays using brushy-DNA origami deposition onto patterned surfaces. Brushy-origami structures are made large enough to occupy the features on the array singly and we developed an efficient process to ensure single protein conjugation to the brushy-origami structures. We hypothesize that our technology will ultimately enable the development of highly scalable proteomics platforms that address the need for both single-molecule sensitivity and high dynamic range.

category: bioengineering


title: Convergence on reduced aggression through shared behavioral traits in multiple populations of Astyanax mexicanus

abstract:
Aggression is a complex behavior that is observed across the animal kingdom, and plays roles in resource acquisition, defense, and reproductive success. While there are many individual differences in propensity to be aggressive within and between populations, the mechanisms underlying differences in aggression between individuals in natural populations are not well understood. We addressed this using the Mexican tetra, Astyanax mexicanus, a powerful model organism to understand behavioral evolution. A. mexicanus exists in two forms: a river-dwelling surface form and multiple populations of a blind cave form. We characterized aggression in surface fish and cavefish in a resident/intruder assay through quantifying multiple behaviors occurring during social interactions. Surface fish, which are aggressive, display multiple social behaviors in this context, which we characterized into two types of behaviors: aggression- associated and escape-associated behaviors. The majority of these behaviors were reduced or lost in Pachon cavefish. Further, both aggression-associated and escape-associated behaviors were not dependent on the presence of light, and both surface fish and cavefish remained aggressive or non-aggressive, respectively, when opposed to fish from a different population. Additionally, we found that within populations, levels of stress response were not correlated with aggression- or escape-associated behaviors. Finally, when we compared aggression- and escape- associated behaviors across four cavefish populations, we found that both types of behaviors are reduced in three cave populations, while still present in one. Together, these results reveal that multiple cavefish populations have repeatedly evolved reduced aggression through shared behavioral components, while other cavefish have retained aggression.

Summary StatementComparison of aggression between surface fish and cavefish demonstrates that multiple complex behaviors compose aggression in surface fish and reveals heterogeneity in loss of aggression in cave populations.

category: evolutionary biology


title: The swan genome and transcriptome: its not all black and white

abstract: The Australian black swan (Cygnus atratus) is an iconic species with contrasting plumage to that of the closely related Northern Hemisphere white swans. The relative geographic isolation of the black swan may have resulted in a limited immune repertoire and increased susceptibility to infectious disease, notably infectious diseases from which Australia has been largely shielded. Indeed, unlike Mallard ducks and the mute swan (Cygnus olor), the black swan is extremely sensitive to severe highly pathogenic avian influenza (HPAI). Understanding this susceptibility has been impaired by the absence of any available swan genome and transcriptome information. Here, we generate the first chromosome-length annotated black and mute swan genomes annotated with transcriptome data, all using long-read based pipelines generated for vertebrate species. We used these genomes and transcriptomes, to show that unlike other wild waterfowl, black swans lack an expanded immune gene repertoire, lack a key viral pattern-recognition receptor in endothelial cells and mount a poorly controlled inflammatory response to HPAI. We also implicate genetic differences in SLC45A2 in the iconic plumage of the Australian black swan. Together, these data suggest that the immune system of the black swan is such that should any avian viral infection become established in its native habitat the survival of the black swan would be in significant peril.

category: genomics


title: Modeling the kinetics of amyloid beta pores and long-term evolution of their Ca2+ toxicity

abstract: The formation of cation-permeable pores due to amyloid beta (A{beta}) in the plasma membrane (PM) and membranes of intracellular organelles has been shown to cause disruption of Ca2+ homeostasis in Alzheimers disease, leading to neuronal malfunction and degeneration. However, detailed information about the formation, function, and time-evolution of A{beta} pores are still lacking. Here we present a comprehensive analysis to show that the Ca2+ toxicity of PM pores formed by A{beta}42 progressively increases over the duration of the experiments that last for more than an hour. This mainly results from a progressively increasing number of pores and activity of individual pores having higher open probability (Po) and Ca2+ conductance. To gain deeper insight into the function and evolution of A{beta}42 pores and establish their role in Ca2+ toxicity beyond experimental times scales, we developed a data-driven computational model that not only mimics the kinetics of these pores at a given time but also tracks their time-evolution over many hours. Our model suggests that the rising toxicity of A{beta}42 pores over time is a combined result of the large number of short-lived pores with low Ca2+ conductance and long-lived higher-conductance pores but fewer in number. Analysis and modeling of the voltage-dependent data show that A{beta}42 pores are most active when the cells membrane potential is near resting values, making the cell at rest especially more prone to Ca2+ disruptions.


title: Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2

abstract: We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly gRNA/library design in custom genomes. GuideScan2 analysis identified widespread confounding effects of low-specificity gRNAs in published CRISPR knockout, interference and activation screens and enabled construction of a ready-to-use gRNA library that reduced off-target effects in a novel gene essentiality screen. GuideScan2 also enabled the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome.


title: Discovery and structure of a widespread bacterial ABC transporter specific for ergothioneine

abstract: Ergothioneine (ET) is the 2-thiourea derivative of trimethylhistidine that is biosynthesized only by select fungi and bacteria, notably Mycobacterium tuberculosis, and functions as a potent scavenger of reactive oxygen species. Although ET is obtained in the diet and accumulates in vertebrate cells via an ET-specific transporter, the extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd_1642-1643 in Streptococcus pneumoniae D39, a Gram-positive respiratory pathogen, encodes a novel ergothioneine uptake ATP-binding cassette (ABC) transporter, which we designate EgtUV. EgtU is a permease-solute binding domain (SBD) fusion protein, and the SBD binds ET with high affinity and exquisite specificity in the cleft between the two subdomains, with cation-{pi} interactions engaging the betaine moiety and a water-mediated hydrogen bonding network surrounding the C2-sulfur-containing imidazole ring. Bioinformatics studies reveal that EgtUV is uniquely strongly conserved among known quaternary amine-specific transporters and widely distributed in firmicutes, including the human pathogens Listeria monocytogenes, as BilEB, Enterococcus faecalis and Staphylococcus aureus. This discovery significantly diversifies the LMW thiol pool in Gram-positive human pathogens that may contribute to antioxidant defenses in the infected host.


title: TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity

abstract: Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/Interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded NADases produce signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR sub-classes. Here we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) via ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of di-ADPR or ADPr-ATP allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A) in vitro and in planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.

category: plant biology


title: Deep multi-omic network fusion for marker discovery of Alzheimer's Disease

abstract:
MotivationMulti-omic data spanning from genotype, gene expression to protein expression have been increasingly explored, with attempt to better interpret genetic findings from genome wide association studies and to gain more insight of the disease mechanism. However, gene expression and protein expression are part of dynamic process changing in various ways as a cell ages. Expression data captured by existing technology is often noisy and only capture a screenshot of the dynamic process. Performance of models built on top of these expression data is undoubtedly compromised. To address this problem, we propose a new interpretable deep multi-omic network fusion model (MoFNet) for predictive modeling of Alzheimers disease. In particular, the information flow from DNA to protein is leveraged as a prior multi-omic network to enhance the signal in gene and protein expression data so as to achieve better prediction power.

ResultsThe proposed model MoFNet significantly outperformed all other state-of-art classifiers when evaluated using genotype, gene expression and protein expression data from the ROS/MAP cohort. Instead of individual markers, MoFNet yielded 3 major multi-omic subnetworks related to innate immune system, clearance of unwanted cells or misfolded proteins, and neurotransmitter release respectively.

AvailabilityThe source code is available through GitHub (https://github.com/yashraj59/MoFNet). Multi-omic data used in this analysis is from the ROS/MAP project and is available upon application through the AMP-AD knowledge portal (https://adknowledgeportal.synapse.org).

category: systems biology


title: Disrupting the ciliary gradient of active Arl3 affects rod photoreceptor nuclear migration

abstract: The small GTPase Arl3 is important for the enrichment of lipidated proteins to primary cilia, including the outer segment of photoreceptors. Human mutations in the small GTPase Arl3 cause both autosomal recessive and dominant inherited retinal dystrophies. We discovered that dominant mutations result in aberrant activity--Arl3-D67V has constitutively activity and Arl3-Y90C is fast cycling--and expression in mouse rods resulted in a displaced nuclear phenotype, similar to the GTP-locked Q71L mutant. Using multiple strategies, we go on to show that removing or restoring the Arl3-GTP gradient within the cilium is sufficient to rescue the nuclear migration defect. Together, our results reveal that a Arl3 ciliary gradient is involved in proper positioning of photoreceptor nuclei during retinal development.


title: Puberty contributes to adolescent development of fronto-striatal functional connectivity supporting inhibitory control

abstract: Adolescence is defined by puberty and represents a neurobiological period characterized by enhanced neuroplasticity facilitating cognitive improvements. Fronto-striatal systems undergo important specialization throughout adolescence, supporting developmental changes in cognition and motivated behaviors. Though studies have characterized age-related changes, the extent to which puberty influences maturation of fronto-striatal networks is less known, limiting our understanding of unique adolescent processes. Here, we combine two longitudinal datasets to characterize the role of puberty in the development of fronto-striatal resting-state functional connectivity (rsFC) and its relationship to inhibitory control in 110 10-18-year-olds. Not surprisingly, age-effects evident with older age ranges were not present in this sample. After controlling for age effects still possibly present, puberty was associated uniquely with late increases rsFC of nucleus accumbens (NAcc) and dorsolateral prefrontal cortex (dlPFC) in both males and females and early puberty increases with ventrolateral PFC (vlPFC) in females. Additionally, greater dlPFC - NAcc rsFC was associated with worse inhibitory control performance in early puberty while vlPFC - NAcc was associated with response latency. Taken together, our findings suggest that mid-late puberty is a crucial period for lateral PFC - NAcc circuitry maturation, which may contribute to critical aspects of developmental improvements in inhibitory control function into adulthood.


title: CaV1 and CaV2 calcium channels mediate the release of distinct pools of synaptic vesicles

abstract: Activation of voltage-gated calcium channels at synapses leads to local increases in calcium and the fusion of synaptic vesicles. However, presynaptic output will be determined by the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at C. elegans neuromuscular junctions, CaV2, and CaV1 mediate the release of two distinct pools of synaptic vesicles. Superresolution microscopy demonstrates that CaV2 channels are concentrated in densely packed clusters [~]300 nm in diameter with active zone proteins Neurexin, -Liprin, SYDE, ELKS, RIMB, -Catulin, and MAGI. The CaV2 channels mediate the fusion of vesicles docked within 100 nm of the dense projection and is colocalized with to the synaptic vesicle priming protein UNC-13L. By contrast, CaV1 channels are dispersed in the synaptic varicosity and are coupled to internal calcium stores via the ryanodine receptor. The CaV1 and ryanodine receptor mediate the fusion of vesicles docked broadly in the synaptic varicosity and are colocalized with the vesicle priming protein UNC-13S. These distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.


title: Axl contributes to efficient migration and invasion of melanoma cells

abstract: Axl, a member of the TAM receptor family has been broadly suggested to play a key role in tumor metastasis. However, the function of Axl in the invasion and metastasis of melanoma, the most lethal skin cancer, remains largely unknown. In the present study, we found that melanoma cell lines present variable protein levels of Axl and Tyro3; interestingly, MerTK is not noted at detectable levels in any of tested MGP (metastatic growth phase) cell lines. Treatment with recombinant human Gas6 significantly activates Akt in the Axl-expressing WM852 and IgR3 lines but just slightly in WM1158. IgR3, WM852 and WM1158 demonstrate different autocrine signaling. Knockdown of Axl by siRNA or the treatment with Axl-specific inhibitor R428 dramatically inhibits the migration and invasion of both IgR3 and WM852 in vitro. These findings suggest that Axl enhances the invasion of melanoma cells.


title: Deeply conserved super-enhancers maintain stem cell pluripotency in placental mammals

abstract:
Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share a deep evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of the transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) which displayed deep conservation in placental mammals and are sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR/Cas9 approach severely impaired the proliferative potential and the ability to form undifferentiated colonies. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.

Significance statementSuper-enhancers (SEs) hold stronger power than regular enhancers to direct gene expression in the regulation of stem cell pluripotency. To dissect how pluripotency-associated SEs have evolved in mammals, we performed a systematic comparison of SEs among pigs, humans, and mice. Our analysis allowed the identification of three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that are highly conserved in Placentalia (accounting for 94% of mammals) as well as many species-specific SEs. All three SEs were sufficient to direct pluripotency-dependent gene expression and disruption of each conserved SE caused the loss of stem cell pluripotency. Our work highlights a small number of highly conserved SEs essential for the maintenance of pluripotency.


title: Pallidin function in drosophila surface glia regulates sleep and is dependent on amino acid availability

abstract: The Pallidin protein is a component of a multimeric complex named the Biogenesis of Lysosome-related Organelles Complex-1 (BLOC1) that regulates specific endosomal function and transmembrane protein trafficking in many different cell types. In the brain, defective BLOC1 function has been linked to schizophrenia, a neuropsychiatric disorder with highly prevalent sleep disruptions, and to impaired cognitive abilities in healthy subjects. In animal models, defective BLOC-1 function also impairs behavior, memory, neurotransmission systems and metabolism. This growing body of experimental evidence suggest an involvement of BLOC1 in sleep/wake regulation. Here, we used Drosophila molecular genetics and conditional, cell-type specific knockdown strategy to address this question. We show that down-regulation of a central subunit of BLOC1, Pallidin, in the surface glia, the Drosophila equivalent of the blood brain barrier, is sufficient to reduce, fragment and delay nighttime sleep at the adult stage and in a circadian clock dependent manner. Other members of the BLOC1 complex appear to be involved in this surface glia-dependent sleep regulation. In agreement with a BLOC1 involvement in amino acid transport, down-regulation of the Large neutral Amino acid Transporter 1 (LAT1)-like transporters JhI-21 and minidiscs, phenocopy the down-regulation of pallidin. Similar results were obtained by inhibiting the TOR amino acid signaling pathway. Supplementing food with essential amino acids normalizes the sleep/wake phenotypes of pallidin and JhI-21 down-regulation. Furthermore, we identify a role for pallidin in the subcellular trafficking of JhI-21 in surface glial cells. Finally, we provide evidence that Pallidin function in surface glia is required for GABAergic neurons activation involved in promoting sleep. Taken together, these data identify a novel role for BLOC1 that, through LAT1-like transporters subcellular trafficking modulates essential amino acid availability and GABAergic sleep/wake regulation.


title: Characterization and mu opioid receptor sensitivity of neuropeptide Y interneurons in the nucleus accumbens

abstract: Inhibitory interneurons represent less than 5% of neurons within the nucleus accumbens, but are critical for proper microcircuit function within this brain region. In the dorsal striatum, neuropeptide Y is expressed by two interneuron subtypes (low-threshold spiking interneurons and neurogliaform interneurons) that exhibit mu opioid receptor sensitivity in other brain regions. However, few studies have assessed the molecular and physiological properties of neuropeptide Y interneurons within the nucleus accumbens. We used a transgenic reporter mouse to identify and characterize neuropeptide Y interneurons in acute nucleus accumbens brain slices. Nearly all cells exhibited electrophysiological properties of low-threshold spiking interneurons, with almost no neurogliaform interneurons observed among neuropeptide Y interneurons. We corroborated this pattern using fluorescent in situ hybridization, and also identified a high level of mu opioid receptor expression by low-threshold spiking interneurons, which led us to examine the functional consequences of mu opioid receptor activation in these cells using electrophysiology. Mu opioid receptor activation caused a reduction in the rate of spontaneous action potentials in low-threshold spiking interneurons, as well as a decrease in optogenetically-evoked GABA release onto medium spiny neurons. The latter effect was more robust in female versus male mice, and when the postsynaptic medium spiny neuron expressed the Drd1 dopamine receptor. This work is the first to examine the physiological properties of neuropeptide Y interneurons in the nucleus accumbens, and show they may be an important target for mu opioid receptor modulation by endogenous and exogenous opioids.


title: Chromosomal inversions can limit adaptation to new environments

abstract: Chromosomal inversions are often thought to facilitate local adaptation and population divergence because they can link multiple adaptive alleles into non-recombining genomic blocks. Selection should thus be more efficient in driving inversion-linked adaptive alleles to high frequency in a population, particularly in the face of maladaptive gene flow. But, what if ecological conditions and hence selection on inversion-linked alleles change? Reduced recombination within inversions could then constrain the formation of optimal combinations of pre-existing alleles under these new ecological conditions. Here, we outline this idea of inversions limiting adaptation and divergence when ecological conditions change across time or space. We reason that the benefit of inversions for local adaptation and divergence under one set of ecological conditions can come with a concomitant constraint for adaptation to novel sets of ecological conditions. This limitation of inversions to adaptation may also provide one possible explanation for why inversions are often maintained as polymorphisms within species.


title: Chromosome-level Genomes Reveal the Genetic Basis of Descending Dysploidy and Sex Determination in Morus Plants

abstract: Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation. Morus notabilis, a dioecious mulberry species, has the fewest chromosomes among Morus spp., but the genetic basis of sex determination and karyotype evolution in this species have not been identified. Three high-quality genome assemblies generated of Morus spp. (including those of dioecious M. notabilis and Morus yunnanensis) were within the range 301-329 Mb in size which were grouped into six pseudochromosomes. Using a combination of genomic approaches, we showed that the putative ancestral karyotype of Morus was close to 14 protochromosomes, and that several chromosome fusion events resulted in descending dysploidy (2n = 2x = 12). We also characterized a [~]6.2-Mb sex-determining region on chromosome 3. The four potential male-specific genes, including a partially duplicated DNA helicase gene orthologue (named MSDH) and three Ty3_Gypsy long terminal repeat retrotransposons (named MSTG), were solely identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation. Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry. In addition, genomic areas containing selective sweeps that distinguish domesticated mulberry trees from wild populations in terms of flowering and disease resistance were identified. Our findings provide an important genetic resource for sex identification and molecular breeding in mulberry.


title: Arc-Expressing Accessory Olfactory Bulb Interneurons Support Chemosensory Social Behavioral Plasticity

abstract:
The accessory olfactory system (AOS) is critical for the development and expression of social behavior. The first dedicated circuit in the AOS, the accessory olfactory bulb (AOB), exhibits cellular and network plasticity in male and female mice after social experience. In the AOB, interneurons called internal granule cells (IGCs) express the plasticity-associated immediate-early gene Arc following intermale aggression or mating. Here, we sought to better understand how Arc-expressing IGCs shape AOB information processing and social behavior in the context of territorial aggression. We used "ArcTRAP" (Arc-CreERT2) transgenic mice to selectively and permanently label Arc-expressing IGCs following male-male resident-intruder interactions. Using whole-cell patch clamp electrophysiology, we found that Arc-expressing IGCs display increased intrinsic excitability for several days after a single resident-intruder interaction. Further, we found that Arc-expressing IGCs maintain this increased excitability across repeated resident-intruder interactions, during which resident mice increase or "ramp" their aggression. We tested the hypothesis that Arc-expressing IGCs participate in ramping aggression. Using a combination of ArcTRAP mice and chemogenetics (Cre-dependent hM4D(Gi)-mCherry AAV injections), we found that disruption of Arc-expressing IGC activity during repeated resident-intruder interactions abolishes the ramping aggression exhibited by resident male mice. This work shows that Arc-expressing AOB IGC ensembles are activated by specific chemosensory environments, and play an integral role in the establishment and expression of sex-typical social behavior. These studies identify a population of plastic interneurons in an early chemosensory circuit that display physiological features consistent with simple memory formation, increasing our understanding of central chemosensory processing and mammalian social behavior.

Significance statementThe accessory olfactory system (AOS) plays a vital role in rodent chemosensory social behavior. We studied experience-dependent plasticity in the accessory olfactory bulb (AOB) and found that internal granule cells (IGCs) expressing the immediate-early gene Arc after the resident-intruder paradigm increase their excitability for several days. We investigated the roles of these Arc-expressing IGCs on chemosensory social behavior by chemogenetically manipulating their excitability during repeated social interactions. We found that inhibiting these cells eliminated intermale aggressive ramping behavior. These studies identify a population of plastic interneurons in an early chemosensory circuit that display physiological features consistent with simple memory formation, increasing our understanding of central chemosensory processing and mammalian social behavior.


title: Protection from Omicron and other VOCs by Bivalent S-Trimer COVID-19 Vaccine

abstract:
The Omicron variant of SARS-COV-2 (GISAID GRA clade [B.1.1.529, BA.1 and BA.2]) is now the single dominant Variant of Concern (VOC). The high number of mutations in the Omicron Spike (S) protein promotes humoral immunological escape. Although a third homologous boost with S, derived from the ancestral strain, was able to increase neutralizing antibody titers and breadth including to Omicron, the magnitude of virus neutralization could benefit from further optimization. Moreover, combining SARS-COV-2 strains as additional valences may address the current antigenicity range occupied by VOCs.

Using Trimer-Tag platform we have previously demonstrated phase 3 efficacy and safety of a prototypic vaccine SCB-2019 in the SPECTRA trial and have submitted applications for licensure. Here, we successfully generated a bivalent vaccine candidate including both Ancestor and Omicron variant S-proteins. Preclinical studies demonstrate this SARS-CoV-2 bivalent S-Trimer subunit vaccine elicits high titers of neutralizing antibodies against all VOCs, with markedly enhanced Omicron specific neutralizing antibody responses.

category: immunology


title: Spatiotemporal landscape of SARS-CoV-2 pulmonary infection reveals Slamf9+Spp1+ macrophages promoting viral clearance and inflammation resolution

abstract: While SARS-CoV-2 pathogenesis has been intensively investigated, the host mechanisms of viral clearance and inflammation resolution are still elusive because of the ethical limitation of human studies based on COVID-19 convalescents. Here we infected Syrian hamsters by authentic SARS-CoV-2 and built an ideal model to simulate the natural recovery process of SARS-CoV-2 infection from severe pneumonia1,2. We developed and applied a spatial transcriptomic sequencing technique with subcellular resolution and tissue-scale extensibility, i.e., Stereo-seq3, together with single-cell RNA sequencing (scRNA-seq), to the entire lung lobes of 45 hamsters and obtained an elaborate map of the pulmonary spatiotemporal changes from acute infection, severe pneumonia to the late viral clearance and inflammation resolution. While SARS-CoV-2 infection caused massive damages to the hamster lungs, including naive T cell infection and deaths related to lymphopenia, we identified a group of monocyte-derived proliferating Slamf9+Spp1+ macrophages, which were SARS-CoV-2 infection-inducible and cell death-resistant, recruiting neutrophils to clear viruses together. After viral clearance, the Slamf9+Spp1+ macrophages differentiated into Trem2+ and Fbp1+ macrophages, both responsible for inflammation resolution and replenishment of alveolar macrophages. The existence of this specific macrophage subpopulation and its descendants were validated by RNAscope in hamsters, immunofluorescence in hACE2 mice, and public human autopsy scRNA-seq data of COVID-19 patients. The spatiotemporal landscape of SARS-CoV-2 infection in hamster lungs and the identification of Slamf9+Spp1+ macrophages that is pivotal to viral clearance and inflammation resolution are important to better understand the critical molecular and cellular players of COVID-19 host defense and also develop potential interventions of COVID-19 immunopathology.


title: Sensitivity of novel SARS-CoV-2 Omicron subvariants, BA.2.11, BA.2.12.1, BA.4 and BA.5 to therapeutic monoclonal antibodies

abstract: As of May 2022, Omicron BA.2 variant is the most dominant variant in the world. Thereafter, Omicron subvariants have emerged and some of them began outcompeting BA.2 in multiple countries. For instance, Omicron BA.2.11, BA.2.12.1 and BA.4/5 subvariants are becoming dominant in France, the USA and South Africa, respectively. In this study, we evaluated the sensitivity of these new Omicron subvariants (BA.2.11, BA.2.12.1 and BA.4/5) to eight therapeutic monoclonal antibodies (bamlanivimab, bebtelovimab, casirivimab, cilgavimab, etesevimab, imdevimab, sotrovimab and tixagevimab). Notably, we showed that although cilgavimab is antiviral against BA.2, BA.4/5 exhibits higher resistance to this antibody compared to BA.2. Since mutations are accumulated in the spike proteins of newly emerging SARS-CoV-2 variants, we suggest the importance of rapid evaluation of the efficiency of therapeutic monoclonal antibodies against novel SARS-CoV-2 variants.

category: microbiology


title: Dietary derived Vitamin B12 protects the nematode Caenorhabditis elegans from thiol reducing agents.

abstract: We describe a novel SAM methyl transferase in the nematode Caenorhabditis elegans that is upregulated by thiol reducing agents and hydrogen sulfide with expression controlled by the hypoxia inducible factor pathway. This methyl transferase, RIPS-1, is expressed in the gut and hypodermis of this nematode with homologues found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. We identified RIPS-1 through forward genetic screens as the only gene that when mutated allowed worms to survive normally lethal concentrations of thiol reducing agents such as dithiothreitol (DTT) and {beta}-mercaptoethanol. The RIPS-1 methyl transferase is an important player in the methionine cycle and its activation consumes methionine in a methionine synthetase and vitamin B12-dependant manner. This requirement limits the availability of vitamin B12 in the mitochondrion. Mitochondrial involvement was also established through a targeted enhancer screen that identified methylmalonyl-CoA epimerase as a strong genetic enhancer of RIPS-1 mutant resistance to DTT. Toxicity associated with thiol reducing agent exposure can be overcome in C. elegans by adding methionine, loss of RIPS-1, or by addition of excess vitamin B12. This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans and defines a new role in countering reductive stress


title: Critical Mechanistic Role of Inositol Hexakisphosphate (IP6) in HIV-1 Viral Capsid Assembly

abstract:
The maturation of HIV-1 capsid protein (CA) into a cone-shaped lattice encasing the condensed RNA genome is critical for viral infectivity. CA can self-assemble into a wide range of capsid morphologies made of [~]175-250 hexamers and exactly 12 pentamers. Most recently, cellular polyanion inositol hexakisphosphate (IP6) has been demonstrated to facilitate conical capsid formation by coordinating a ring of arginine residues within the central cavity of capsid hexamers and pentamers. However, the precise interplay of events during IP6 and CA co-assembly is unclear. In this work, we use Coarse-Grained Molecular Dynamics (CGMD) simulations to elucidate the underlying molecular mechanism that allows IP6 to have crucial roles in HIV-1 maturation. We show that IP6 promotes curvature generation by trapping pentameric defects in the growing lattice and shifts assembly behavior towards kinetically favored outcomes. Our analysis suggests that IP6 can stabilize metastable capsid intermediates and may induce structural pleomorphism in mature capsids.

TeaserCoarse Grained simulations reveal that IP6 promote fullerene-like capsid formation by stabilizing high curvature regions of the capsid.

category: biophysics


title: Actuating tension-loaded DNA clamps drives membrane tubulation

abstract: Membrane dynamics in living organisms can arise from proteins adhering to, assembling on, and exerting force on cell membranes. Programmable synthetic materials, such as self-assembled DNA nanostructures, offer the capability to drive membrane remodeling events in a way that resembles protein-mediated dynamics, but with user-defined outcomes. An example showcasing this capability is the tubular deformation of liposomes by DNA nanostructures with purposely designed shapes, surface modifications, and self-assembling properties. However, stimulus-responsive membrane tubulation mediated by DNA structure reconfiguration remains challenging. Here we present the triggered formation of membrane tubes in response to specific DNA signals that actuate membrane-bound DNA clamps from an open state to various predefined closed states, releasing pre-stored energy to activate membrane deformation. Using giant unilamellar vesicles (GUVs) as a model system, we show that the timing and efficiency of tubulation, as well as the width of membrane tubes, are modulated by the conformational change of DNA clamps, marking a solid step toward spatiotemporal control of membrane dynamics in an artificial system.

category: synthetic biology


title: FoxP3-mediated blockage of ryanodine receptor 2 is the molecular basis for the contact-based suppression by regulatory T cells

abstract:
The suppression mechanism of regulatory T cells is an intensely investigated topic. As our focus has shifted towards a model centered on indirect inhibition of dendritic cells, a universally applicable effector mechanism controlled by FoxP3 expression has not been found. Here, we report that FoxP3 blocks the transcription of ER Ca2+-release channel ryanodine receptor 2. Reduced RyR2 shuts down basal Ca2+ oscillation in Tregs, which reduces m-Calpain activities that is needed for T cells to disengage from DCs, suggesting a persistent blockage of DC antigen presentation. RyR2 deficiency renders the CD4+ T cell pool to become immune suppressive, and behave in the same manner as FoxP3+ Tregs in viral infection, asthma, hypersensitivity, colitis and tumor development. In the absence of FoxP3, RyR2-deficient CD4+ T cells rescue the systemic autoimmunity associated with Scurfy mice. Therefore, FoxP3-mediated Ca2+ signaling inhibition may be a central effector mechanism of Treg immune suppression.

One Sentence SummaryCalcium channel RyR2 dictates Treg adhesion-based suppression


title: The making of the oral microbiome in Agta hunter-gatherers

abstract: Ecological and genetic factors have influenced the composition of the human microbiome during our evolutionary history. We analyzed the oral microbiota of the Agta, a hunter-gatherer population where part of its members is adopting an agricultural diet. We show that age is the strongest factor modulating the microbiome, likely through immunosenescence as there is an increase of pathogenicity with age. Biological and cultural processes generate sexual dimorphism in the oral microbiome. A small subset of oral bacteria is influenced by the host genome, linking host collagen genes to bacterial biofilm formation. Our data also suggests that shifting from a fish/meat to a rice-rich diet transforms their microbiome, mirroring the Neolithic transition. All these factors have implications in the epidemiology of oral diseases. Thus, the human oral microbiome is multifactorial, and shaped by various ecological and social factors that modify the oral environment.


title: Hunter-gatherer oral microbiomes are shaped by contact network structure

abstract: Ancestral humans evolved a complex social structure still observed in extant hunter-gatherers. Here we investigate the effects of extensive sociality and mobility on the oral microbiome of 138 Agta hunter-gatherers from the Philippines. Comparisons of microbiome composition showed that the Agta are more similar to Central African Bayaka hunter-gatherers than to neighboring farmers. We also defined the Agta social microbiome as a set of 137 oral bacteria (only 7% of 1980 amplicon sequence variants) significantly influenced by social contact (quantified through wireless sensors of short-range interactions). We show that interaction networks covering large areas, and their strong links between close kin, spouses, and even unrelated friends, can significantly predict bacterial transmission networks across Agta camps. Finally, more central individuals to social networks are also bacterial supersharers. We conclude that hunter-gatherer social microbiomes, which are predominantly pathogenic, were shaped by evolutionary tradeoffs between extensive sociality and disease spread.


title: Carbapenem heteroresistance of KPC-producing Klebsiella pneumoniae results from tolerance, persistence and resistance.

abstract:
Carbapenemase-producing Klebsiella pneumoniae (CPKp) have disseminated globally and represent a major threat in hospitals with few therapeutic options and high mortality rates. Isolates producing the carbapenemase KPC (KPC-Kp) might be classified as susceptible according to clinical breakpoints by antibiotic susceptibility testing (AST), allowing the use of imipenem or meropenem for treatment of infections. However, some KPC-Kp show heteroresistance (HR) to carbapenems, with colonies growing in the inhibition halo of agar-based AST. HR KPC-Kp have been associated with a higher risk of treatment failure. Here, we characterized the diversity of mechanisms behind HR to imipenem of these isolates. By analyzing a diverse collection of CPKp, we showed that HR is frequent among KPC-Kp. By monitoring single HR colony appearance using the ScanLag setup, we discriminated surviving cells in two subpopulations leading to a Gaussian-like distribution of early-appearing colonies, with a delayed emergence compared to colonies arising in the absence of antibiotics, and a long tail of late-appearing colonies. A subset of colonies showed a reduced growth rate. Characterization of surviving populations by AST and whole-genome sequencing of 333 colonies revealed a majority of parental genotypes and a broad landscape of genetic alterations in 28% of the colonies, including gene loss, DNA amplification and point mutations. This unveils the complexity of imipenem HR among KPC-Kp isolates, which involves tolerant and persistent cells, but also resistant bacteria. These observations contribute to a better understanding of reasons behind carbapenem treatment failure of KPC-Kp isolates.

AUTHOR SUMMARYIn the course of an antibiotic treatment, bacteria will encounter varying drug concentrations with different potential for their elimination and resistance selection. The ability of a bacterium to defeat antibiotics not only depends upon resistance, but also on tolerance and persistence, which allow a bacterial population to temporarily survive high drug doses. Carbapenems are antibiotics of last resort and Klebsiella pneumoniae isolates producing the carbapenemase KPC are a threat to hospitals, although they might remain susceptible to carbapenems. However, seemingly homogeneous populations of KPC-K. pneumoniae isolates frequently show varying degrees of susceptibility to carbapenem, i.e., a phenomenon called heteroresistance. We characterized bacteria surviving a high dose of imipenem, progressively degraded by the released carbapenemase, by monitoring the growth of the resulting colonies using the ScanLag system, their genome sequence and carbapenem susceptibility. We show that the observed phenotypic diversity combines tolerance, persistence and resistance making the treatment with high doses of carbapenems frequently inefficient.


title: The dually localized EF-hand domain-containing protein TgEFP1 regulates the lytic cycle of Toxoplasma gondii

abstract: The propagation of the obligate intracellular parasite Toxoplasma gondii is tightly regulated by calcium signaling. However, the mechanisms by which calcium homeostasis and fluxes are regulated in this human pathogen are not fully understood. To identify Toxoplasmas calcium homeostasis network, we have characterized a novel EF-hand domain-containing protein, which we have named TgEFP1. We have determined that TgEFP1 localizes to a previously described compartment known as the plant-like vacuole or the endo-lysosomal compartment (PLV/ELC), which harbors several proteins related to ionic regulation. Interestingly, partial permeabilization techniques showed that TgEFP1 is also secreted into the parasitophorous vacuole (PV), within which the parasite divides. Ultrastructure expansion microscopy confirmed the unusual dual localization of TgEFP1 at the PLV/ELC and the PV. Furthermore, we determined that the localization of TgEFP1 to the PV, but not to the PLV/ELC, is affected by disruption of Golgi-dependent transport with Brefeldin A. Knockout of TgEFP1 results in faster propagation in tissue culture, hypersensitivity to calcium ionophore-induced egress, and premature natural egress. Thus, our work has revealed an interplay between the PV and the PLV/ELC and a role for TgEFP1 in the regulation of calcium-dependent events.


title: Intermittent antibiotic treatment of bacterial biofilms favors the rapid evolution of resistance

abstract: The rise of antibiotic resistance in bacterial pathogens is a major health concern and the determinants of this emergence are actively studied. By contrast, although biofilms are an important cause of infections due to their high tolerance to a broad range of antimicrobials, much less is known on the development of antibiotic resistance within the biofilm environment, an issue potentially aggravating the current antibiotic crisis. Here, we compared the occurrence of resistance mutations in pathogenic Escherichia coli planktonic and biofilm populations exposed to clinically relevant cycles of lethal treatments with the aminoglycoside antibiotic amikacin. This experimental evolution approach revealed that mutations in sbmA and fusA are rapidly selected in biofilm but not in planktonic populations. The apparition of these bona fide resistance --and not tolerance-- mutations is favored by the biofilm preexisting tolerance and high mutation rate. Moreover, we showed that while fusA mutations displayed a high fitness cost in planktonic conditions, these mutations were maintained in biofilms, a phenomenon further possibly amplified by the selection of fimH mutations favoring biofilm formation itself. Our study therefore provides new insights into the dynamic evolution of antibiotic resistance in biofilms, which could lead to clinically practical antibiotic regimen limiting biofilm-associated infections, while mitigating the emergence of worrisome antibiotic resistance mutations.


title: Antibiotic-induced accumulation of lipid II sensitizes bacteria to antimicrobial fatty acids

abstract: Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is of major clinical importance for the treatment of Gram-positive bacterial infections. Here we show that unsaturated fatty acids (UFAs) and vancomycin act synergistically to rapidly kill S. aureus, including vancomycin tolerant and resistant populations. Our results suggest that antibiotic-mediated accumulation of lipid II at the septum facilitates membrane invasion by antimicrobial UFAs. UFA-vancomycin dual treatment generates large fluid patches of flexible lipids in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. This mechanism of synergy may be exploited for the development of new antibiotic therapies that target lipid II to combat both antibiotic tolerance and resistance.


title: Viruses inhibit TIR gcADPR signaling to overcome bacterial defense

abstract: The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants, and animals. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signaling molecule whose molecular structure remained elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function. We identified a large family of phage-encoded proteins, denoted here Thoeris anti-defense 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are "sponges" that bind and sequester the immune signaling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. A high-resolution crystal structure of Tad1 bound to the signaling molecule revealed that its chemical structure is 1'-2' glycocyclic ADPR (gcADPR), a unique molecule not previously described in other biological systems. Our results define the chemical structure of a central immune signaling molecule, and reveal a new mode of action by which pathogens can suppress host immunity.


title: High-efficiency HDR in immortalized cell lines by crude rAAV mediated donor template delivery

abstract: Owing to low efficiency of homology-directed repair (HDR), precise knock-in (KI) of large DNA fragments is a challenge in genome editing. High-efficiency HDR has been reported for primary cells in preclinical gene therapy by combining CRISPR/Cas9 mediated induction of double-strand breaks (DSB) with delivery of a single-stranded DNA HDR-donor-template via highly purified recombinant adeno-associated virus (rAAV). Due in part to the labor and expense of rAAV particle purification, rAAV-mediated HDR-template delivery has been underutilized used to generate large KIs in cultured cell lines. Here, we report application of crude preparations of rAAV to deliver HDR-templates for the KI of large [~]2kb fragments at various genomic loci in several -human as well as mouse cell lines at high efficiency. Our approach should facilitate experiments necessitating KI of large DNA fragments to tag endogenous loci for visualization and/or conditional protein degradation.


title: Semi-field evaluation of the space spray efficacy of Fludora Co-Max EW against wild insecticide-resistant Aedes aegypti and Culex quinquefasciatus mosquito populations from Abidjan, Cote d'Ivoire

abstract:
BackgroundSpace spraying of insecticides is still an important mean of controlling Aedes and Culex mosquitoes and arboviral diseases. This study evaluated the space spray efficacy of Fludora Co-Max EW (a combination of flupyradifurone and transfluthrin, with Film Forming Aqueous Spray Technology (FFAST)) against wild, insecticide-resistant Aedes aegypti and Culex quinquefasciatus populations from Abidjan, Cote dIvoire, against K-Othrine EC (deltamethrin-only product), through small-scale field trials.

MethodologyWild Ae. aegypti and Cx. quinquefasciatus mosquito larvae were collected in Abidjan, Cote dIvoire from August to December 2020. Mosquito larvae were reared until adult stage. Emerged adult females were tested against Fludora Co-Max EW and K-Othrine EC using ultra-low volume cold fogging (ULV) and thermal fogging (TF) both outdoors and indoors in Agboville, Cote dIvoire. Cages containing 20 mosquitoes each were placed at 10, 25, 50, 75 and 100 m from the spraying line for outdoor spraying, and at ceiling, mid-height and floor levels for indoor house spraying. Knockdown and mortality were recorded at each checkpoint and compared by treatments.

Principal findingsOverall, Fludora Co-Max EW induced significantly higher knockdown and mortality effects in the wild insecticide-resistant Ae. aegypti and Cx. quinquefasciatus compared with K-Othrine EC. With both species, Fludora Co-Max EW mortality rates were above 80% (up to 100%) for outdoor ULV spray at each distance checkpoint (i.e. 10 to 100 m), and 100% for indoor ULV and TF sprays at all level checkpoints (i.e. ceiling, mid-height and floor). K-Othrine EC induced high mortality indoors (97.9-100%), whereas outdoor mortality rapidly declined in Ae. aegypti from 96.7% to 36.7% with ULV, and 85.0% to 38.3% with TF, from 10 to 100 m. For outdoor Fludora Co-Max EW spray, ULV showed both higher knockdown and killing performance Ae. aegypti and Cx. quinquefasciatus compared with TF. Fludora Co-Max EW performed better against Cx. quinquefasciatus compared with Ae. aegypti.

Conclusion/significanceFludora Co-Max EW induced high mortality and knockdown effects against wild insecticide-resistant Ae. aegypti and Cx. quinquefasciatus Abidjan strains and performed better than K-Othrine EC. The presence of flupyradifurone and transfluthrin (with new and independent modes of action) and FFAST technology in the current Fludora Co-Max EW formulation appears to have broadened its killing capacity. Fludora Co-Max EW is thus an effective adulticide and may be a useful tool for Aedes and Culex mosquito and arbovirus control in endemic areas.

Author SummarySpace spraying of insecticides is an important tool to control Aedes and Culex mosquitoes and prevent the viral diseases (i.e. dengue, yellow fever, etc.) that they transmit. We studied the efficacy of the product Fludora Co-Max EW (a new space spray insecticide) against adult wild insecticide-resistant populations of Aedes aegypti and Culex quinquefasciatus mosquitoes from Abidjan, Cote dIvoire. We compared Fludora Co-Max EW knockdown and mortality effects in these mosquitoes with the local insecticide K-Othrine EC using ultra-low volume (ULV) and thermal fogging (TF) spraying outdoors and indoors. The product Fludora Co-Max EW induced high rates of knockdown and mortality (i.e. 80-100%) in these wild insecticide-resistant mosquitoes and performed better than the product K-Othrine EC. Additionally, ULV sprays of Fludora Co-Max EW demonstrated higher knockdown and killing efficacy at larger distances (i.e. up to 100 m) compared with TF. The higher efficacy of Fludora Co-Max EW may be due to the interaction of two unrelated insecticides, flupyradifurone and transfluthrin, in combination with Film Forming Aqueous Spray Technology (FFAST). Fludora Co-Max EW therefore appears to be an effective and useful tool to control adult populations of wild insecticide-resistant Aedes and Culex mosquitoes and may be recommended for preventing related mosquito-transmitted viral diseases.

category: ecology


title: Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation

abstract: Whereas techniques to map chromatin-bound proteins are well-developed, mapping chromatin-associated RNAs remains a challenge. Here we describe Reverse Transcribe & Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tag-mented for sequencing by Tn5. We demonstrate the utility of RT&Tag in Drosophila cells for capturing the noncoding RNA roX2 with the dosage compensation complex and maturing transcripts associated with silencing histone modifications. We also show that RT&Tag can detect N6-methyladenosine (m6A)-modified mRNAs, and show that genes producing methylated transcripts are characterized by extensive promoter pausing of RNA polymerase II. The high efficiency of in situ antibody tethering and tagmentation makes RT&Tag especially suitable for rapid low-cost profiling of chromatin-associated RNAs from small samples.


title: Thousands of human non-AUG extended proteoforms lack evidence of evolutionary selection among mammals

abstract: The synthesis of most proteins begins at AUG codons, yet a small number of non-AUG initiated proteoforms are also known. Here we used publicly available ribo-seq data with phylogenetic approaches to identify novel, previously uncharacterised non-AUG proteoforms. Unexpectedly we found that the number of non-AUG proteoforms identified with ribosome profiling data greatly exceeds those with strong phylogenetic support. We identified an association between proteoforms with alternative N-termini and multiple compartmentalisation of corresponding gene products. In dozens of genes N-terminal extensions encode localisation signals, including mitochondrial presequence and signal peptides. While the majority of non-AUG initiated proteoforms occur in addition to AUG initiated proteoforms, in few cases non-AUG appears to be the only start. This suggests that alternative compartmentalisation is not the only function of non-AUG initiation. Taking a conservative approach, we updated annotation of several genes in the latest GENCODE version in human and mouse where non-AUG initiated proteofoms are supported by both, ribosome profiling and phylogenetic evidence. Yet, the number of such extensions is likely much higher. Thousands of non-AUG proteoforms supported only by ribosome profiling suggest that they may evolve neutrally. Indeed, expression of some may not be consequential, i.e. when N-termini is processed or they have identical biochemical properties. Nonetheless they may contribute to immune response as antigen sources. It is also possible that some proteoforms accrued useful functions only recently and evolved under purifying selection in a narrow phylogenetic group. Thus, further characterisation is important for understanding their phenotypical and clinical significance.


title: HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation

abstract: Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, on the kinase specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on a Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5, in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity, reveal how PP5 is activated by recruitment to HSP90 complexes to comprehensively dephosphorylate client proteins.


title: Variations of a theme - crystal forms of the amino acid transporter MhsT

abstract:
The bacterial amino acid transporter MhsT of the SLC6A family was crystallized in complex with different substrates in order to understand the determinants of substrate specificity of the transporter. Surprisingly, crystals of the different MhsT-substrate complexes showed interrelated, but different crystal packing arrangements. Space group assignment and structure determination of these different crystal forms presented challenging combinations of pseudosymmetry, twinning and tNCS.

SynopsisAn unusual case of protein-substrate complexes obtained in similar conditions, but containing different packing arrangements. The crystals exhibit a combination of various crystal imperfections (pseudosymmetry, twinning and tNCS) masking the true crystal symmetries and challenging data processing and structure determination.


title: Nuclear receptor ligand screening in an iPSC-derived in vitro blood-brain barrier model identifies new contributors to leptin transport

abstract:
BackgroundThe peptide hormone leptin exerts its function in the brain to reduce food intake and increase energy expenditure to prevent obesity. However, most obese subjects reflect the resistance to leptin even with elevated serum leptin. Considering that leptin must cross the blood-brain barrier (BBB) in several regions to enter the brain parenchyma, altered leptin transport through the BBB might play an important role in leptin resistance and other biological conditions. Here, we report the use of a human induced pluripotent stem cell (iPSC)-derived BBB model to explore mechanisms that influence leptin transport.

MethodsiPSCs were differentiated into brain microvascular endothelial cell (BMEC)-like cells using standard methods. BMEC-like cells were cultured in Transwell filters, treated with ligands from a nuclear receptor agonist library, and assayed for leptin transport using an enzyme-linked immune sorbent assay. RNA sequencing was further used to identify differentially regulated genes and pathways. The role of a select hit in leptin transport was tested with the competitive substrate assay and after gene knockdown using CRISPR techniques.

ResultsFollowing a screen of 73 compounds, 17{beta}-estradiol was identified as a compound that could significantly increase leptin transport. RNA sequencing revealed many differentially expressed transmembrane transporters after 17{beta}-estradiol treatment. Of these, cationic amino acid transporter-1 (CAT-1, encoded by SLC7A1) was selected for follow-up analyses due to its high and selective expression in BMECs in vivo. Treatment of BMEC-like cells with CAT-1 substrates, as well as knockdown of CAT-1 expression via CRISPR-mediated epigenome editing, yielded significant increases in leptin transport.

ConclusionsA major female sex hormone, as well as an amino acid transporter, were revealed as regulators of leptin BBB transport in the iPSC-derived BBB model. Outcomes from this work provide insights into regulation of peptide hormone transport across the BBB.


title: Bacterial Infection of the Placenta Induces Sex-Specific Responses in the Fetal Brain

abstract:
BACKGROUNDEpidemiological data indicate that prenatal infection is associated with an increased risk of several neurodevelopmental disorders in the progeny. These disorders display sex differences in presentation. The role of the placenta, which is a target of prenatal infection, in the sex-specificity of neurodevelopmental abnormalities is unknown. We used an imaging-based animal model of the bacterial pathogen Listeria monocytogenes to identify sex-specific effects of placental infection on neurodevelopment of the fetus.

METHODSPregnant CD1 mice were infected with a bioluminescent strain of Listeria on embryonic day 14.5 (E14.5). Excised fetuses were imaged on E18.5 to identify the infected placentas. The associated fetal brains were analyzed for gene expression and altered brain structure due to infection. The behavior of adult offspring affected by prenatal Listeria infection was analyzed.

RESULTSPlacental infection induced sex-specific alteration of gene expression patterns in the fetal brain and resulted in abnormal cortical development correlated with placental infection levels. Furthermore, male offspring exhibited abnormal social interaction, whereas females exhibited elevated anxiety.

CONCLUSIONPlacental infection by Listeria induced sex-specific abnormalities in neurodevelopment of the fetus. Prenatal infection also affected the behavior of the offspring in a sex-specific manner.

ImpactO_LIPlacental infection with Listeria monocytogenes induces sexually dichotomous gene expression patterns in the fetal brain.
C_LIO_LIAbnormal cortical lamination is correlated with placental infection levels.
C_LIO_LIPlacental infection results in autism related behavior in male offspring and heightened anxiety level in female offspring.
C_LI


title: Endogenous sterol synthesis is dispensable for Trypanosoma cruzi epimastigote growth but not stress tolerance

abstract: In addition to scavenging exogenous cholesterol, the parasitic kinetoplastid Trypanosoma cruzi can endogenously synthesize sterols. Similar to fungal species, T. cruzi synthesizes ergostane type sterols and is sensitive to a class of azole inhibitors of ergosterol biosynthesis that target the enzyme lanosterol 14-demethylase (CYP51). In the related kinetoplastid parasite Leishmania donovani, CYP51 is essential, yet in Leishmania major, the cognate enzyme is dispensable for growth; but not heat resistance. The essentiality of CYP51 and the specific role of ergostane-type sterol products in T. cruzi has not been established. To better understand the importance of this pathway, we have disrupted the CYP51 gene in T. cruzi epimastigotes ({Delta}CYP51). Disruption of CYP51 leads to accumulation of 14-methylated sterols and a concurrent absence of the final sterol product ergosterol. While{Delta} CYP51 epimastigotes have slowed proliferation compared to wild type parasites, the enzyme is not required for growth; however,{Delta} CYP51 epimastigotes exhibit sensitivity to elevated temperature, an elevated mitochondrial membrane potential and fail to establish growth as intracellular amastigotes in vitro. Further genetic disruption of squalene epoxidase ({Delta}SQLE) results in the absence of all endogenous sterols and sterol auxotrophy, yet failed to rescue tolerance to stress in{Delta} CYP51 parasites, suggesting the loss of ergosterol and not accumulation of 14-methylated sterols modulates stress tolerance.


title: Cell detoxification of secondary metabolites by P4-ATPase mediated vesicle transport

abstract: Mechanisms for cellular detoxification of drug compounds are of significant interest in human health. Cyclosporine A (CsA) and tacrolimus (FK506) are widely known antifungal and immunosuppressive microbial natural products. However, both compounds can result in significant side effects when used as immunosuppressants. The insect pathogenic fungus Beauveria bassiana shows resistance to CsA and FK506. However, the mechanisms underlying the resistance have remained unknown. Here, we identify a P4-ATPase gene, BbCRPA, from the fungus, which confers resistance via a unique vesicle mediated transport pathway that targets the compounds into detoxifying vacuoles. Interestingly, the expression of BbCRPA in plants promotes resistance to the phytopathogenic fungus Verticillium dahliae via detoxification of the mycotoxin cinnamyl acetate using a similar pathway. Our data reveal a new function for a subclass of P4-ATPases in cell detoxification. The P4-ATPases conferred cross-species resistance can be exploited for plant disease control and human health protection.


title: The contribution of Kaposis sarcoma-associated herpesvirus ORF7 and its zinc-finger motif to viral genome cleavage and capsid formation

abstract:
Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of endothelial and B cell malignancies. During KSHV lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in - and {beta}-herpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring non-spherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Thus, our data revealed that ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation.

IMPORTANCEIn herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficent KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.


title: The LEPIS-HuR-TMOD4 axis regulates hepatic cholesterol homeostasis and accelerates atherosclerosis

abstract:
Long noncoding RNAs (lncRNAs) play an important role in the entire progression of atherosclerosis. In this study, we identified an uncharacterized lncRNA, Liver Expressions by PSRC1 Induce Specifically (LEPIS). The expression of LEPIS and its potential target tropomodulin 4 (TMOD4) in the liver of ApoE-/- mice fed a high-fat diet was increased. An ApoE-/- mouse model with the overexpression of LEPIS or TMOD4 in liver was established, and we found that both LEPIS and TMOD4 increased the burden of atherosclerosis and reduced hepatic cholesterol levels. Further study revealed that LEPIS and TMOD4 affect the expression of genes related to hepatic cholesterol homeostasis, including proprotein convertase subtilisin/kexin type9 (PCSK9) and low-density lipoprotein receptor (LDLR), which are closely related to hypercholesterolemia. Mechanistically, human antigen R (HuR), an RNA-binding protein, was shown to be critical for the regulation of TMOD4 by LEPIS. Further, we found that overexpression of LEPIS promoted the shuttling of HuR from the nucleus to the cytoplasm, enhanced the stability of TMOD4 mRNA, and in turn promoted the expression of TMOD4. In addition, TMOD4 was found to affect intracellular cholesterol levels through PCSK9. These results suggest that the LEPIS-HuR-TMOD4 axis is a potential intervention target for hepatic cholesterol homeostasis and atherosclerosis.

Graphical abstract

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category: molecular biology


title: One-Step Making of DNA Dumbbells Allows Tunneling of Short-Read Libraries into a Long-Read Format

abstract: DNA dumbbells have many applications. One of the useful features is to have DNA amplified in rolling circles. Repeated iterations allow long and accurate sequencing from single molecules. We have developed a highly efficient and simple method for  tunneling short-read Illumina sequencing libraries into long-read dumbbells. A 5-exonuclease used in Gibson Assembly converted fixed Illumina P5/P7 sequences into sticky ends. Self-looping oligonucleotides with complementary 3 overhangs were ligated to form dumbbells. The reaction proceeded in one pot and modified only end nucleotides of input libraries. Resultant dumbbells were ready for Phi29-dependent rolling circle amplifications. Demonstrative readouts of a standard microbial community on the platform of Pacific Biotechnology confirmed successful tunneling of the original P5/P7-ended sequencing libraries.


title: Defective subviral particles modify ecological equilibria and enhance viral coexistence

abstract: Cooperation is a main driver of biological complexity at all levels. In the viral world, gene sharing among viral genomes, complementation between genomes or interactions within quasispecies are frequently observed. In this contribution, we explore the advantages that flexible associations between fully fledged viruses and subviral entities, such as virus satellites, might yield. We devise a mathematical model to compare different situations of competition between two viruses and to quantify how the association with a satellite qualitatively modifies dynamical equilibria. The relevant parameter is the invasion fitness of each virus or of the virus-satellite tandem, which in the model depends on the transmission rate of viruses and on their effect on host survival. While in a virus-virus competition one of the viruses becomes eventually extinct, an association with a satellite might change the outcome of the competition to favor the less competitive virus (regardless of whether it is the helper virus or not) or to allow for the stable coexistence of the two viruses and the satellite. We hypothesize that the latter scenario, in particular, constitutes a parsimonious evolutionary pathway towards more stable cooperative associations, such as bipartite viral forms.


title: Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation

abstract: The mammalian NF-{kappa}B p52:p52 homodimer together with its cofactor Bcl3 activates transcription of {kappa}B sites with a central G/C base pair (bp), while it is inactive toward {kappa}B sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined its structure in complex with a P-selectin(PSel)-{kappa}B DNA (5-GGGGTGACCCC-3) (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-{kappa}B-DNA complex structures, which have a central A/T bp. Molecular dynamics (MD) simulations show free DNAs exist in distinct preferred conformations, and p52:p52 homodimer induces the least amount of conformational changes on the more transcriptionally active natural PSel-{kappa}B DNA in the bound form. Our binding assays further demonstrate that the fast kinetics driven by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for {kappa}B DNA in complex with NF-{kappa}B and suggest the importance of binding kinetics, dictated by free DNA conformational and dynamic states, in controlling transcriptional activation for NF-{kappa}B.


title: A molecular plugin rescues GroEL/ES substrates from pre-folding oxidation

abstract: Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10) to GroEL induces CnoX release. Cryo-electron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal -helix. Furthermore, the identification of complexes in which CnoX, bound to GroEL, forms mixed-disulfides with GroEL substrates indicates that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, which suggests that Hsp60 molecular plugins have been conserved through evolution.


title: Describing Inhibitor Specificity for the Amino Acid Transporter LAT1 from Metainference Simulations

abstract:
The human L-type amino acid transporter 1 (LAT1; SLC7A5) is a membrane transporter of amino acids, thyroid hormones, and drugs such as the Parkinsons disease drug L-Dopa. LAT1 is found in the blood-brain-barrier (BBB), testis, bone marrow, and placenta, and its dysregulation has been associated with various neurological diseases such as autism and epilepsy as well as cancer. In this study, we combine metainference molecular dynamics (MD) simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. We first conducted a series of molecular docking experiments to identify the most relevant interactions between LAT1s substrate binding site and ligan ds, including both inhibitors and substrates. We then performed metainference MD simulations using cryo-EM structures in different conformations of LAT1 with the electron density map as a spatial restraint, to explore the inherent heterogeneity in the structures. We analyzed the LAT1 substrate binding site to map important LAT1-ligand interactions as well as newly described druggable pockets. Finally, this analysis guided the discovery of previously unknown LAT1 ligands using virtual screening and cellular uptake experiments. Our results improve our understanding of LAT1-inhibitor recognition, providing a framework for rational design of future lead compounds targeting this key drug target.

Statement of SignificanceLAT1 is a membrane transporter of amino acids, thyroid hormones, and therapeutic drugs, that is primarily found in the BBB and placenta, as well as in tumor cells of several cancer types. We combine metainference MD simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. Our computational analysis predicts S66, G67, F252, G255, Y259, W405 are critical residues for inhibitor binding and druggable sub-pockets in the outward-occluded conformation that are ideal for LAT1 inhibitor discovery. Using virtual screening and functional testing, we discovered multiple LAT1 inhibitors with diverse scaffolds and binding modes. Our results improve our understanding of LAT1s structure and function, providing a framework for development of future therapeutics targeting LAT1 and other SLC transporters.


title: A transcriptional cycling model recapitulates chromatin-dependent features of noisy inducible transcription

abstract:
Activation of gene expression in response to environmental cues results in substantial phenotypic heterogeneity between cells that can impact a wide range of outcomes including differentiation, viral activation, and drug resistance. An important source of gene expression noise is transcriptional bursting, or the observation that transcripts are produced during infrequent bursts of promoter activity. Chromatin accessibility, which regulates assembly of polymerase complexes on promoters, impacts transcriptional bursting, suggesting that how an activating signal affects transcriptional noise will depend on the initial chromatin state at the promoter. To explore this possibility, we simulated transcriptional activation using a transcriptional cycling model with three promoter states that represent chromatin remodeling, polymerase binding and pause release. We initiated this model over a large parameter range representing target genes with different chromatin environments, and found that, upon increasing the polymerase pause release rate to activate transcription, changes in gene expression noise varied significantly across initial promoter states. This model captured phenotypic differences in activation of latent HIV viruses integrated at different chromatin locations and mediated by the transcription factor NF-{kappa}B. Activating transcription in the model via increasing one or more of the transcript production rates, as occurs following NF-{kappa}B activation, reproduced experimentally measured transcript distributions for four different latent HIV viruses, as well as the bimodal pattern of HIV protein expression that leads to a subset of reactivated virus. Importantly, the parameter  activation path differentially affected gene expression noise, and ultimately viral activation, in line with experimental observations. This work demonstrates how upstream signaling pathways can be connected to biological processes that underlie transcriptional bursting, resulting in target gene-specific noise profiles following stimulation of a single upstream pathway.

Author SummaryMany genes are transcribed in infrequent bursts of mRNA production through a process called transcriptional bursting, which contributes to variability in responses between cells. Heterogeneity in cell responses can have important biological impacts, such as whether a cell supports viral replication or responds to a drug, and thus there is an effort to describe this process with mathematical models to predict biological outcomes. Previous models described bursting as a transition between an "OFF" state or an "ON" state, an elegant and simple mathematical representation of complex molecular mechanisms, but one which failed to capture how upstream activation signals affected bursting. To address this, we added an additional promoter state to better reflect biological mechanisms underlying bursting. By fitting this model to variable activation of quiescent HIV infections in T cells, we showed that our model more accurately described viral expression variability across cells in response to an upstream stimulus. Our work highlights how mathematical models can be further developed to understand complex biological mechanisms and suggests ways to connect transcriptional bursting to upstream activation pathways.


title: An in-vitro investigation of effects of acetylcholine on attenuate and low motile diluted rooster semen quality

abstract: This study was conducted to evaluate the effects of acetylcholine (ACh) on 24 hours preserved diluted rooster semen at 4{degrees}C with a low motility percentage during 2 hours at room temperature incubation. Ten indigenous same-aged roosters were used in this study, adapted by Dorso-abdominal massage for semen collection for one month before starting main work. Semen was collected weekly twice by the Dorso-abdominal massage technique, collected semen transferred to the laboratory in less than 10 minutes, and took a previous qualifying examination. Qualify semen from each rooster polled together and diluted by the Lake extender and preserved for 24 hours at 4{degrees}C in the refrigerator. Different ACh concentrations (10mM-100mM) were added to Low motile semen and stored for 24 h at 4{degrees}C, and the quality parameters such as motility, viability, acrosome, and plasma membrane integrity were measured evaluated. Adding ACh in 10mM for the low motile semen significantly increased semen motility from 50% to 78.5% (P<0.05) at time zero, but other ACh concentrations did not have any significant differences compared to control. During two hours incubation of recovered semen at room temperature, 10mM ACh prevented declining semen motility compared to control and other ACh concentrations significantly (P<0.05), which motility in 10mM ACh concentration 59%. In contrast, control, 1mM ACh, and 100M ACh group was 2.5%, 4%, and 1%, respectively. Semen viability after two hours of recovery at room temperature significantly 3.5% was less than the control group (P<0.05), but acrosome and plasma membrane integrity has not had any differences between all experimental groups (P>0.05). We can conclude that 10mM ACh can recover semen motility and not have toxicity and side effects on semen quality.


title: Selection and validation of reference genes for quantitative real-time polymerase chain reaction in Serratia ureilytica DW2

abstract: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacterium isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula. However, no validated reference genes from the genus Serratia for use in quantitative real-time polymerase chain reaction (RT-qPCR) normalization have been reported. To screen stable reference genes in S. ureilytica DW2, the expression of eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT-qPCR. The expression stability of these genes was analyzed with different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the most stably expressed reference gene was used to quantify expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels. The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing National Botanical Research Institute phosphate (NBRIP) growth medium. Therefore, this study provides a stable and reliable reference gene for Serratia, which is vital for the accurate quantification of functional gene expression in future studies.


title: Sensory responses in dorsolateral striatum are modulated by motor activity in a dopamine-dependent manner

abstract: The dorsolateral striatum (DLS) receives excitatory inputs from both sensory and motor cortical regions and is involved in sensory and motor functions. In cortical regions, sensory responses are altered by motor activity, however, it is not known if such sensorimotor interactions also occur in the striatum and how they are modulated by dopamine (DA). To determine the impact of motor activity on striatal sensory processing, we performed in vivo whole-cell recordings in the DLS of awake mice during the presentation of tactile stimuli. Striatal medium spiny neurons (MSNs) were activated by both whisker stimulation and spontaneous whisking, however, responses to whisker deflection during ongoing whisking were attenuated. DA depletion reduced the representation of whisking in direct-pathway MSNs, but not in those of the indirect-pathway. Furthermore, DA depletion impaired the discrimination between ipsi- and contralateral sensory stimulation in both direct- and indirect- pathway MSNs. Our results show that sensory responses in basal ganglia circuits are modulated by motor activity and that both processes are dopamine- and cell type-dependent.