title: Monitoring conservation effectiveness in tropical forests: bushmeat offtake parameters vary more quickly compared to parameters related to the living wildlife community

abstract: Evaluating the effectiveness of conservation actions in tropical forests is essential. Generally based on the monitoring of living wildlife communities, combination with bushmeat extraction indicators is seldom used. It is therefore interesting to carry out a monitoring of indices related to these two categories of indicators in order to identify indices presenting more temporal variation and allowing for a rapid detection of real changes. Between 2017 and 2020, data on bushmeat offtake were recorded and surveys of wildlife and human activity signs were conducted in a conservation zone in Dja Biosphere Reserve in Cameroon. Data were collected around villages where human pressure is high. Our results show a decline in total biomass and number of animals killed. An increase in numbers of traps set was observed, but there was no notable difference in the mean biomass of a carcass and mean number of hunting trips. Overall, wildlife abundance decreased slightly in 2020, mainly reflecting changes for three species--bay duiker (Cephalophus dorsalis), yellow-backed duiker (Cephalophus sylvicultor) and red river hog (Potamocherus porcus)--which were more abundant in 2018. Regarding keystone species, we noted a slight increase in the abundance of chimpanzees. Patterns of species richness in both study years were similar. These results suggest that bushmeat offtake data may be more informative for early evaluations of the effectiveness of wildlife conservation efforts. This underlines the importance of monitoring bushmeat for the evaluation of wildlife conservation projects in contexts where hunting occurs.


title: Habitat Suitability Modeling and Ecological Forecasting of Northern Goshawk Nesting Habitat

abstract: Northern Goshawks (Accipiter gentilis) are dynamic forest raptors often used as a management indicator species in forest management planning. Despite their use as indicators, there is a limited understanding of habitat requirements for goshawks, especially in dry forested landscapes as climate change occurs. We examined goshawk nesting habitat on the Helena-Lewis and Clark National Forest (HLCNF) in central Montana to further understand climate change on goshawk nesting habitat. The HLCNF receives low annual precipitation, making this forest useful to understand climate change in dry forest in structure and ecology. We measured characteristics of nest sites throughout the HLCNF and used principle component analysis to predict important variables for nest trees and nesting habitat for goshawks in this landscape. We also used remote sensing and GIS to create a composite map predicting areas of high, medium and low suitability for nesting habitat for goshawks in the HLCNF. We used the composite map to predict potential impacts of mountain pine beetle (Dendroctonus ponderosae), fire, and climate change to Northern Goshawk habitat in the HLCNF. The best indicators of a chosen nest tree were diameter at breast height, tree height, and tree status, which can be considered indicative of trees preferred for nesting by goshawks. The best indicators for nest forest were canopy closure, slope angle, and slope position, which indicate habitat areas of high suitability for goshawk nesting. Two forecast models where developed based on climate change predictions. After overlapping a projected mountain pine beetle risk map and fire frequency risk map onto the modeled of high suitability goshawk nesting habitat, we calculated that 52 % of modeled high suitability habitat may be at risk for mountain pine beetle blight and 66 % high suitability habitat at risk for frequent wildfires, which will reduce goshawk habitat quality.

category: ecology


title: Ambra1 haploinsufficiency results in metabolic alterations and exacerbates age-associated retinal degeneration

abstract: Autophagy is a key process in the maintenance of cellular homeostasis. The age-dependent decline in retinal autophagy has been associated with photoreceptor degeneration. Retinal dysfunction can also result from damage to the retinal pigment epithelium (RPE), as the RPE-retina constitutes an important metabolic ecosystem that must be finely tuned to preserve visual function. While studies of mice lacking essential autophagy genes have revealed a predisposition to retinal degeneration, the consequences of a moderate reduction in autophagy, similar to that which occurs during physiological aging, remain unclear. Here, we describe a retinal phenotype consistent with accelerated aging in mice carrying a haploinsufficiency for Ambra1, a pro-autophagic gene. These mice show protein aggregation in the retina and RPE, metabolic underperformance, and premature vision loss. Moreover, Ambra1+/gt mice are more prone to retinal degeneration after RPE stress. These findings indicate that autophagy provides crucial support to RPE-retinal metabolism and protects the retina against stress and physiological aging.

category: cell biology


title: Human XIST RNA acts early to condense architecture which facilitates A-repeat density-dependent initiation of gene silencing

abstract: XIST RNA triggers gene silencing chromosome-wide and transforms a euchromatic chromosome into a condensed Barr body. XIST is heavily studied in mouse ES cells, but here an inducible iPSC system allows analysis of initial steps in human chromosome silencing, revealing key points not known in either system. XIST RNA distribution was examined relative to biochemical and transcriptional changes directly within architecture of individual chromosome territories. Within a few hours of induction, XIST transcripts distribute as a large "sparse zone" and a smaller "dense zone", which, importantly, exhibit different effects on chromatin. Very sparse transcripts immediately trigger bright staining for H2AK119ub and CIZ1, a structural matrix protein. In contrast, H3K27me3 enrichment comes hours later and is much more restricted to the smaller dense RNA zone, which enlarges as the chromosome condenses. Importantly, silencing of several genes examined occurred well after architectural condensation, suggesting a possibly separable step. Surprisingly, we show the small A-repeat fragment of XIST can alone silence endogenous genes; however, results indicate this requires high local RNA density for effective histone deacetylation. Results support a concept whereby XIST RNA acts directly to condense the chromosome territory, comprised largely of non-coding DNA, which facilitates a required step to initiate gene silencing by the A-repeat. Hence, compacted architecture is not a consequence of collective gene silencing, but an early step required for chromosome-wide gene silencing.


title: A novel 5' DNA binding site in RFC facilitates PCNA loading for gap DNA repair

abstract: RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {gamma}. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures revealing a novel 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located in the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. It appears 5' DNA binds after 3' DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. We propose that the 5' site facilitates RFCs PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5' DNA site in longer gaps including during lagging strand DNA synthesis.

category: biochemistry


title: A novel 5 prime DNA binding site in RFC facilitates PCNA loading for gap DNA repair


title: Single-Objective Evanescent Scattering Microscopy for Imaging Single Proteins and Binding Kinetics

abstract:
Plasmonic scattering microscopy has advanced the evanescent detection approaches by offering wide-field single-molecule imaging capability. However, two limitations prevent the broader application of plasmonic single-molecule imaging. One is the heating effect accompanying the plasmonic enhancement, and the other is the complicated system structure resulting from the two-objective optical arrangement. Here, we report single-objective evanescent scattering microscopy. The evanescent field is created by total internal reflection instead of the surface plasmon resonance on the gold film. As a result, the sensing substrate without gold film produces little heat, and allows excitation and observation using one objective. In addition, this system enables quantification of protein binding kinetics by simultaneously counting the binding of individual molecules and recording their binding sites with nanometer precision, providing a digital method to measure binding kinetics with high spatiotemporal resolution. This work may pave a road for label-free single protein analysis in conventional microscopy.

TeaserLabel-free single-molecule imaging on a total internal reflection fluorescence objective.

category: biophysics


title: Understanding the Separation of Timescales in Rhodococcus erythropolis Proteasome Core Particle Assembly

abstract: The 20S proteasome Core Particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form, but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits, 14  and 14 {beta}, arranged in four stacked 7-member rings (7{beta}7{beta}77). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HP: 7{beta}7) dimerize to form the CP. In the bacterium Rhodococcus erythropolis, experiments have shown that the formation of the HP is completed within minutes, while the dimerization process takes hours. The N-terminal propeptide of the {beta} subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of time scales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used Molecular Dynamics (MD) simulations to characterize HP conformations and found that the HP exists in two states: one where the propeptide interacts with key residues in the HP dimerization interface and likely blocks dimerization, and one where this interface is free. We found that a propeptide mutant that dimerizes extremely slowly is essentially always in the non-dimerizable state, while the WT rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in MD simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than WT. Our work thus provides unprecedented insight into how this critical step in CP assembly is regulated, with implications both for efforts to inhibit proteasome assembly and for the evolution of hierarchical assembly pathways.


title: Oligomerization processes limit photoactivation and recovery of the Orange Carotenoid Protein.

abstract:
The Orange Carotenoid Protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection, by quenching of the excess of light harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which structural changes in the protein take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy quenching activity. Here, we probed photo-induced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in the regulation of OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation, a numbed-state forms, which remains in a non-photoexcitable structural state for at least [~]0.5 {micro}s after absorption of a first photon.

Significance StatementThe orange carotenoid protein (OCP) is a photoactivatable protein involved in cyanobacterial photoprotection. Upon photoactivation, OCP becomes able to quench the excess of energy uptaken by the light-harvesting antennae, thereby evading damage to the cells. It remains unclear, however, what is the exact OCP photoactivation mechanism, and whether or not oligomerization partakes in the regulation of the OCP function. Here, we investigated these two issues by combining static and time-resolved (TR) X-ray scattering and optical spectroscopy. Our results show that OCP oligomerizes both in the dark-adapted inactive and light-adapted active states, suggesting a role for oligomerization. TR scattering data furthermore reveal that the first large-scale conformational changes associated with the photoactivation of OCP take place on the {micro}s time scale.


title: Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury

abstract:
The complement component C3 is a fundamental plasma protein for host defense. However, recent work has demonstrated the critical importance of local C3 expression in cell survival. Here we analyzed the effects of local versus peripheral sources of C3 expression in a model of bacterial pneumonia. While mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient in liver-deficient C3 remain protected, comparable to wildtype mice.

Human lung transcriptome analysis showed secretory epithelial cells are a major source of C3. Mice with a C3 gene ablation from lung epithelial cells had worse pulmonary injury compared to wild type, despite maintaining normal circulating C3 levels. Finally, in human cellular and mouse pneumonia models, we show that C3 reduces epithelial cell death mediated through the alternative pathway component Factor B. Thus, our findings suggest that a locally-derived C3-Factor B pathway protects the lung mucosal barrier.

One Sentence SummaryLung-derived C3 mitigates severe bacterial pneumonia suggesting a novel cytoprotective role at mucosal barrier surfaces independent of circulating C3.

category: immunology


title: IgD+ Age-Associated B cells are the progenitors of the main T-independent B cell response to infection that generates protective Ab and can be induced by an inactivated vaccine in the aged

abstract: Age-associated B cells (ABC) accumulate with age and are associated with autoimmunity and chronic infection. However, their contributions to acute infection in the aged and their developmental pathways are unclear. We find that the response against influenza A virus infection in aged mice is dominated by a Fas+GL7- effector B cell population we call infection-induced-ABC (iABC). Most iABC express IgM and include antibody-secreting cells in the spleen, lung and bone marrow. We find that in response to influenza, IgD+CD21-CD23-ABC are the precursors of iABC. These IgD+ABC develop in germ free mice, so are independent of foreign antigen recognition, suggesting they arise from an intrinsic age-associated developmental program. The response of ABC to influenza infection, resulting in iABC, is T cell independent and requires both extrinsic TLR7 and TLR9 signals. In response to influenza infection, IgD+ABC can induce a faster recovery of weight and higher total anti-influenza IgG and IgM titers that can neutralize virus. Immunization with whole inactivated virus also generates iABC in aged mice. Thus, in unimmunized aged mice, whose other B and T cell responses have waned, IgD+ABC are likely the naive B cells with the potential to become Ab-secreting cells and to provide protection from infection in the aged.


title: Frequency of a nucleotide overhang at the 5' end of hemorrhagic fever mammarenavirus genomes in public sequence data

abstract: Mammarenaviruses, such as Lassa virus and South American hemorrhagic fever (SAHF) virus, cause severe hemorrhagic fevers in humans, and pose major threats to public health. Mammarenaviruses consist of a bi-segmented negative-sense RNA genome in which the 5 and 3 ends form complementary strands that serve as a replication promoter. Some mammarenaviruses have a nucleotide overhang at the 5 genome end. By examining the complementarity of 5 and 3 genome ends using public mammarenavirus genome sequences, we found that the 5 guanine overhang (5-G overhang) was present more frequently in Lassa and SAHF viruses than in other viruses. The 5-G overhang in the Lassa and SAHF virus sequences was found to be restricted to the L and S segments, respectively. If the genome end sequence data in the public database are accurate, the 5-G overhang may be related to the high pathogenicity of mammarenaviruses in humans.

category: microbiology


title: Endothelial beta-arrestins Regulate Mechanotransduction by the Type II Bone Morphogenetic Protein Receptor in Primary Cilia

abstract:
RationaleModulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined.

ObjectiveWe sought to better understand how {beta}-arrestins, intracellular proteins that regulate agonist-mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress.

Methods and ResultsIn vitro studies with primary endothelial cells subjected to {beta}-arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of {beta}-arrestin 1 and {beta}-arrestin 2 were conducted. We found that {beta}-arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of {beta}-arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. {beta}-arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, {beta}-arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR-II), whose disruption similarly led to an impaired endothelial shear response. {beta}-arrestins also regulated Smad transcription factor phosphorylation by BMPR-II. Mice with endothelial specific deletion of {beta}-arrestin 1 and {beta}-arrestin 2 were found to have impaired retinal angiogenesis.

ConclusionWe have identified a novel role for endothelial {beta}-arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR-II and contribute to vascular development.

NOVELTY AND SIGNIFICANCE

What Is Known?O_LIEndothelial cells respond to flow-induced shear stress with biochemical changes, such as phosphorylation of endothelial nitric oxide synthase, that promote morphological changes, such as cell alignment.
C_LIO_LIThe endothelial response to shear stress can involve primary cilia, microtubule-based sensory organelles that detect extracellular stimuli and generates intracellular signals.
C_LIO_LIThe specific ciliary signaling pathways that regulate endothelial mechanotransduction have not been fully elucidated.
C_LI

What New Information Does This Article Contribute?O_LI{beta}-arrestins directly interact with the ciliary protein intraflagellar transport protein 81 (IFT81), which is present in the primary cilia of endothelial cells, and are required for the morphological response to flow-induced shear stress.
C_LIO_LI{beta}-arrestins regulates type II bone morphogenetic protein receptor signaling, which is required for the endothelial response to shear stress, and is required for the phosphorylation of Smad transcription factors.
C_LIO_LI{beta}-arrestins are required for endothelial nitric oxide synthase-mediated flow-induced shear stress response in endothelial cells.
C_LIO_LIEndothelial cell-specific knockout of {beta}-arrestins results in abnormal vascular development, with a loss of vessel length and branchpoints.
C_LI

category: molecular biology


title: Dynamic gain decomposition reveals functional effects of dendrites, ion channels and input statistics in ultrafast population encoding

abstract:
The information processing capabilities of large neuronal circuits form the basis of higher-level cognition. It is unknown how the constituent cells properties interact with the external stimuli to shape the networks collective activity patterns and the associated computations. The dynamic gain function, a spectrally resolved approximation of a populations coding capability, is sensitive to the properties of stimuli and cells and could provide a mechanistic link from cell-level to network-level performance. However, the dynamic gains shape is co-determined in a complex manner by axonal and somatodendritic parameters and the populations operating regime, which so far precluded an understanding of any individual parameters impact. Here, we decomposed the dynamic gain function into three components corresponding to separate signal transformations. This allowed attribution of network-level encoding features to specific cell-level parameters. Applying the method to data from real neurons and biophysically plausible models, we found: 1. The encoding bandwidth of real neurons, approximately 400 Hz, is constrained by the voltage dependence of axonal currents during an early stage of action potential initiation. 2. The state-of-the-art models only achieve encoding bandwidths around 100 Hz and are mostly limited by subthreshold processes instead. 3. Large dendrites and low-threshold potassium currents modulate the bandwidth by shaping the subthreshold stimulus-to-voltage transformation. Dynamic gain decomposition can physiologically interpret changes in the dynamic gain curve, which is particularly desirable in studies of cellular effects in spectrinopathies and neurodegeneration. By pinpointing shortcomings of current models, it also guides inference of neuron models best suited for large-scale network simulations.

ImpactThe dynamic gain function quantifies how neurons can engage in collective, network-level activity, shaping brain rhythms and corresponding information encoding capabilities. Its shape results from a complex interaction between properties of different molecules (ion channels) and cell compartments (morphology, resistance), and is so far only understood for the simplest neuron models. Here we provide an interpretable analysis, decomposing dynamic gain based on the stimulus transformation steps in individual neurons. It is applicable to models of any complexity and real neurons, and allows attribution of dynamic gain changes to specific sub- and suprathreshold processes. Using this decomposition method, we reveal the relevance of subthreshold potassium channels for ultrafast information encoding and expose the shortcomings of even the state-of-the-art neuron models.

category: neuroscience


title: Dynamic gain decomposition reveals functional effects of dendrites, ion channels and input statistics in population coding


title: Sensory Schwann cells are required for mechanical nociception and touch perception

abstract: That sensory neurons alone transduce mechanical stimuli was challenged by the discovery of nociceptive Schwann cells that can initiate pain. Consistent with the existence of inherently mechanosensitive sensory Schwann cells, we found that mechanosensory function of almost all nociceptors, including those signaling fast pain were critically dependent on sensory Schwann cells. Furthermore, in polymodal nociceptors, sensory Schwann cells signal mechanical, but not cold or heat pain. Terminal Schwann cells also surround mechanoreceptor nerve-endings forming Meissners corpuscles that signal vibrotactile touch, raising the possibility that touch sensation is also dependent on such cells. Using optogenetics we show that Meissners corpuscle Schwann cells are necessary for the detection and perception of vibrotactile stimuli. Our results show that sensory Schwann cells within diverse glio-neural mechanosensory end-organs are sensors for mechanical pain as well as touch perception. These results place specialized sensory Schwann cells at the center of somatic sensation to all types of mechanical stimuli.


title: A centromeric RNA-associated protein complex affects germ line development in Drosophila melanogaster.

abstract: In many metazoans, centromeres are embedded in large blocks of highly repetitive (peri-) centromeric heterochromatin from which non-coding RNAs emanate that have been assigned diverse functions in different species. However, little is known about their functional details or regulation. The pericentromere of the X chromosome in Drosophila melanogaster contains a multi mega-base array of the 359 bp satellite repeats from the 1.688 family, which is transcribed into a lncRNA (SAT III RNA). We performed a SAT III RNA pulldown assay and identified a SAT III RNA-associated complex of four previously uncharacterized proteins and show that they affect germline development. These factors not only interact with each other and with SAT III RNA but also co-regulate each other. RNAi depletion of any of the factors leads to severe defects in the developing germline and sterility. Moreover, we show that the complex plays a crucial role in SAT III RNA repression, as RNAi depletion of the factors leads to a drastic increase of SAT III RNA levels. Importantly, genetic reduction of SAT III RNA level in the RNAi-depleted flies partially rescued the germ line defects and infertility phenotype. Based on our results we hypothesize that the identified complex functions in the germline to regulate SAT III RNA levels, possibly to offset effects of chromatin remodelling taking place in the developing germline.


title: Interferon receptor gene dosage determines multiple hallmarks of Down syndrome

abstract:
Trisomy 21 causes Down syndrome, a condition characterized by cognitive impairments, immune dysregulation, and atypical morphogenesis. Using whole blood transcriptome analysis, we demonstrate that specific overexpression of four interferon receptors encoded on chromosome 21 associates with chronic interferon hyperactivity and systemic inflammation in Down syndrome. To define the contribution of interferon receptor overexpression to Down syndrome phenotypes, we used genome editing to correct interferon receptor gene dosage in mice carrying triplication of a large genomic region orthologous to human chromosome 21. Normalization of interferon receptor copy number attenuated lethal antiviral responses, prevented heart malformations, decreased developmental delays, improved cognition and normalized craniofacial anomalies. Therefore, interferon receptor gene dosage determines major hallmarks of Down syndrome, indicating that trisomy 21 elicits an interferonopathy amenable to therapeutic intervention.

One-Sentence SummaryCorrection of interferon receptor gene dosage rescues multiple key phenotypes in a mouse model of trisomy 21.

category: genetics


title: DNA opening during transcription initiation by RNA polymerase II in atomic detail

abstract: RNA polymerase II (RNAP II) is a macro-molecular complex that synthesizes RNA by reading the DNA code, a process called transcription. During transcription initiation, RNAP II opens the double-stranded DNA to expose the DNA template to the active site. The molecular interactions driving and controlling the DNA opening are not well understood. We used all-atom molecular dynamics (MD) simulations to obtain a continuous atomistic pathway for the DNA opening process in human RNAP II. To achieve such large-scale and highly nonlinear transition, we steered the MD simulations along a combination of collective variables involving a guided DNA rotation and a set of path collective variables. The simulations reveal extensive interactions of the DNA with three protein loops near the active site, namely the rudder, fork loop 1, and fork loop 2. According to the simulations, these DNA-protein interactions support DNA opening by attacking Watson-Crick hydrogen bonds, and they stabilize the open DNA bubble by the formation of a wide set of DNA-protein salt bridges.


title: Progerin triggers a phenotypic switch in vascular smooth muscle cells that causes replication stress and an aging-associated secretory signature

abstract: Hutchinson Gilford Progeria Syndrome is a premature aging disease caused by LMNA gene mutation and the production of a truncated lamin A protein "progerin" that elicits cellular and organismal toxicity. Progerin accumulates in the vasculature, being especially toxic for vascular smooth muscle cells (VSMC). Patients autopsies show that vessel stiffening, and aortic atherosclerosis is accompanied by VSMC depletion in the medial layer, altered extracellular matrix (ECM), and thickening of the adventitial layer. Mechanisms whereby progerin causes massive VSMC loss and vessel alterations remain poorly understood. Mature VSMC retain phenotypic plasticity and can switch to a synthetic/proliferative phenotype. Here we show that progerin expression in human and mouse VSMC causes a switch towards the synthetic/proliferative phenotype. This switch elicits some level of replication stress in normal cells, which is exacerbated in the presence of progerin, leading to telomere fragility, genomic instability, and ultimately VSMC death. Importantly, calcitriol prevents replication stress, telomere fragility, and genomic instability, reducing VSMC death. In addition, RNAseq analysis shows induction of a profibrotic and proinflammatory aging-associated secretory phenotype upon progerin expression in human primary VSMC. Our data suggest that phenotypic switch-induced replication stress might be an underlying cause of VSMC loss in progeria, which together with loss of contractile features and gain of profibrotic and proinflammatory signatures contribute to vascular stiffness in HGPS. Preventing the phenotypic switch-induced replication stress with compounds such as calcitriol might ameliorate CVD in HGPS patients.


title: Coexistence between similar invaders: The case of two cosmopolitan exotic insects

abstract: Biological invasions have become widespread, threatening native biodiversity and ecosystem services. However, most studies have focused on the impacts of exotic species on natives, and fewer have examined dynamics between invaders, when multiple adventive species successfully coexist in a novel environment. Exotic lady beetle species were introduced into North America for biological control in agricultural systems and have since become dominant species within many native communities. Competitive interactions have been primarily investigated among native and exotic lady beetle species to assess mechanisms of decline and the impact of invasion, and yet two species of invaders, Harmonia axyridis and Coccinella septempunctata have become co-dominant in many of these communities. In this study, we investigated coexistence via spatial and temporal niche partitioning among H. axyridis and C. septempunctata using a 31-year dataset from southwestern Michigan, USA. We found evidence of long-term coexistence of these two invaders through a combination of small-scale environmental, habitat, and seasonal mechanisms. Across years, H. axyridis and C. septempunctata experienced patterns of cyclical dominance likely related to yearly variation in temperatures and precipitation. Within years, populations of C. septempunctata peaked early in the growing season around 550 degree days, while H. axyridis populations peaked later in the season around 1220 degree days. Habitat preferences for C. septempunctata were primarily crops such as soybean, wheat, and alfalfa, whereas H. axyridis did not display strong habitat preferences. These findings suggest that within this region H. axyridis is a broad habitat and environmental generalist, while C. septempunctata has more specific environmental niche preferences. These ecological differences have contributed to the continued coexistence among these two invaders, seemingly via the storage effect. Understanding mechanisms that allow coexistence and success of dominant exotic species contributes to native biodiversity conservation management of invaded ecosystems.


title: Investigations into the emergent properties of gene-to-phenotype networks across cycles of selection: A case study of shoot branching in plants

abstract: Predictive breeding is now widely practised in crop improvement programs and has accelerated selection response (i.e., the amount of genetic gain between breeding cycles) for complex traits. However, world food production needs to increase further to meet the demands of the growing human population. The prediction of complex traits with current methods can be inconsistent across different genetic, environmental, and agronomic management contexts because the complex relationships between genomic and phenotypic variation are not well accounted for. Therefore, developing gene-to-phenotype network models for traits that integrate the knowledge of networks from systems biology, plant and crop physiology with population genomics has been proposed to close this gap in predictive modelling. Here, we develop a gene-to-phenotype network for shoot branching, a critical developmental pathway underpinning harvestable yield for many crop species, as a case study to explore the value of developing gene-to-phenotype networks to enhance understanding of selection responses. We observed that genetic canalization is an emergent property of the complex interactions among shoot branching gene-to-phenotype network components, leading to the accumulation of cryptic genetic variation, reduced selection responses, and large variation in selection trajectories across populations. As genetic canalization is expected to be pervasive in traits, such as grain yield, that result from interactions among multiple genes, traits, environments, and agronomic management practices, the need to model traits in crop improvement programs as outcomes of gene-to-phenotype networks is highlighted as an emerging opportunity to advance our understanding of selection response and the efficiency of developing resilient crops for future climates.


title: Monotreme-specific conserved proteins derived from retroviral reverse transcriptase

abstract: Endogenous retroviruses (ERVs) have played an essential role in the evolution of mammals. Many ERV-derived genes are reported in the therians that are involved in placental development. However, the contribution of the ERV-derived genes in monotremes, which are oviparous mammals, remains to be uncovered. Here, we conducted a comprehensive search for possible ERV-derived genes in platypus and echidna genomes and identified three reverse transcriptase-like genes named RTOM1, 2, and 3. They were found to be clustered in the GRIP2 intron. Phylogenetic analysis revealed that RTOM1, 2, and 3 are strongly conserved between these species, and they were generated by tandem duplications before the divergence of platypus and echidna. The RTOM transcripts were specifically expressed in the testis, suggesting the physiological importance of RTOM genes. This is the first study reporting monotreme-specific de novo gene candidates derived from ERVs, which provides new insights into the unique evolution of monotremes.

category: genomics


title: Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

abstract: Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins (RIPs). When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.


title: A scalable and unbiased discordance metric with H+

abstract:
A standard unsupervised analysis is to cluster observations into discrete groups using a dissimilarity measure, such as Euclidean distance. If there does not exist a ground-truth label for each observation necessary for external validity metrics, then internal validity metrics, such as the tightness or consistency of the cluster, are often used. However, the interpretation of these internal metrics can be problematic when using different dissimilarity measures as they have different magnitudes and ranges of values that they span. To address this problem, previous work introduced the  scale-agnostic G+ discordance metric, however this internal metric is slow to calculate for large data. Furthermore, we show that G+ varies as a function of the proportion of observations in the predicted cluster labels (group balance), which is an undesirable property.

To address this problem, we propose a modification of G+, referred to as H+, and demonstrate that H+ does not vary as a function of group balance using a simulation study and with public single-cell RNA-sequencing data. Finally, we provide scalable approaches to estimate H+, which are available in the fasthplus R package.

category: bioinformatics


title: Profiling Unbinding Events to Identify Near-Native RNA-Ligand Poses

abstract: Predicting the structure (or pose) of RNA-ligand complexes is an important problem in RNA structural biology and drug discovery. Although one could use molecular docking procedures to rapidly sample putative poses of RNA-ligand complexes, accurately dis-criminating the native-like poses from non-native, decoy structures remains a formidable challenge. Here, we started from the assumption that native-like RNA-ligand poses are less likely to dissociate during molecular dynamics simulations, and then we used enhanced simulations to promote ligand for diverse poses of a handful of RNA-ligand complexes. By fitting unbinding profiles obtained from the simulations to a single-exponential, we identified the characteristic decay time ({tau}) as particularly effective at resolving native poses from decoys. Remarkably, a simple regression model trained to predict the simulation-derived parameters directly from structure could also discriminate poses. As such, when molecular dynamics simulations are feasible, one could use them to simulate ligand unbinding to aid in the identification of near-native poses. On the other hand, when speed is more important, one could use a simple structure-based regression model, like the ones described in this study, to analyze and filter RNA-ligand poses.


title: Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

abstract: Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses. Therefore, there is a critical need to identify early non-invasive biomarkers of response. We performed plasma proteomic analysis (>700 proteins) at three timepoints on 174 metastatic melanoma patients treated with ICB. We leverage independent training and testing cohorts to build a predictor of immunotherapy response that outperforms several tissue-based approaches. We found 217 differentially expressed proteins between ICB responders (R) and non-responders (NR), including a co-regulated module of proteins enriched in certain NR patients. By analyzing single-cell RNA-sequencing data of tumor biopsies from 32 patients, we dissected the relative contribution of cells in the tumor to proteins in circulation. The majority of proteins in the co-regulated NR module derived from tumor and myeloid cells. Amongst myeloid cells, we identified a subset of tumor-associated macrophages (TAMs) with a suppressive phenotype that expressed high levels of the co-regulated NR module, thus suggesting they are key drivers of non-response signatures. Together, our data demonstrates the utility of plasma proteomics in biomarker discovery and in understanding the biology of host response to tumors.

category: cancer biology


title: A scalable pipeline for SARS-CoV-2 replicon construction based on de-novo synthesis

abstract: Replicons are synthetic viral RNA molecules that recapitulate the self-replicating activities of the virus but are missing its infectivity potential. Here, we report on a scalable pipeline to generate a replicon of any SARS-CoV-2 strain using de-novo synthesis. Our pipeline relies only on publicly available sequencing data without requiring access to any material, simplifying logistical and bureaucratic issues of sample acquisition. In addition, our system retains the nucleotide sequence of most of the SARS-CoV-2 full genome and therefore better captures its underlying genomic and biological functions as compared to the popular pseudotypes or any replicon system published to-date. We utilized our system to synthesize a SARS-CoV-2 non-infectious version of the Beta strain. We then confirmed that the resulting RNA molecules are non-infectious and safe to handle in a BSL2/CL2 facility. Finally, we show that our replicon can be specifically inhibited by molnupiravir and RNAi treatments, demonstrating its utility for drug research and development.

category: synthetic biology


title: Whole-Brain Map of Top-down afferent Inputs to Nucleus Accumbens of the Mouse

abstract: Nucleus accumbens (NAc) is an important part of basal ganglia and receives major transsynaptic inputs from medial prefrontal cortex (mPFC), basolateral amygdala nucleus (BL) and hippocampus (HPC). The neural circuits formed by mPFC, BL, HPC with NAc are closely related to diverse brain functions such as decision-making, social behavior, reward seeking behavior and aggressive behavior. However, a lack of information about network structure of NAc top-down afference has limited our understanding of mechanisms of these functions. Here, we systematically analyzed whole brain patterns of inputs to mPFC/HPC/BL-NAc projectors via using (TRIO) labeling strategy. We found that the upstream input patterns between mPFC-core and mPFC-shell projections, BL-core and BL-shell projections, HPC-core and HPC-shell projections were similar. The projections from mPFC and BL to NAc receive a wide range of inputs, while the projections from HPC to NAc receive convergent inputs. Furthermore, all of mPFC/HPC/BL-NAc projectors receive considerable inputs from respective local regions. This study lays a foundation for further analysis of complex NAc functional mechanisms.


title: Enhanced excitability of the hippocampal CA2 region and its contribution to seizure generation in a mouse model of temporal lobe epilepsy

abstract: The hippocampal CA2 region, an area important for social memory, has been suspected to play a role in temporal lobe epilepsy (TLE) because of its resistance to the degeneration observed in neighboring CA1 and CA3 regions in both human and rodent models of TLE. However, little is known about whether alterations in CA2 properties serve to promote seizure generation or propagation. Here we have used the pilocarpine-induced status epilepticus (PILO-SE) model of TLE to explore the role of CA2. Ex vivo electrophysiological recordings from acute hippocampal slices revealed a set of coordinated changes that enhance CA2 intrinsic excitability, reduce CA2 local inhibitory input, and increase CA2 excitatory output to its major CA1 synaptic target. Moreover, selective silencing of CA2 pyramidal cells using a chemogenetic approach caused a significant decrease in the frequency of spontaneous seizures. These findings provide the first evidence that CA2 actively contributes to TLE seizure activity and may thus be a promising therapeutic target.


title: Interaction of an α-synuclein epitope with HLA-DRB1*15:01 initiates early enteric features of Parkinson's disease in humanized mice

abstract:
Parkinsons disease (PD) patients possess circulating T cells that recognize specific -synuclein-(-syn)-derived epitopes. One epitope, -syn32-46, interacts strongly with the HLA-DRB1*15:01 allele implicated in multiple autoimmune diseases. Whether this interaction and -syn-specific T cells play roles in PD pathogenesis remains unknown. We report that -syn32-46 immunization of a humanized mouse that expresses HLA-DRB1*15:01 triggers intestinal inflammation. This enteric pathology is characterized by activation of innate and adaptive immune responses and type I interferon signaling as well as loss of enteric dopaminergic neurons in the submucosal plexus, and results in constipation and weight loss. Depletion of CD4+, but not CD8+, T cells partially rescues enteric neurodegeneration. Thus, an interaction between -syn32-46 and HLA-DRB1*15:01 induces gut inflammation and CD4+ T cell-mediated loss of enteric dopaminergic neurons in the humanized mice. These findings suggest a mechanism for the prodromal enteric features of PD and indicate future directions for disease screening and treatment.

HIGHLIGHTS AND eTOC BlurbO_LI-syn32-46 immunization of a HLA-DRB1*15:01 mouse triggers weight loss and constipation.
C_LIO_LI-syn32-46 immunizations induce gut inflammation and loss of enteric dopaminergic neurons.
C_LIO_LIDepletion of CD4+, but not CD8+, T cells partially rescues enteric neurodegeneration.
C_LIO_LIInteraction between -syn32-46 and HLA-DRB1*15:01 are critical for the prodromal phase of PD.
C_LI

Parkinsons disease (PD) patients have circulating T cells that recognize -synuclein-(-syn)-epitopes. One epitope -syn32-46, interacts with the HLA-DRB1*15:01; however, its role in PD pathogenesis remains unknown. Garretti et al. show that -syn32-46 immunization of a humanized mouse expressing HLA-DRB1*15:01 triggers intestinal inflammation, loss of enteric dopaminergic neurons, constipation and weight loss, suggesting a critical role in the prodromal PD.


title: Comparative small RNA and degradome sequencing provide new insights into antagonistic interactions in the biocontrol fungus Clonostachys rosea

abstract:
Necrotrophic mycoparasitism is an intricate process involving recognition, physical mycelial contact and killing of host fungi (mycohosts). During such interactions, mycoparasites undergo a complex developmental process involving massive regulatory changes of gene expression to produce a range of chemical compounds and proteins that contribute to the parasitism of the mycohosts. Small-RNAs (sRNAs) are vital components of post-transcriptional gene regulation, although their role in gene expression regulation during mycoparasitism remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tags sequencing of the mycoparasitic fungus Clonostachys rosea interacting with the plant pathogenic mycohosts Botrytis cinerea and Fusarium graminearum at two time points. The majority of differentially expressed sRNAs were down-regulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing de-suppression (up-regulation) of mycohost-responsive genes. Degradome analysis showed a positive correlation between high degradome counts and antisense sRNA mapping and led to the identification of 201 sRNA-mediated gene targets for 282 differentially expressed sRNAs. Analysis of sRNA gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. While the regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum highlighting common and mycohosts-specific gene regulation of C. rosea. By combining these results with transcriptome data collected in similar experimental conditions during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism.

ImportanceSmall-RNAs (sRNAs) are emerging as key players in pathogenic and symbiotic fungus-plant interactions, however, their role in fungal-fungal interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and plant pathogenic mycohots Botrytis cinerea and Fusarium graminearum and investigated the sRNA-mediated gene regulation in mycoparasitic interactions. The combined approach of sRNA and degradome tag sequencing identified 201 sRNA-mediated putative gene targets for 282 differentially expressed sRNAs highlighting the role of sRNA-mediated regulation of mycoparasitism in C. rosea. We also identified 36 known and 13 novel miRNAs and their potential gene targets at endogenous level, and at a cross-species level in B. cinerea and F. graminearum indicating a role of cross-species RNAi in mycoparasitism, representing a novel mechanism in biocontrol interactions. Furthermore, we showed that C. rosea adapts its transcriptional response, and thereby its interaction mechanisms, based on the interaction stages and identity of the mycohost.


title: Evidence for evolutionary adaptation of mixotrophic nanoflagellates to warmer temperatures

abstract: Mixotrophs, organisms that combine photosynthesis and heterotrophy to gain energy, play an important role in global biogeochemical cycles. Metabolic theory predicts that mixotrophs will become more heterotrophic with rising temperatures, potentially creating a positive feedback loop that accelerates carbon dioxide accumulation in the atmosphere. Studies testing this theory have focused on phenotypically plastic (short-term) thermal responses of mixotrophs. However, as small organisms with short generation times and large population sizes, mixotrophs may rapidly evolve in response to climate change. Here we present data from a 3-year experiment quantifying the evolutionary response of two mixotrophic nanoflagellates to temperature. We found evidence for adaptive evolution through increasing growth rates in the obligately mixotrophic strain, but not in the facultative mixotroph. All lineages showed trends of increased carbon use efficiency, flattening of thermal reaction norms, and a return to homeostatic gene expression. Generally, mixotrophs evolved reduced photosynthesis and higher grazing with increased temperatures, suggesting that evolution may act to exacerbate mixotrophs effects on global carbon cycling.


title: Quantifying the Adaptive Potential of a Nascent Bacterial Community

abstract: The fitness effects of all possible mutations available to an organism largely shapes the dynamics of evolutionary adaptation. Tremendous progress has been made in quantifying the strength and abundance of selected mutations available to single microbial species in simple environments, lacking strong ecological interactions. However, the adaptive potential of strains that are part of multi-strain communities remains largely unclear. We sought to fill this gap for a stable community of two closely related ecotypes ("L" and "S") shortly after they emerged within the E. coli Long-Term Evolution Experiment (LTEE). To this end, we engineered genome-wide barcoded transposon libraries and developed a computational inference pipeline to measure the fitness effects of all possible gene knockouts in the coexisting strains as well as their ancestor, for many different conditions. We found that the fitness effect of most gene knockouts sensitively depends on the genetic background and the ecological conditions, as set by environmental perturbations and the relative frequency of both ecotypes. Despite the idiosyncratic behavior of individual knockouts, we still see consistent statistical patterns of fitness effect variation across both genetic background and community composition. The background dependence of mutational effects appears to reflect widespread changes in which gene functions are important for determining fitness, for all but the most strongly interacting genes. Additionally, fitness effects are correlated with evolutionary outcomes for a number of conditions, possibly revealing shifting patterns of adaptation. Together, our results reveal how ecological and epistatic effects combine to drive adaptive potential in recently diverged, coexisting ecotypes.

category: evolutionary biology


title: Correlated responses of energy metabolism, development and reproduction to evolution for increasing heat tolerant in Drosophila subobscura.

abstract:
O_LIAdaptation to increasing environmental temperatures implies an increase of the upper thermal limit (CTmax), but also an enhanced capacity of the organisms to perform fundamental biological functions at high temperatures.
C_LIO_LIEvolution of stress resistance is companied with a metabolic depression, which allows to organisms have reduced maintaining costs and allocate the energy surplus to energy demanding resistance mechanisms and reproduction.
C_LIO_LIHeat tolerance evolution depends on the heat intensity selection and it could have different consequences on the evolution of correlated traits such as energy metabolism and fitness-related traits. Thus, we evaluated the correlated responses of metabolic rate, metabolism-related enzymes (G6P branchpoint enzymes), reproductive and preadult survival in control and selected lines for increasing heat tolerance in Drosophila subobscura.
C_LIO_LIWe did not find a correlated response of metabolic rate associated with heat tolerance evolution, but we detected a reduced hexokinase activity in the slow-ramping selected lines. On the other hand, we found an increase in fecundity in the fast- and slow-ramping selected lines, but an increase in egg-to-adult viability was only found in the slow-ramping selected lines.
C_LIO_LIOur results show that the evolution of heat tolerance does not imply an increase in energy demand neither a tradeoff with reproduction and preadult survival, which suggests that heat tolerance evolution induces correlated response to increase the performance at high temperatures. Therefore, spatial and temporal variability of thermal stress intensity should be taking into account in future studies if we want to understand and predict the adaptive response to ongoing and future climatic conditions.
C_LI


title: Genomic prediction with whole-genome sequence data in intensely selected pig lines

abstract:
BackgroundEarly simulations indicated that whole-genome sequence data (WGS) could improve prediction accuracy and its persistence across generations and breeds. However, results in real datasets have been ambiguous so far. Large data sets that capture most of the genome diversity in a population must be assembled so that allele substitution effects are estimated with higher accuracy. The objectives of this study were to use a large pig dataset to assess the benefits of using WGS for genomic prediction compared to using commercial marker arrays, to identify scenarios in which WGS provides the largest advantage, and to identify potential pitfalls for its effective implementation.

MethodsWe sequenced 6,931 individuals from seven commercial pig lines with different numerical size. Genotypes of 32.8 million variants were imputed for 396,100 individuals (17,224 to 104,661 per line). We used BayesR to perform genomic prediction for 8 real traits and 9 simulated traits with different genetic architectures. Genomic predictions were performed using either data from a marker array or variants preselected from WGS based on linkage disequilibrium, functional annotation, or association tests. Both single and multi-line training sets were explored.

ResultsUsing WGS improved prediction accuracy relative to the marker array, provided that training sets were sufficiently large, especially for traits with high heritability and low number of quantitative trait nucleotides. The performance of each set of predictor variants was not robust across traits and lines. The most robust results were obtained when preselected variants with statistically significant associations were added to the marker array. Under this method, average improvements of prediction accuracy of 2.5 and 4.2 percentage points were observed in within-line and multi-line scenarios, respectively, with training sets of around 80k individuals.

ConclusionsOur results evidenced the potential for WGS to improve genomic prediction accuracy in intensely selected pig lines. Although the prediction accuracy improvements achieved so far were modest at best, we would expect that more robust improvements could be attained with a combination of larger training sets and optimised pipelines.


title: Curvelet Transform-based Sparsity Promoting Algorithm for Fast Ultrasound Localization Microscopy

abstract: Ultrasound localization microscopy (ULM) based on microbubble (MB) localization was recently introduced to overcome the resolution limit of conventional ultrasound. However, ULM is currently challenged by the requirement for long data acquisition times to accumulate adequate MB events to fully reconstruct vasculature. In this study, we present a curvelet transform-based sparsity promoting (CTSP) algorithm that improves ULM imaging speed by recovering missing MB localization signal from data with very short acquisition times. CTSP was first validated in a simulated microvessel model, followed by the chicken embryo chorioallantoic membrane (CAM), and finally, in the mouse brain. In the simulated microvessel study, CTSP robustly recovered the vessel model to achieve an 86.94% vessel filling percentage from a corrupted image with only 4.78% of the true vessel pixels. In the chicken embryo CAM study, CTSP effectively recovered the missing MB signal within the vasculature, leading to marked improvement in ULM imaging quality with a very short data acquisition. Taking the optical image as reference, the vessel filling percentage increased from 2.7% to 42.2% using 50ms of data acquisition after applying CTSP. CTSP used 80% less time to achieve the same 90% maximum saturation level as compared with conventional MB localization. We also applied CTSP on the microvessel flow speed maps and found that CTSP was able to use only 1.6s of microbubble data to recover flow speed images that have similar qualities as those constructed using 33.6s of data. In the mouse brain study, CTSP was able to reconstruct the majority of the cerebral vasculature using 1-2s of data acquisition. Additionally, CTSP only needed 3.2s of microbubble data to generate flow velocity maps that are comparable to those using 129.6s of data. These results suggest that CTSP can facilitate fast and robust ULM imaging especially under the circumstances of inadequate microbubble localizations.

category: bioengineering


title: High-performance Flexible Microelectrode Array with PEDOT:PSS Coated 3D Micro-cones for Electromyographic Recording

abstract: High signal-to-noise ratio (SNR) electromyography (EMG) recordings are essential for identifying and analyzing single motor unit activity. While high-density electrodes allow for greater spatial resolution, the smaller electrode area translates to a higher impedance and lower SNR. In this study, we developed an implantable and flexible 3D microelectrode array (MEA) with low impedance that enables high-quality EMG recording. With polyimide micro-cones realized by standard photolithography process and PEDOT:PSS coating, this design can increase effective surface area by up to 250% and significantly improve electrical performance for electrode sites with various geometric surface areas, where the electrode impedance is at most improved by 99.3%. Acute EMG activity from mice was recorded by implanting the electrodes in vivo, and we were able to detect multiple individual motor units simultaneously and with high resolution (SNR >> 100). The charge storage capacity was measured to be 34.2 mC/cm2, indicating suitability of the electrodes for stimulation applications as well.


title: Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA reveals novel spatial biology

abstract: Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We propose a novel machine learning algorithm, CELESTA, which uses both cells protein expression and spatial information to identify cell type of individual cells. We demonstrate the performance of CELESTA on multiplexed immunofluorescence in situ images of colorectal cancer and head and neck cancer. Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, which we validate in an independent cohort. By coupling our in situ spatial analysis with single-cell RNA-sequencing data on proximal sections of the same tissue specimens, we identify and validate cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of spatial biology to reveal clinically-relevant cellular interactions.


title: An expanded classification of active, inactive, and druggable RAS conformations

abstract:
For many human cancers and tumor-associated diseases, mutations in the RAS isoforms (KRAS, NRAS, and HRAS) are the most common oncogenic alterations, making these proteins high-priority therapeutic targets. Effectively targeting the RAS isoforms requires an exact understanding of their active, inactive, and druggable conformations. However, there is no structure-guided catalogue of RAS conformations to guide therapeutic targeting or examining the structural impact of RAS mutations. We present an expanded classification of RAS conformations based on analyzing their catalytic switch 1 (SW1) and switch 2 (SW2) loops. From all 721 available human KRAS, NRAS, and HRAS structures in the Protein Data Bank (PDB) (206 RAS-protein complexes, 190 inhibitor-bound, and 325 unbound, including 204 WT and 517 mutated structures), we created a broad conformational classification based on the spatial positions of residue Y32 in SW1 and residue Y71 in SW2. Subsequently, we defined additional conformational subsets (some previously undescribed) by clustering all well modeled SW1 and SW2 loops using a density-based machine learning algorithm with a backbone dihedral-based distance metric. In all, we identified three SW1 conformations and nine SW2 conformations, each which are associated with different nucleotide states (GTP-bound, nucleotide-free, and GDP-bound) and specific bound proteins or inhibitor sites. The GTP-bound SW1 conformation can be further subdivided based on the hydrogen (H)-bond type made between residue Y32 and the GTP {gamma}-phosphate: water-mediated, direct, or no H-bond. Further analyzing these structures clarified the catalytic impact of the G12D and G12V RAS mutations, and the inhibitor chemistries that bind to each druggable RAS conformation. To facilitate future RAS structural analyses, we have created a web database, called Rascore, presenting an updated and searchable dataset of human KRAS, NRAS, and HRAS structures in the PDB, and which includes a page for analyzing user uploaded RAS structures by our algorithm (http://dunbrack.fccc.edu/rascore/).

SignificanceAnalyzing >700 experimentally determined RAS structures helped define an expanded landscape of active, inactive and druggable RAS conformations, the structural impact of common RAS mutations, and previously uncharacterized RAS-inhibitor binding modes.


title: Defining An Expanded RAS Conformational Landscape Based on Over 700 Experimentally Determined Structures of KRAS, NRAS, and HRAS


title: Bi-steric mTORC1-selective Inhibitors activate 4EBP1 reversing MYC-induced tumorigenesis and synergize with immunotherapy

abstract: The MYC oncogene is causally involved in the pathogenesis of most types of human cancer but it remains therapeutically untargeted. The mTORC1 protein complex regulates cap-dependent translation through 4EBP1 and S6K and thereby, downstream MYC protein expression. However, to date, agents such as rapalogs that selectively target mTORC1 (as compared to mTORC2) fail to reactivate 4EBP1 and thus, to block MYC in vivo. In contrast, agents that nonselectively inhibit both protein complexes of the mTOR pathway, mTORC1 and mTORC2, can activate 4EBP1, but often suffer from a lack of tolerability including in vivo hepatotoxicity and immunosuppression. Here, we report the anti-tumor activity of bi-steric mTORC1-selective inhibitors, including Revolution Medicines clinical candidate RMC-5552, that potently and selectively target mTORC1 over mTORC2. In an autochthonous transgenic mouse model of MYC-amplified and MYC-driven hepatocellular carcinoma (HCC), representative bi-steric mTORC1-selective inhibitors suppress translation initiation via activation of 4EBP1, thereby suppressing MYC protein expression and blocking tumor growth. Furthermore, in human HCC samples, the low levels of 4EBP1 and MYC is correlated with immune reactivation. Immunohistochemistry, CIBERSORT, and CODEX reveal that selective mTORC1 inhibition results in activation of both CD4+ T cell- and NKp46+ NK cell-mediated immune surveillance. Moreover, bi-steric mTORC1-selective inhibitors synergize with -PD-1 to induce sustained tumor regression, with immune cell degranulation and release of perforins and granzyme B. These agents also exhibit anti-tumor activity in human patient-derived xenografts of HCC, colorectal cancer, head and neck cancer, and ovarian cancer harboring genomic amplifications in MYC. We infer that selective mTORC1 inhibition is a potential therapeutic strategy to drive effective MYC inactivation in cancer, and the consequent restoration of immune surveillance against neoplasia.


title: A heterogeneous drug tolerant persister state in BRAF-mutant melanoma is characterized by ion channel dysregulation and susceptibility to ferroptosis

abstract: There is increasing interest in cancer cell subpopulations that can withstanding treatment via non-genetic mechanisms, such as tumor cell plasticity and adaptation. These cell populations may be comprised of cells with diverse phenotypes, e.g., quiescent or slow cycling. Such populations have been broadly termed "drug-tolerant persisters" (DTPs) and may be responsible for minimal residual disease following anticancer treatment and acquired resistance. Understanding molecular mechanisms that drive emergence of DTPs could lead to new strategies to improve therapeutic outcomes. Recently, we reported that BRAF-mutant melanoma cells under prolonged BRAF inhibition enter a DTP state with balanced cell death and division, which we termed "idling." Here, we apply single cell barcoding to show that idling DTP populations emerge via cell state transitions, rather than selection of a few pre-existing drug-tolerant clones. Within the time frame of our experiments, DTPs exhibit varying proportions of fast- and slow-cycling cells within each lineage, suggesting that entry into the DTP state is a stochastic process. Furthermore, single-cell transcriptomics and bulk epigenomics reveal common gene expression and ontology signatures in DTP lineages that are consistent with rebalancing of ion channels. Calcium flux experiments uncover a reduction of divalent cation reserves in intracellular organelles, likely leading to endoplasmic reticulum stress. Accordingly, idling DTPs are more prone to ferroptotic cell death, as indicated by increased sensitivity to inhibition of glutathione peroxidase 4 (GPX4), which prevents removal of toxic lipid peroxides. In summary, we propose that ion channel homeostasis is a central process underlying idling DTP emergence in BRAF-mutated melanoma. Future studies will investigate translational aspects of this insight.

category: systems biology


title: SPATIAL ORGANIZATION OF NEURON-ASTROCYTE INTERACTIONS IN THE SOMATOSENSORY CORTEX

abstract: Microcircuits in the neocortex are functionally organized along layers and columns, which are the fundamental modules of cortical information processing. While the function of cortical microcircuits has focused on neuronal elements, much less is known about the functional organization of astrocytes and their bidirectional interaction with neurons. Here we show that CB1R-mediated astrocyte activation by neuron-released endocannabinoids elevate astrocyte Ca2+ levels, stimulate ATP/adenosine release as gliotransmitters, and transiently depress synaptic transmission in layer 5 pyramidal neurons at relatively distant synapses (>20 {micro}m) from the stimulated neuron. This astrocyte-mediated heteroneuronal synaptic depression occurred between pyramidal neurons within a cortical column and was absent in neurons belonging to adjacent cortical columns. Moreover, this form of heteroneuronal synaptic depression occurs between neurons located in particular layers, following a specific connectivity pattern that depends on a layer-specific neuron-to-astrocyte signaling. These results unravel the existence of astrocyte-mediated non-synaptic communication between cortical neurons, and that this communication is column- and layer-specific, which adds further complexity to the intercellular signaling processes in the cortex.


title: Regulation of mannitol metabolism in Enterococcus faecalis and association with parEF0409 toxin-antitoxin locus function.

abstract:
The parEF0409 type I toxin-antitoxin locus is situated between genes for two paralogous mannitol-family phosphoenolpyruvate phosphotransferase systems (PTS). In order to address the possibility that parEF0409 function was associated with sugar metabolism, genetic and phenotypic analyses were performed on the flanking genes. It was found that the genes were transcribed as two operons; the downstream operon essential for mannitol transport and metabolism and the upstream operon performing a regulatory function. In addition to genes for the PTS components, the upstream operon encodes a gene similar to mtlR, the key regulator of mannitol metabolism in other Gram-positive bacteria. We confirmed that this gene is essential for regulation of the downstream operon and identified putative phosphorylation sites required for carbon catabolite repression and mannitol-specific regulation. Genomic comparisons revealed that this dual operon organization of mannitol utilization genes is uncommon in enterococci and that association with a toxin-antitoxin system is unique to E. faecalis. Finally, we consider possible links between parEF0409 function and mannitol utilization.

ImportanceEnterococcus faecalis is both a common member of the human gut microbiota and an opportunistic pathogen. Its evolutionary success is partially due to its metabolic flexibility: in particular, its ability to import and metabolize a wide variety of sugars. While a large number of phosphoenolpyruvate phosphotransferase sugar transport systems have been identified in the E. faecalis genome bioinformatically, the specificity and regulation of most of these systems remains undetermined. Here we characterize a complex system of two operons flanking a type I toxin-antitoxin system required for the transport and metabolism of the common dietary sugar mannitol. We also determine the phylogenetic distribution of mannitol utilization genes in the enterococcal genus and discuss the significance of association with toxin-antitoxin systems.


title: Priority effects determine how dispersal affects biodiversity in seasonal metacommunities

abstract: The arrival order of species frequently determines the outcome of their interactions. This phenomenon, called the priority effect, is ubiquitous in nature and determines local community structure, but we know surprisingly little about how it influences biodiversity across different spatial scales. Here, we use a seasonal metacommunity model to show that biodiversity patterns and the homogenizing effect of high dispersal depend on the specific mechanisms underlying priority effects. When priority effects are driven by a numeric advantage, diversity measurements are highly sensitive to initial conditions and high dispersal homogenizes communities. However, when local priority effects arise from phenological shifts that alter species interaction strength (trait-mediated priority effects), local, regional, and temporal diversity are higher and are surprisingly robust to variation in dispersal and initial conditions, given spatiotemporal variations in phenology. Our results highlight that accounting for the mechanisms underlying priority effects is fundamental to understanding the maintenance of regional biodiversity.


title: Physiological causes and biogeographic consequences of thermal optima in the hypoxia tolerance of marine ectotherms

abstract:
The minimum O2 needed to fuel the demand of aquatic animals is commonly observed to increase with temperature, driven by accelerating metabolism. However, recent measurements of critical O2 thresholds ( Pcrit) reveal more complex patterns, including those with a minimum at an inter-mediate thermal  optimum. To discern the prevalence, physiological drivers, and biogeographic manifestations of such curves, we analyze new experimental and biogeographic data using a general dynamic model of aquatic water breathers. The model simulates the transfer of oxygen from ambient water, through a boundary layer and into animal tissues driven by temperature-dependent rates of metabolism, diffusive gas exchange, and ventilatory and circulatory systems with O2-protein binding. We find that a thermal optimum in Pcrit can arise even when all physiological rates increase steadily with temperature. This occurs when O2 supply at low temperatures is limited by a process that is more temperature sensitive than metabolism, but becomes limited by a less sensitive process at warmer temperatures. Analysis of species respiratory traits suggests this scenario is not uncommon in marine biota, with ventilation and circulation limiting supply under cold conditions and diffusion limiting supply at high temperatures. Using biogeographic data, we show that species with these physiological traits inhabit lowest O2 waters near the optimal temperature for hypoxia tolerance, and are restricted to higher O2 at temperatures above and below this optimum. Our results imply that O2 tolerance can decline under both cold and warm conditions, and thus may influence both poleward and equatorward species range limits.

Significance StatementPhysiology shapes the ecology, biogeography, and climate responses of marine species. In aquatic ectotherms, accelerating metabolism and lowered oxygen availability generally result in increasing oxygen limitation with warming. Here we present evidence for thermal optima in hypoxia tolerance of diverse species that is explained by a dynamical model of organismal physiology. Our results indicate that this potentially widespread bidirectional pattern explains species biogeographic limits in cold and warm waters. It can be understood using a generalized Metabolic Index of O2 supply to demand, which captures the variable observed trends between temperature and species hypoxia sensitivity. Oxygen limitation of aerobic metabolism in cold water has far-reaching implications for marine biogeography and species migrations under climate change.


title: Laboratory and field trials reveal the potential of a gel formulation of entomopathogenic nematodes as biocontrol against the fall armyworm (Spodoptera frugiperda)

abstract:
1.The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae) can cause tremendous yield losses in maize. Its invasion into Africa and Asia has led to an enormous influx of insecticides into maize agro-ecosystems. Safe, effective and readily available alternatives are desperately needed. Entomopathogenic nematodes (EPN) are commonly used against soil insect pests, but can also control some above-ground pests. We explored the possibility to control FAW by incorporating EPN into a formulation that can be easily applied into the whorl of maize plants, where the caterpillars mostly feed. We tested this approach in laboratory cage experiments as well as in field trials. In the laboratory, treating maize plants with a low dose of EPN in a carboxymethyl cellulose (CMC) gel formulation (about 3000 infective juveniles per plant), caused 100% mortality of FAW caterpillars and prevented plant damage considerably, whereas EPN applied in water or a surfactant-polymer-formulation (SPF) caused 72% and 94% mortality, respectively. Under field conditions, one-time treatments with EPN applied in water, SPF or CMC gel were all able to prevent significant plant damage, but only the EPN-gel formulation significantly reduced FAW infestation. Notably, the gel formulation was as effective as a standard dose of cypermethrin, an insecticide commonly used against FAW. Repeated applications may be needed to reduce re-infestations by FAW across a whole cropping season depending on the local maize phenology and pest dynamics. These findings demonstrate that EPN are excellent candidates for the biological control of FAW and are a safe and sustainable alternative to chemical insecticides.

HighlightsO_LIEntomopathogenic nematodes are highly lethal to fall armyworm caterpillars.
C_LIO_LIAppropriate formulation of the nematodes is crucial for above-ground application.
C_LIO_LIA gel formulation of entomopathogenic nematodes was as effective as chemical insecticides.
C_LIO_LIEntomopathogenic nematodes can be used for the control of fall armyworm in maize.
C_LI


title: High rates of spontaneous chromosomal duplications are compensated by translational regulation in a photosynthetic unicellular eukaryote

abstract: While duplications have long been recognized as a fundamental process driving major evolutionary innovations, direct estimations of spontaneous chromosome duplication rates are scarce. Here, we provide the first estimations of spontaneous whole chromosome duplication rates in four green algae and in one diatom species from mutation accumulation (MA) experiments. The spontaneous whole duplication events are 7 to 70 times less frequent than spontaneous point mutations per cell division. Comparative transcriptome analyses between the control versus a MA line with a whole chromosome duplication indicate a [~]1.94-fold higher relative level of mRNAs in genes located within the duplicated regions. However, comparative analyses of the translation rate of mRNAs demonstrate that the excess of mRNAs is compensated by a proportional decrease to [~]0.67 in translation rates of genes located on duplicated chromosomes. This provides evidence that translation regulation mechanisms orchestrate dosage compensation in duplicated chromosomes. We further investigate the role of the poly(A) tail length in tempering mRNA translation and suggest that poly(A) tail length is regulated and shorter for transcripts linked to the chromosome duplication. These results point out the existence of a post-transcriptional mechanism co-ordinating the translation of hundreds of transcripts from genes located on duplicated regions in eukaryotes.


title: Interactions between genes altered during cardiotoxicity and neurotoxicity in zebrafish revealed using induced network modules analysis

abstract: Zebrafish has become a prominent model organism to study toxicology due to its genomic similarity to humans, optical clarity, well defined developmental stages, short generation time and cost-effective maintenance. It also provides a shorter time frame for in vivo toxicology evaluation compared to mammalian experimental systems. As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health and the effects of toxicant exposure are emerging as critical public health concerns. Owing to this, in this study, we analyzed the impact of such chemical induced toxicity in zebrafish. We first searched the literature comprehensively for genes that were modulated during neurotoxicity and cardiotoxicity in zebrafish and then studied the interactions between those genes using induced network modules analysis of the database ConsensusPathDB. The induced network modules analysis helps to study gene interactions through various types of interactions like gene regulatory, biochemical, genetic and protein interactions. A constant communication between the heart and the brain is an important physiological phenomenon. Therefore, we also analyzed the interactions among genes that are modulated simultaneously during cardiotoxicity and neurotoxicity. This study has led us to identify some potential predictive biomarkers for neurotoxicity and cardiotoxicity in zebrafish.

category: pharmacology and toxicology


title: The Ca2+-gated Cl- channel TMEM16A amplifies capillary pericyte contraction reducing cerebral blood flow after ischemia

abstract: Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca2+ concentration ([Ca2+]i) in pericytes activates chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplifies the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slows the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction and pericyte death, reduces neutrophil stalling and improves cerebrovascular reperfusion. Genetic analysis implicates altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function, and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimers disease and vascular dementia.

category: physiology


title: Developmental emergence of sleep rhythms enables long-term memory capabilities in Drosophila

abstract: In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day (1-4). The mechanism through which sleep rhythms emerge, and the consequent advantage conferred to a juvenile animal, are unknown. In 2nd instar Drosophila larvae (L2), like human infants, sleep is not under circadian control (5). Here, we identify the precise developmental timepoint when the circadian clock begins to regulate sleep in Drosophila, leading to the emergence of sleep rhythms at the early 3rd instar stage (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousalpromoting Dh44 neurons, bringing arousal under clock control to drive the emergence of circadian sleep. Finally, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of an aversive cue, and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.


title: Reward contingency gates selective cholinergic suppression of amygdala neurons

abstract: Basal forebrain cholinergic neurons are thought to modulate how organisms process and respond to environmental stimuli through impacts on arousal, attention, memory, and motivated behavior. We questioned whether basal forebrain cholinergic neurons are directly involved in conditioned behavior, independent of ancillary roles in stimulus processing. We found that cholinergic neurons are active during behavioral responding for a reward - even in the absence of reward or discrete stimuli. Photostimulation of cholinergic neurons in the basal forebrain or their terminals in the basolateral amygdala (BLA) selectively drove conditioned responding (licking), but not unconditioned licking nor innate motor outputs. In vivo electrophysiological recordings revealed reward-contingency-dependent-gating of cholinergic suppression of BLA neural activity during cholinergic photostimulation, but not dorsomedial prefrontal cortex (dmPFC). Finally, cholinergic terminals suppressed BLA projection neuron activity via monosynaptic muscarinic receptor signaling and facilitation of firing in GABAergic interneurons. Taken together, we show that cholinergic effects are modulated by reward contingency in a target-specific manner to promote conditioned responding. Given that the effects cholinergic photostimulation were modulated by rewards, our results constrain clinical goals of augmenting cholinergic function to improve neuropsychiatric symptoms.


title: Multiple mechanisms mediate the suppression of motion vision during escape maneuvers in flying Drosophila.

abstract: Animals must be able to discriminate self-generated (reafferent) from external (exafferent) sensory input. Otherwise, the former could interfere with perception and behavioral actions. The way this can be achieved is through an efference copy, which suppresses reafferent sensory input. An example for this is the optomotor response of the fly. With the optomotor response, flies stabilize a straight flight path by correcting for unintended deviations, which they sense as visual motion of their surrounding or optic flow. HS cells of the fly are tuned to rotational optic flow and are thought to mediate optomotor responses to horizontal motion. It has been shown that during spontaneous turns, an efference copy influences the membrane potential of HS cells. Here we investigate the influence of an efference copy during looming-elicited evasive turns combined with a subsequent optomotor stimulus in Drosophila. We show that looming stimuli themselves can influence the processing of preferred-direction motion in HS cells. In addition, an efference copy can influence visual processing during saccades in both directions, but only in a subset of cells. Our study supports the notion that processing of sensory information is finely tuned and dependent on both stimulus history and behavioral context.


title: The conserved Pelado/ZSWIM8 protein regulates actin dynamics by promoting linear actin filament polymerization

abstract: Actin filament polymerization can be branched or linear, which depends on the associated regulatory proteins. Competition for Actin monomers occurs between proteins that induce branched or linear actin polymerization. Cell specialization requires the regulation of actin filaments to allow the formation of cell-type specific structures, like cuticular hairs in Drosophila, formed by linear actin filaments. Here, we report the functional analysis of CG34401/pelado, a gene encoding a SWIM domain containing protein, conserved throughout the animal kingdom. Mutant pelado epithelial cells display actin hair elongation defects. This phenotype is reversed by increasing Actin monomer levels or by either pushing linear actin polymerization or reducing branched actin polymerization. The same behavior occurs in hemocytes, where Pelado is essential to induce filopodia, a linear actin-based structure. We further show that this function of Pelado, ZSWIM8 in mammals, is conserved in human cells, where Pelado inhibits branched actin polymerization in a cell migration context. In summary, our data indicate that the function of Pelado/ZSWIM8 in regulating actin cytoskeletal dynamics is conserved, favoring linear actin polymerization at the expense of branched filaments.

category: cell biology


title: Pharmacogenetic and whole-brain activity analyses uncover integration of distinct molecular and circuit programs that drive learning

abstract: Habituation is a foundational learning process critical for animals to adapt their behavior to changes in their sensory environment. Although habituation is considered a simple form of learning, the identification of a multitude of molecular pathways including several neurotransmitter systems that regulate this process suggests an unexpected level of complexity. How the vertebrate brain integrates these various pathways to accomplish habituation learning, whether they act independently or intersect with one another, and whether they act via divergent or overlapping neural circuits has remained unclear. To address these questions, we combined pharmacogenetic pathway analysis with unbiased whole-brain activity mapping using the larval zebrafish. This approach revealed five distinct molecular and circuit modules that regulate habituation learning and identified a set of molecularly defined brain regions associated with four of the five modules. Moreover, we find that in module 1 the palmitoyltransferase Hip14 cooperates with dopamine and NMDA signaling to drive plasticity, while in module 3 the adaptor protein complex subunit Ap2s1 drives habituation by antagonizing dopamine signaling, revealing two distinct and opposing roles for dopaminergic neuromodulation in the regulation of learning. Combined, our results define a core set of distinct modules that act in concert to regulate learning-associated plasticity, and provide compelling evidence that even seemingly simple learning behaviors in a compact vertebrate brain are regulated by a complex and overlapping set of molecular and circuit mechanisms.


title: GSK2556286 is a novel antitubercular drug candidate effective in vivo with the potential to shorten tuberculosis treatment

abstract:
As a result of a high-throughput compound screening campaign of Mycobacterium tuberculosis infected macrophages, a new preclinical drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (IC50 = 0.07 {micro}M), is active against extracellular bacteria in cholesterol-containing culture media and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug or extensively-resistant (M/XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens.

One Sentence SummaryGSK2556286 is a novel preclinical drug candidate for the treatment of tuberculosis with a new mode of action potentially able to contribute to the shortening of TB chemotherapy.


title: Chronic Alcohol Consumption Alters Home-Cage Behaviors and Responses to Ethologically Relevant Predator Tasks in Mice

abstract: Alcohol use disorders (AUD) are the most prevalent substance use disorders worldwide. Considering recent reports indicating an increase in alcohol use particularly in females, it is vital to understand how alcohol history impacts behavior. Animal model research on withdrawal-associated affective states tends to focus on males, forced alcohol paradigms, and a few traditional anxiety/stress tests. While this has been essential, heavy alcohol use triggers adverse withdrawal-related affective states that can influence how people respond to a large variety of life events and stressors. To this end, we show that behaviors in the home-cage, open field, looming disc, and robogator predator threat task, which vary in task demand and intensity, are altered in mice with a history of voluntary alcohol consumption. In alcohol-exposed males, behaviors in the home cage, a low anxiety baseline environment, suggest increased vigilance/exploration. However, in the open field and robogator task, which induce heightened arousal and task demands, a more hesitant/avoidant phenotype is seen. Female alcohol mice show no behavioral alterations in the home cage and open field test, however, in the looming disc task, which mimics an overhead advancing predator and forces a behavioral choice, we see greater escape responses compared to water controls, indicative of active stress coping behaviors. This suggests females may begin to show alcohol-induced alterations as task demands increase. To date, few drugs have advanced past clinical trials for the treatment of AUD, and those that have are predominately used in life-threatening situations only. No treatments exist for ameliorating negative withdrawal related states, which could aid in harm reduction related to heavy alcohol use. Understanding how withdrawal alters a variety of behavioral responses that are linked to stress coping can widen our understanding of alcohol abuse and lead us closer to better therapeutics to help individuals with AUD.


title: Tissue specific dual RNA-seq defines host-parasite interplay in murine visceral leishmaniasis caused by Leishmania donovani and Leishmania infantum

abstract:
Visceral leishmaniasis is associated with hepato-splenomegaly and altered immune and haematological parameters in both pre-clinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFN{gamma} signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1{beta} and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFN{gamma} signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and [~]10% of host DEGs vary in infections with either parasite species.

ImportanceVisceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.


title: Insights in the complex DegU, DegS, Spo0A regulation system of Paenibacillus polymyxa by CRISPR-Cas9-based targeted point mutations

abstract:
Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated: the single mutants DegU Q218*, DegS L99F, Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild type and the engineered strains revealed differences in swarming behavior, genetic competence, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed significant increase in regard to the genetic competence as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences of the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights in the complex regulatory system of P. polymyxa DSM 365.

ImportanceTo date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate three regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations are introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, with the model bacterium Bacillus subtilis in regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance in comparison to the wild type strain. Overall, our study provides novel insights which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.