title: Analyses of cephalic and non-cephalic sensory cell types provide insight into joint photo- and mechanoreceptor evolution

abstract:
Rhabdomeric Opsins (r-Opsins) are light-sensors in cephalic eye photoreceptors, but also function in additional sensory organs. This has prompted questions on the evolutionary relationship of these cell types, and if ancient r-Opsins cells were non-photosensory. Our profiling of cephalic and non-cephalic r-opsin1-expressing cells of the marine bristleworm Platynereis dumerilii reveals shared and distinct features. Non-cephalic cells possess a full set of phototransduction components, but also a mechanosensory signature. We determine that Pdu-r-Opsin1 is a Gq-coupled blue-light receptor. Profiling of cells from r-opsin1 mutants versus wild-types, and a comparison under different light conditions reveals that in the non-cephalic cells, light - mediated by r-Opsin1 - adjusts the expression level of a calcium transporter relevant for auditory mechanosensation in vertebrates. We establish a deep learning-based quantitative behavioral analysis for animal trunk movements, and identify a light-and r-Opsin-1-dependent fine-tuning of the worms undulatory movements in headless trunks, which are known to require mechanosensory feedback.

Our results suggest an evolutionary concept in which r-Opsins act as ancient, light-dependent modulators of mechanosensation, and suggest that light-independent mechanosensory roles of r-Opsins likely evolved secondarily.


title: Mechanisms shaping the synergism of zeaxanthin and PsbS in photoprotective energy dissipation in the photosynthetic apparatus of plants

abstract: Safe operation of photosynthesis is vital to plants and is ensured by the activity of numerous processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is believed to be the main photoprotective mechanism and is most effective with the simultaneous presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle activity. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, the wild-type and the mutants lacking PsbS (npq4) and affected in the xanthophyll cycle (npq1), with the application of multiple molecular spectroscopy and imaging techniques. Research results lead to the conclusion that PsbS interferes with the formation of tightly packed aggregates of thylakoid membrane proteins, thus enabling the incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.

category: biophysics


title: Differential expression of striatal proteins in a mouse model of DOPA-responsive dystonia reveals shared mechanisms among dystonic disorders

abstract: Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting movements and postures. Although basal ganglia dysfunction is implicated in many forms of dystonia, the underlying mechanisms are unclear. Therefore, to reveal abnormal striatal cellular processes and pathways implicated in dystonia, we used an unbiased proteomic approach in a knockin mouse model of DOPA-responsive dystonia, a model in which the striatum is known to play a central role in the expression of dystonia. Fifty-seven of the 1805 proteins identified were differentially regulated in DOPA-responsive dystonia mice compared to control mice. Most differentially regulated proteins were associated with gene ontology terms that implicated either mitochondrial or synaptic dysfunction whereby proteins associated with mitochondrial function were generally over-represented whereas proteins associated with synaptic function were largely under-represented. Remarkably, nearly 20% of the differentially regulated proteins identified in our screen are associated with pathogenic variants that cause inherited dystonic disorders in humans suggesting shared mechanisms across many different forms of dystonia.

category: neuroscience


title: Deficiency of intellectual disability-related gene Brpf1 reduced inhibitory neurotransmission and Map2k7 expression in GABAergic interneurons

abstract: Intellectual disability is closely related to impaired GABA neurotransmission. Brpf1 was specifically expressed in medial ganglionic eminence (MGE), a developmental niche of GABAergic interneurons, and patients with BRPF1 mutations were mentally retarded. To test its role in development and function of MGE-derived GABAergic interneurons, we performed immunofluorescence staining, whole-cell patch-clamp, MGE transplantation and mRNA-Seq to understand its effect on neuronal differentiation, dendritic morphology, electrophysiology, migration and gene regulation, using mouse MGE-derived GABAergic interneurons infected with AAV-shBrpf1. We found a decreasing trend on parvalbumin+ interneuron differentiation. Moreover, increased firing threshold, decreased number of evoked APs, and a reduced amplitude of mIPSCs were observed before any significant change of MAP2+ dendritic morphology and in vivo migration appeared. Finally, mRNA-Seq analysis revealed that genes related to neurodevelopment and synaptic transmission such as Map2k7 were dysregulated. Our results demonstrated a key role of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic interneurons.


title: Voxel-wise and spatial modelling of binary lesion masks: A review and comparison of methods

abstract:
ObjectivesWhite matter lesions are a very common finding on MRI in older adults and their presence increases the risk of stroke and dementia. Accurate and computationally efficient modelling methods are necessary to map the association of lesion incidence with risk factors, such as hypertension. However, there is no consensus in the brain mapping literature whether a voxel-wise modelling approach is better for binary lesion data than a more computationally intensive spatial modelling approach that accounts for voxel dependence.

MethodsWe review three regression approaches for modelling binary lesion masks including massunivariate probit regression modelling with either maximum likelihood estimates, or mean bias-reduced estimates, and spatial Bayesian modelling, where the regression coefficients have a conditional autoregressive model prior to account for local spatial dependence. We design a novel simulation framework of artificial lesion maps to compare the three alternative lesion mapping methods. The age effect on lesion probability estimated from a reference data set (13,680 individuals from the UK Biobank) is used to simulate a realistic voxel-wise distribution of lesions across age. To mimic the real features of lesion masks, we propose matching brain lesion summaries (total lesion volume, average lesion size and lesion count) across the reference data set and the simulated data sets. Thus, we allow for a fair comparison between the modelling approaches, under a realistic simulation setting.

ResultsOur findings suggest that bias-reduced estimates for voxel-wise binary-response generalized linear models (GLMs) overcome the drawbacks of infinite and biased maximum likelihood estimates and scale well for large data sets because voxel-wise estimation can be performed in parallel across voxels. Contrary to the assumption of spatial dependence being key in lesion mapping, our results show that voxel-wise bias-reduction and spatial modelling result in largely similar estimates.

ConclusionBias-reduced estimates for voxel-wise GLMs are not only accurate but also computationally efficient, which will become increasingly important as more biobank-scale neuroimaging data sets become available.


title: Voxel-wise and spatial modelling of binary lesion masks: Comparison of methods with a realistic simulation framework


title: Male sex and age biases viral burden, viral shedding, and type 1 and 2 interferon responses during SARS-CoV-2 infection in ferrets

abstract: SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and the elderly. Here we investigated the impact of male sex and age by infecting adult male, aged male, and adult female ferrets with SARS-CoV-2. Aged male ferrets had a decrease in temperature which was accompanied by prolonged viral replication with increased pathology in the upper respiratory tract after infection. Transcriptome analysis of the nasal turbinates and lungs indicated that female ferrets had significant increases in interferon response genes (OASL, MX1, ISG15, etc.) on day 2 post infection which was delayed in aged males. In addition, genes associated with taste and smell such as RTP1, CHGA, and CHGA1 at later time points were upregulated in males but not in females. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.

category: microbiology


title: Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases

abstract: Pro-inflammatory fibroblasts are critical to pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjogrens syndrome, and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited the understanding of which pathways are shared by multiple diseases. To investigate, we profiled patient-derived fibroblasts from inflamed and non-inflamed synovium, intestine, lung, and salivary glands with single-cell RNA-sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts were expanded in all inflamed tissues and additionally mapped to dermal analogues in a public atopic dermatitis atlas. We further confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. This work represents the first cross-tissue, single-cell fibroblast atlas revealing shared pathogenic activation states across four chronic inflammatory diseases.

category: genomics


title: Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the liver

abstract: Reconstruction of heterogeneity through single-cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we implement Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.


title: srnaMapper: an optimal mapping tool for sRNA-Seq reads

abstract:
MotivationSequencing is the key method to study the impact of short RNAs, which include micro RNAs, tRNA-derived RNAs, and piwi-interacting RNA, among other. The first step to make use of these reads is to map them to a genome. Existing mapping tools have been developed for the long RNAs in mind, and, so far, no tool has been conceived for short RNAs. However, short RNAs have several distinctive features which make them different from messenger RNAs: they are shorter (not greater than 200bp), they often redundant, they can be produced by duplicated loci, and they may be edited at their ends.

ResultsIn this work, we present a new tool, srnaMapper, that maps these reads with all these objectives in mind. We show on two data sets that srnaMapper is more efficient considering computation time and edition error handling: it quickly retrieves all the hits, with arbitrary number of errors.

AvailabilitysrnaMapper source code is available at https://github.com/mzytnicki/srnaMapper.

Contactmatthias.zytnicki@inrae.fr

category: bioinformatics


title: Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody

abstract:
The recently reported "UK variant" of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-[A] resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments.

Short summaryThe N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.


title: Cryo-EM Structures of the N501Y SARS-CoV-2 Spike Protein in Complex with ACE2 and Two Potent Neutralizing Antibodies


title: Mapping of m6A and Its Regulatory Targets in Prostate Cancer Reveals a METTL3-low Induction of Therapy Resistance

abstract: Recent evidence has highlighted the role of N6-methyladenosine (m6A) in the regulation of mRNA expression, stability and translation, supporting a potential role for post-transcriptional regulation mediated by m6A in cancer. Here we explore prostate cancer as an exemplar and demonstrate that low levels of N6-adenosine-methyltransferase (METTL3) is associated with advanced metastatic disease. To explore this relationship, we generated the first prostate m6A maps, and further examined how METTL3 regulates expression at the level of transcription, translation, and protein. Significantly, transcripts encoding extracellular matrix proteins are consistently upregulated with METTL3 knockdown. We also examined the relationship between METTL3 and androgen signaling and discovered the upregulation of a hepatocyte nuclear factor-driven gene signature that is associated with therapy resistance in prostate cancer. Significantly, METTL3 knockdown rendered the cells resistant to androgen receptor antagonists, implicating changes in m6A as a mechanism for therapy resistance in metastatic prostate cancer.

category: cancer biology


title: -actinin-4 drives invasiveness by regulating myosin IIB expression and myosin IIA localization

abstract: The mechanisms by which the mechanoresponsive actin crosslinking protein -actinin-4 (ACTN4) regulates cell motility and invasiveness remains incompletely understood. Here we show that in addition to regulating protrusion dynamics and focal adhesion formation, ACTN4 transcriptionally regulates expression of non-muscle myosin IIB (NMM IIB), which is essential for mediating nuclear translocation during 3D invasion. We further demonstrate association between NMM IIA and ACTN4 at the cell front ensures retention of NMM IIA at the cell periphery. A protrusion-dependent model of confined migration recapitulating experimental observations predicts a dependence of protrusion forces on the degree of confinement and on the ratio of nucleus to matrix stiffness. Together, our results suggest that ACTN4 is a master regulator of cancer invasion that regulates invasiveness by controlling NMM IIB expression and NMM IIA localization.

category: cell biology


title: The Necroptosis Effector MLKL drives Small Extracellular Vesicle Release and Tumour Growth in Glioblastoma

abstract: Extracellular vesicles (EVs) are lipid-based nano-sized particles that convey biological material from donor to recipient cells. They play key roles in tumour progression, notably in glioblastoma in which the subpopulation of Glioblastoma Stem-like Cells (GSCs) might represent a meaningful source of tumour-derived EVs. However, the mechanisms involved in the production and release of EVs by GSCs are still poorly understood. Here, we report the identification of MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of small EVs from GSCs. The targeting of MLKL by genetic, protein depletion or chemical approaches alters endosomal trafficking and EV release and reduces GSC expansion in vitro. This function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL triggers a reduction of both the tumour burden in xenografted mice and of the level of plasmatic EVs. This work reinforces the idea of a non-deadly role for MLKL in endosomal trafficking and suggests that interfering with EV biogenesis is a promising therapeutic option to sensitize glioblastoma cells to death.


title: Restore mitophagy is essential to prevent cardiac oxidative stress during hypertrophy

abstract: Heart failure, mostly associated with cardiac hypertrophy, is still a major cause of illness and death. Oxidative stress causes contractile failure and the accumulation of reactive oxygen species leads to mitochondrial dysfunction, associated with aging and heart failure, suggesting that mitochondria-targeted therapies could be effective in this context. The purpose of this work was to characterize how mitochondrial oxidative stress is involved in cardiac hypertrophy development and to determine if mitochondria-targeted therapies could improve cardiac phenotypes. We used neonatal and adult rat cardiomyocytes (NCMs and ACMs) hypertrophied by isoproterenol (Iso) to induce an increase of mitochondrial superoxide anion. Superoxide dismutase 2 activity and mitochondrial biogenesis were significantly decreased after 24h of Iso treatment. To counteract the mitochondrial oxidative stress induced by hypertrophy, we evaluated the impact of two different anti-oxidants, mitoquinone (MitoQ) and EUK 134. Both significantly decreased mitochondrial superoxide anion and hypertrophy in hypertrophied NCMs and ACMs. Conversely to EUK 134 which preserved cell functions, MitoQ impaired mitochondrial function by decreasing maximal mitochondrial respiration, mitochondrial membrane potential and mitophagy (particularly Parkin expression) and altering mitochondrial structure. The same decrease of Parkin was found in human cardiomyocytes but not in fibroblasts suggesting a cell specificity deleterious effect of MitoQ. Our data showed the importance of mitochondrial oxidative stress in the development of cardiomyocyte hypertrophy. Interestingly, we observed that targeting mitochondria by an anti-oxidant (MitoQ) impaired metabolism specifically in cardiomyocytes. Conversely, the SOD mimic (EUK 134) decreased both oxidative stress and cardiomyocyte hypertrophy and restored impaired cardiomyocyte metabolism and mitochondrial biogenesis.


title: Convergent extension requires adhesion-dependent biomechanical integration of cell crawling and junction contraction

abstract: Convergent extension is an evolutionarily conserved collective cell movement that elongates the body axis of almost all animals and is required for the morphogenesis of several organ systems. Decades of study have revealed two distinct mechanisms of cell movement during CE, one based on cell crawling and the other on junction contraction. How these two behaviors collaborate during CE is not understood. Here, using quantitative live cell imaging we show that these two modes act both independently and in concert during CE, but that cell movement is more effective when the two modes are integrated via mechano-reciprocity. Based on these findings, we developed a novel computational model that for the first time treats crawling and contraction independently. This model not only confirmed the biomechanical efficacy of integrating the two modes, but also revealed for the first time how the two modes -and their integration- are influenced by cell adhesion. Finally, we use these new insights to further understand the complex CE phenotype resulting from loss of the C-cadherin interacting catenin Arvcf. These data are significant for providing new biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.

category: developmental biology


title: Coregulated assembly of actin-like FtsA polymers with FtsZ during Z-ring formation and division in Escherichia coli

abstract: In Escherichia coli, the actin homolog FtsA localizes the cell division machinery, beginning with the Z-ring, to the cytoplasmic membrane through direct interaction with FtsZ. FtsZ polymers are first to assemble at the Z-ring at midcell, where they direct constriction and septation. While FtsZ polymerization is critical for establishing a functional Z-ring that leads to constriction, the assembly state of FtsA and the role of FtsA ATP utilization during division in E. coli remain unclear. Here, we show that ATP hydrolysis, FtsZ interaction, and phospholipid vesicle remodeling by FtsA are impaired by a substitution mutation at the predicted active site for hydrolysis. This mutation, Glu 14 to Arg, also impairs Z-ring assembly and division in vivo. To further investigate the role of phospholipid engagement and ATP utilization in regulating FtsA function, we characterized a truncated E. coli FtsA variant, FtsA({Delta}MTS), which lacks the region at the C-terminus important for engaging the membrane and is defective for ATP hydrolysis. We show that E. coli FtsA({Delta}MTS) forms ATP-dependent actin-like filaments and assembly is antagonized by FtsZ. Polymerization of FtsZ with GTP, or a non-hydrolyzable analog, blocks inhibition of ATP-dependent FtsA assembly, and instead favors coassembly of stable FtsA/FtsZ polymers. In the cell, FtsA/FtsZ coassembly is favored at midcell, where FtsZ polymerizes, and inhibited at regions where FtsZ polymers are destabilized by regulators, such as MinC at the poles or SlmA at the nucleoid. We show that MinC prevents recruitment of FtsZ, via FtsA, to phospholipids, suggesting that local interactions of MinC with FtsZ block membrane tethering and uncouple the Z-ring from its major membrane contact. During Z-ring formation, the coassembly of FtsZ polymers with FtsA is coordinated and is a critical early step in division. This step also serves as a checkpoint by responding to the suite of FtsZ assembly regulators in the cell that modulate Z-ring position and dynamics prior to initiating cell wall synthesis.


title: ADF and cofilin-1 collaborate to promote cortical actin flow and the leader bleb-based migration of confined cells

abstract: Melanoma cells have been shown to undergo fast amoeboid (leader bleb-based) migration, requiring a single large bleb for migration. In leader blebs, is a rapid flow of cortical actin that drives the cell forward. Using RNAi, we find that co-depleting cofilin-1 and ADF led to a large increase in cortical actin, suggesting that both proteins regulate cortical actin. Furthermore, severing factors can promote contractility through the regulation of actin architecture. However, RNAi of cofilin-1 but not ADF led to a significant decrease in cell stiffness. We found cofilin-1 to be enriched at leader bleb necks, whereas RNAi of cofilin-1 and ADF reduced bleb sizes and the frequency of motile cells. Strikingly, cells without cofilin-1 and ADF had blebs with abnormally long necks. Many of these blebs failed to retract and displayed slow actin turnover. Collectively, our data identifies cofilin-1 and ADF as actin remodeling factors required for fast amoeboid migration.


title: Cell cycle exit and stem cell differentiation are coupled through regulation of mitochondrial activity in the Drosophila testis

abstract: Stem cells maintain tissue homeostasis by proliferating to replace cells lost to damage or natural turnover. Whereas stem and progenitor cells proliferate, fully differentiated cells exit the cell cycle. How cell identity and cell cycle state are coordinated during this process is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs), which are the only proliferating somatic cells in the testis. CySCs give rise to post-mitotic cyst cells and therefore provide a tractable model to ask how stem cell identity is linked to proliferation. We show that the G1/S cyclin, Cyclin E, is required for CySC self-renewal; however, its canonical transcriptional regulator, a complex of the E2f1 and Dp transcription factors is dispensable for self-renewal and cell cycle progression. Nevertheless, we demonstrate that E2f1/Dp activity must be silenced to allow CySCs to differentiate. We show that E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial activity or biogenesis is sufficient to rescue the differentiation of CySCs with ectopic E2f1/Dp activity but not their exit from the cell cycle. Our findings together indicate that E2f1/Dp coordinates cell cycle progression with stem cell identity by regulating the metabolic state of CySCs.


title: Quantitative analysis of signaling responses during mouse primordial germ cell specification

abstract: During early mammalian development, the pluripotent cells of the embryo are exposed to a combination of signals that drive exit from pluripotency and germ layer differentiation. At the same time, a small population of pluripotent cells give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. Despite the importance of PGCs, it remains unclear how they are first segregated from the soma, and if this involves distinct responses to their signaling environment. To investigate this question, we mapped BMP, MAPK and WNT signaling responses over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution. We showed that, in the mouse embryo, early PGCs exhibit lower BMP and MAPK responses compared to neighboring extraembryonic mesoderm cells, suggesting the emergence of distinct signaling regulatory mechanisms in the germline versus soma. In contrast, PGCs and somatic cells responded comparably to WNT, indicating that this signal alone is not sufficient to promote somatic differentiation. Finally, we investigated the requirement of a BMP response for these cell fate decisions. We found that cell lines with a mutation in the BMP receptor (Bmpr1a-/-), which exhibit an impaired BMP signaling response, can efficiently generate PGC-like cells revealing that canonical BMP signaling is not cell autonomously required to direct PGC-like differentiation.


title: Conserved and unique transcriptional features of pharyngeal arches in the skate (Leucoraja erinacea) and evolution of the jaw

abstract: The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers bapx1 and gdf5 and pro-chondrogenic transcription factors barx1 and gsc, and the dorsalising transcription factor pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.


title: Maternal Biological Age Assessed in Early Pregnancy is Associated with Gestational Age at Birth

abstract: AO_SCPLOWBSTRACTC_SCPLOWMaternal age is an established predictor of preterm birth independent of other recognized risk factors. The use of chronological age makes the assumption that individuals age at a similar rate. Therefore, it does not capture interindividual differences that may exist due to genetic background and environmental exposures. As a result, there is a need to identify biomarkers that more closely index the rate of cellular aging. One potential candidate is biological age (BA) estimated by the DNA methylome. This study investigated whether maternal BA, estimated in either early and/or late pregnancy, predicts gestational age at birth. BA was estimated from a genome-wide DNA methylation platform using the Horvath algorithm. Linear regression methods assessed the relationship between BA and pregnancy outcomes, including gestational age at birth and perceived stress during pregnancy, in a primary and replication cohort. Prenatal BA estimates from early pregnancy explained variance in gestational age at birth above and beyond the influence of other recognized preterm birth risk factors. Sensitivity analyses indicated that this signal was driven primarily by self-identified African American participants. This predictive relationship was sensitive to small variations in the BA estimation algorithm. Benefits and limitations of using BA in translational research and clinical applications for preterm birth are considered.


title: Local brainstem circuits contribute to reticulospinal output in the mouse

abstract:
Glutamatergic reticulospinal neurons in the gigantocellular reticular nucleus (GRN) of the medullary reticular formation can function as command neurons, transmitting motor commands to spinal cord circuits. Recent advances in our understanding of this neuron-dense region have been facilitated by the discovery of expression of the transcriptional regulator, Chx10, in excitatory reticulospinal neurons. Here, we address the capacity of local circuitry in the GRN to contribute to reticulospinal output. We define two sub-populations of Chx10-expressing neurons in this region, based on distinct electrophysiological properties and somata size (small and large), and show that these correspond to local interneurons and reticulospinal neurons, respectively. Using focal release of caged-glutamate combined with patch clamp recordings, we demonstrated that Chx10 neurons form microcircuits in which the Chx10 interneurons project to and facilitate the firing of Chx10 reticulospinal neurons. We discuss the implications of these microcircuits in terms of movement selection.

SIGNIFICANCE STATEMENTReticulospinal neurons in the medullary reticular formation play a key role in movement. The transcriptional regulator Chx10 defines a population of glutamatergic neurons in this region, a proportion of which have been shown to be involved in stopping, steering, and modulating locomotion. While it has been shown that these neurons integrate descending inputs, we asked whether local processing also ultimately contributes to reticulospinal outputs. Here, we define Chx10-expressing medullary reticular formation interneurons and reticulospinal neurons, and demonstrate how the former modulate the output of the latter. The results shed light on the internal organization and microcircuit formation of reticular formation neurons.


title: Toward Optimized and Predictive Connectomics at Scale

abstract:
Probabilistic MRI diffusion tractography is a sophisticated technique to investigate structural connectomes, but its steep computational cost prevents application to broader research and clinical settings. Major speedup can be achieved by reducing the number of tractography streamlines. To ensure this does not degrade connectome quality, we calculate the identifiability of connectomes between test and retest MRI as a proxy for information content. We find that reducing streamline count by up to two orders of magnitude from prevailing levels in literature has no significant impact on identifiability. Incidentally, we also observe that Jaccard similarity is more effective than Pearson correlation in achieving identifiability.

This document was prepared as an account of work sponsored by an agency of the United States government. Neither the United States government nor Lawrence Livermore National Security, LLC, nor any of their employees makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States government or Lawrence Livermore National Security, LLC. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States government or Lawrence Livermore National Security, LLC, and shall not be used for advertising or product endorsement purposes.


title: Maternal behavior and the neonatal HPA axis in the Wistar Audiogenic Rat (WAR) strain: early life implications for a genetic animal model in epilepsy

abstract: Epileptogenesis is a multistage process and seizure susceptibility can be influenced by stress early in life. Wistar Audiogenic Rat (WAR) strain is an interesting model to study the association between stress and epilepsy, since it is naturally susceptible to seizures and present changes in the hypothalamus-pituitary-adrenal (HPA) axis activity. All these features are related to the pathogenic mechanisms usually associated to psychiatric comorbidities present in epilepsy. Therefore, the current study aimed to evaluate the neonate HPA axis function and maternal care under control and stress conditions in the WAR strain. Maternal behavior and neonate HPA axis were evaluated in Wistar and WAR strains under rest and after the presence of stressors. We observed that WAR pups present higher plasmatic corticosterone concentration as compared to Wistar pups. Although WAR dams do not show significant altered maternal behavior at rest, there is a higher latency to recover the litter in the pup retrieval test, while some did not recover all the litter. WAR dams presented similar behaviors to Wistar dams to a female intruder and maternal care with the pups in the maternal defense test. Taken together, these findings indicate that the WAR strain could show HPA axis disruption early in life and dams present altered maternal behavior under stressful events. Those alterations make the WAR strain an interesting model to evaluate vulnerability to epilepsy and its associated neuropsychiatric comorbidities.


title: Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

abstract: Stress related disorders lead to serious psychiatric disabilities and are comorbid with anxiety and depression. Current therapies targeting several neurotransmitter systems are only able to mitigate symptoms partially. It is well recognized that stress and trauma related disorders lead to a prominent inflammatory response in humans, and in several animal models a robust neuroinflammatory response has been observed. However, the therapeutic potential of targeting specific components of the inflammatory response has not been adequately studied in this context. The current study investigated the NLRP3 - Caspase1-IL-1{beta} pathway, which recent research has identified as a major contributor to exacerbated inflammatory response in several peripheral and central nervous system pathological conditions. Using two different models of stress, first - single prolonged restraint stress followed by brief underwater submersion and second - predator odor exposure in mice, we demonstrate heightened anxious behavior in mice one-week after stress. Females in both models display an exacerbated anxiety response than males within the stressed group. Consistent with this data stressed animals demonstrate upregulation of IL-1{beta}, IL-6, Caspase1 activity and NLRP3 inflammasome activation in brain, with female animals showing a stronger neuroinflammatory phenotype. Pharmacological inhibition of NLRP3 inflammasome activation led to a rescue in terms of anxious behavior as well as attenuated neuroinflammatory response, both of which were significantly more prominent in female animals. Further, we observed induction of activated Brutons Tyrosine Kinase (BTK), an upstream positive regulator of NLRP3 inflammasome activation, in hippocampus and amygdala of stressed mice. Next, we conducted proof-of-concept pharmacological BTK inhibitor studies with Ibrutinib, a drug that is already FDA approved for use in certain types of lymphomas and leukemias, as well as a second inhibitor of BTK, LFM-A13. In both sets of experiments, we found inhibition of BTK significantly reduced the anxious behavior in stressed mice and attenuated the induction of NLRP3 inflammasome, Caspase 1 and IL1{beta}. Our results suggest that BTK inhibition can be further investigated in context of human stress and trauma related disorders as a therapeutic strategy.


title: Evolving cryo-EM structural approaches for GPCR drug discovery

abstract: G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted Falcon 4 yielded a 3.2 [A] map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

category: biochemistry


title: A flexible micro-piston device for mechanical cell stimulation and compression in microfluidic settings

abstract: Evidence continues to emerge that cancer is not only a disease of genetic mutations, but also of altered mechanobiological profiles of the cells and microenvironment. This mutation-independent element might be a key factor in promoting development and spread of cancer. Biomechanical forces regulate tumor microenvironment by solid stress, matrix mechanics, interstitial pressure and flow. Compressive stress by tumor growth and stromal tissue alters the cell deformation, and recapitulates the biophysical properties of cells to grow, differentiate, spread or invade. Such a solid stress can be introduced externally to change the cell response and to mechanically induce cell lysis by dynamic compression. In this work we report a microfluidic cell-culture platform with an integrated, actively-modulated actuator for the application of compressive forces on cancer cells. Our platform is composed of a control microchannel in a top layer for introducing external force and a polydimethylsiloxane (PDMS) membrane with monolithically integrated actuators. The integrated actuator, herein called micro-piston, was used to apply compression on SKOV-3 ovarian cancer cells in a dynamic and controlled manner by modulating applied gas pressure, localization, shape and size of the micro-piston. We report fabrication of the platform, characterization of the mechanical actuator experimentally and computationally, as well as cell loading and culture in the device. We further show use of the actuator to perform both, repeated dynamic cell compression at physiological pressure levels, and end-point mechanical cell lysis, demonstrating suitability for mechanical stimulation to study the role of compressive forces in cancer microenvironments.

category: bioengineering


title: CoSTA: Unsupervised Convolutional Neural Network Learning for Spatial Transcriptomics Analysis

abstract: The rise of spatial transcriptomics technologies is leading to new insights about how gene regulation happens in a spatial context. Here, we present CoSTA: a novel approach to learn spatial similarities between gene expression matrices via convolutional neural network (ConvNet) clustering. By analyzing simulated and previously published spatial transcriptomics data, we demonstrate that CoSTA learns spatial relationships between genes in a way that emphasizes whole patterns rather than pixel-level correlation. CoSTA provides a quantitative measure of how similar each pair of genes are by their spatial pattern rather than only classifying genes into categories. We find that CoSTA identifies narrower, but biologically relevant, sets of significantly related genes as compared to other approaches.


title: Snake venom phospholipases A2 possess a strong virucidal activity against SARS-CoV-2 in vitro and block the cell fusion mediated by spike glycoprotein interaction with the ACE2 receptor

abstract: A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the absence of specific therapeutic and prophylactic agents, the virus has infected almost hundred million people, of whom nearly two million have died from the viral disease COVID-19. The ongoing COVID-19 pandemic is a global threat requiring new therapeutic strategies. Among them, antiviral studies based on natural molecules are a promising approach. The superfamily of phospholipases A2 (PLA2s) consists of a large number of members that catalyze the hydrolysis of phospholipids at a specific position. Here we show that secreted PLA2s from the venom of various snakes protect to varying degrees the Vero E6 cells widely used for the replication of viruses with evident cytopathic action, from SARS-CoV-2 infection PLA2s showed low cytotoxicity to Vero E6 cells and the high antiviral activity against SARS-CoV-2 with IC50 values ranged from 0.06 to 7.71 g/ml. Dimeric PLA2 HDP-2 from the viper Vipera nikolskii, as well as its catalytic and inhibitory subunits, had potent virucidal (neutralizing) activity against SARS-CoV-2. Inactivation of the enzymatic activity of the catalytic subunit of dimeric PLA2 led to a significant decrease in antiviral activity. In addition, dimeric PLA2 inhibited cell-cell fusion mediated by SARS-CoV-2 spike glycoprotein. These results suggest that snake PLA2s, in particular dimeric ones, are promising candidates for the development of antiviral drugs that target lipid bilayers of the viral envelope and may be good tools to study the interaction of viruses with host cell membranes.


title: An SR protein is essential for the recovery of malaria parasites from DNA damage and exposure to artemisinin

abstract: Plasmodium falciparum, the parasite responsible for the deadliest form of human malaria, maintains a complex life cycle with a relatively small number of genes. PfSR1 is an alternative splicing factor that regulates expansion of the P. falciparum protein repertoire. To further investigate PfSR1 functions, we set to unveil its interactome. We found that PfSR1 interacts with proteins, which are linked to various processes of RNA metabolism in a stage-dependent manner. These include: chromatin re-modeling, transcription, splicing and translation. Intriguingly, some of the PfSR1 interacting proteins are orthologues of proteins implicated in the DNA damage response. We demonstrate that PfSR1 expression is important for preventing the accumulation of DNA damage in proliferating parasites. In addition, following parasites exposure to a source of DNA damage, PfSR1 is recruited to damaged foci where it interacts with the phosphorylated core histone PfH2A, which marks damaged chromatin. Furthermore, PfSR1 expression was found to be essential for the ability of the parasite to activate the DNA repair machinery and recover from DNA damage caused by either irradiation or exposure to artemisinin, the first line anti-malarial drug. These findings unveil a novel role of PfSR1 in protecting P. falciparum from DNA damage and artemisinin exposure.


title: Experimental corticosterone manipulation increases mature feather corticosterone content: implications for inferring avian stress history from feather analyses

abstract: Feathers incorporate plasma corticosterone (CORT) during their development and, because feathers lack blood supply at maturity, the CORT content of feathers (CORTf) is presumed to represent an integrated average of plasma CORT levels during feather growth. We tested this assumption by quantifying CORTf in feathers of house sparrows (Passer domesticus) that were plucked before and after being implanted with either a corticosterone-filled, metyrapone-filled, or empty (sham) silastic capsule. Two of the seven flight feathers collected from each bird were fully grown when birds received implants. We found that CORTf of all seven feathers corresponded with treatment type, and with plasma CORT levels of non-moulting reference sparrows also given these implants. We also found that CORTf of the two mature feathers of each bird were 4 to 10-fold higher than values measured in new feathers. Given the avascular nature of mature feathers, and the fact that we did not wash the feathers prior to analysis, the most plausible explanation for our results is that CORT was externally deposited on feathers after implant. This outcome emphasises the need for follow-up studies to identify the external sources of CORT that may affect the CORTf of feathers. We hope this study will stimulate discussions and further much needed studies on the mechanism of CORT deposition in feathers and open exciting opportunities for application of such methods in ecological research, such as measuring multiple time scales in such a non-invasive manner.

category: physiology


title: The three-dimensional landscape of chromatin accessibility in Alzheimer's disease

abstract: Much is still unknown about the neurobiology of Alzheimers disease (AD). To better understand AD, we generated 636 ATAC-seq libraries from cases and controls to construct detailed genomewide chromatin accessibility maps of neurons and non-neurons from two AD-affected brain regions, the entorhinal cortex and superior temporal gyrus. By analyzing a total of 19.6 billion read pairs, we expanded the known repertoire of regulatory sequences in the human brain. Multi-omic data integration associated global patterns of chromatin accessibility with gene expression and identified cell-specific enhancer-promoter interactions. Using inter-individual variation in chromatin accessibility, we define cis-regulatory domains capturing the 3D structure of the genome. Multifaceted analyses uncovered disease associated perturbations impacting chromatin accessibility, transcription factor regulatory networks and the 3D genome, and implicated transcriptional dysregulation in AD. Overall, we applied a systematic approach to understand the role of the 3D genome in AD and to illuminate novel disease biology that can advance diagnosis and therapy.


title: Cortical responses to vagus nerve stimulation are modulated by ongoing oscillatory activity associated with different brain states in non-human primates

abstract: Vagus nerve stimulation (VNS) has been tested as therapy for several brain disorders and as a means to modulate cortical excitability and brain plasticity. Cortical effects of VNS, manifesting as vagal-evoked potentials (VEPs), are thought to arise from activation of ascending cholinergic and noradrenergic systems. However, it is unknown whether those effects are modulated by brain state at the time of stimulation. In 2 freely behaving macaque monkeys, we delivered trains of left cervical VNS at different frequencies (5-300 Hz) while recording local field potentials (LFPs) from sites in contralateral prefrontal, sensorimotor and parietal cortical areas. Brain states were inferred from spectral components of LFPs and the presence of overt movement: active awake, resting awake, REM sleep and NREM sleep. VNS elicited VEPs comprising early (<70 ms), intermediate (70-250 ms) and late (>250 ms) components in all sampled cortical areas. The magnitude of only the intermediate and late components was modulated by brain state and pulsing frequency. These findings have implications for the role of ongoing cortical activity and brain state in shaping cortical responses to peripheral stimuli, for the modulation of vagal interoceptive signaling by cortical states, and for the calibration of VNS therapies.


title: Cortical responses to vagus nerve stimulation are modulated by brain state in non-human primates


title: Acute inflammation, mediated by lung neutrophils, confers enhanced protection against Mycobacterium tuberculosis infection in mice

abstract: Inflammation plays a crucial role in the control of Mycobacterium tuberculosis (M.tb) infection. In this study, we demonstrate that an inflammatory pulmonary environment at the time of infection mediated by liposaccharide (LPS) treatment in mice confers enhanced protection against M.tb for up to 6 months post infection. This transient protective inflammatory environment was associated with a neutrophil and monocyte/macrophage influx as well as increased inflammatory cytokines. In vitro infection of neutrophils from LPS treated mice demonstrated that LPS neutrophils exhibited increased recognition of M.tb, and had a greater innate capacity for killing M.tb. Finally, partial depletion of neutrophils in LPS treated mice showed an increase in M.tb burden, suggesting neutrophils conferred the enhanced protection observed in LPS treated mice. These results indicate a positive role of an inflammatory environment during initial M.tb infection, and suggests that acute inflammation at the time of M.tb infection can positively alter disease outcome.

category: immunology


title: Low oxygen arrests Babesia duncani schizonts and leads to increased drug susceptibilities in hamster erythrocytes

abstract:
We studied the effect of oxygen concentrations on the in vitro growth and drug susceptibility of Babesia duncani. We found that the growth of B. duncani required high level oxygen and the culture condition at ambient aerobic condition (21% O2) was optimal. Compared with ambient air, our results further showed that low oxygen (6-16%) could arrest B. duncani schizonts and lead to high susceptibilities to antiparasitic drugs atovaquone, pyrimethamine, quinine, and chloroquine at certain concentrations in vitro. Drug susceptibilities of other Babesia spp impacted by O2 levels need to be studied in the future, and this study indicates that culturing conditions of Babesia spp should be considered and reestablished for generating more comparable and reliable results in drug research in the future.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/426147v1_ufig1.gif" ALT="Figure 1">
View larger version (54K):
org.highwire.dtl.DTLVardef@6414b2org.highwire.dtl.DTLVardef@8ad845org.highwire.dtl.DTLVardef@17cd3bcorg.highwire.dtl.DTLVardef@30f1f2_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIThe in vitro growth of B. duncani requires high level oxygen and the culture condition of 5% CO2 ambient air is optimal
C_LIO_LILow level oxygen results B. duncani in low growth rate and high schizont ratio in vitro
C_LIO_LICompared with 5% CO2 ambient air, in vitro drug susceptibilities of B. duncani can be significantly increased when cultured in microaerobic condition
C_LI


title: Microbial retention and resistances in stormwater quality improvement devices treating road runoff

abstract: Current knowledge about the microbial communities that occur in in urban road runoff is scarce. Road runoff of trafficked roads can be heavily polluted and is treated by stormwater quality improvement devices (SQIDs). However, microbes may influence the treatment process of these devices or could lead to stress resistant opportunistic microbial strains. In this study, the microbial community in the influent, effluent and the filter materials for the removal of dissolved heavy metals of two different SQIDs were analyzed to determine the microbial load, retention, composition, and mobile resistance genes. Although the microbes were replaced by new taxa in the effluent, there was no major retention of microbial genera. Further, the bacterial abundance of the SQIDs effluent was relatively stable over time. The heavy metal content correlated with intl1 and with microbial genera. The filter media itself was enriched with Intl1 gene cassettes, carrying several heavy metal and multidrug resistance genes (e.g. czrA, czcA, silP, mexW and mexI), indicating that this is a hot spot for horizontal gene transfer. Overall, the results shed light on road runoff microbial communities, and pointed to distinct bacterial communities within the SQIDs, which subsequently influence the microbial community and the genes released with the treated water.


title: Saccharomyces cerevisiae gene expression during fermentation of Pinot noir wines at industrially relevant scale

abstract:
During a wine fermentation, Saccharomyces cerevisiae transforms grape must through metabolic activities that generate ethanol and other compounds. Thousands of genes change expression over the course of a wine fermentation to allow S. cerevisiae to adapt to and dominate the fermentation environment. Investigations into these gene expression patterns have previously revealed genes that underlie cellular adaptation to the grape must and wine environment involving metabolic specialization and ethanol tolerance. However, the vast majority of studies detailing gene expression patterns have occurred in controlled environments that do not recapitulate the biological and chemical complexity of fermentations performed at production scale. Here, we present an analysis of the S. cerevisiae RC212 gene expression program across 40 pilot-scale fermentations (150 liters) using Pinot noir grapes from 10 California vineyards across two vintages. We observe a core gene expression program across all fermentations irrespective of vintage similar to that of laboratory fermentations, in addition to novel gene expression patterns likely related to the presence of non-Saccharomyces microorganisms and oxygen availability during fermentation. These gene expression patterns, both common and diverse, provide insight into Saccharomyces cerevisiae biology critical to fermentation outcomes at industry-relevant scales.

ImportanceThis study characterized Saccharomyces cerevisiae RC212 gene expression during Pinot noir fermentation at pilot scale (150 liters) using production-relevant conditions. The reported gene expression patterns of RC212 is generally similar to that observed in laboratory fermentation conditions, but also contains gene expression signatures related to yeast-environment interactions found in a production setting (e.g., presence of non-Saccharomyces microorganisms). Key genes and pathways highlighted by this work remain under-characterized, raising the need for further research to understand the roles of these genes and their impact on industrial wine fermentation outcomes.


title: A point mutation in HIV-1 integrase redirects proviral integration into centromeric repeats

abstract: Retroviruses utilize the viral integrase (IN) protein to integrate a DNA copy of their genome into the host chromosomal DNA. HIV-1 integration sites are highly biased towards actively transcribed genes, likely mediated by binding of the IN protein to specific host factors, particularly LEDGF, located at these gene regions. We here report a dramatic redirection of integration site distribution induced by a single point mutation in HIV-1 IN. Viruses carrying the K258R IN mutation exhibit more than a 25-fold increase in integrations into centromeric alpha satellite repeat sequences, as assessed by both deep sequencing and qPCR assays. Immunoprecipitation studies identified host factors that uniquely bind to the mutant IN protein and thus may account for the novel bias for integration into centromeres. Centromeric integration events are known to be enriched in the latent reservoir of infected memory T cells, as well as in patients who control viral replication without intervention (so-called elite controllers). The K258R point mutation in HIV-1 IN reported in this study has also been found in databases of latent proviruses found in patients. The altered integration site preference induced by this mutation has uncovered a hidden feature of the establishment of viral latency and control of viral replication.


title: High-content imaging to phenotype antimicrobial effects on individual bacteria at scale

abstract:
High-content imaging (HCI) is technique for screening multiple cells in high resolution to detect subtle morphological and phenotypic variation. The method has been commonly deployed on model eukaryotic cellular systems, often for screening new drugs and targets. HCI is not commonly utilised for studying bacterial populations but may be powerful tool in understanding and combatting antimicrobial resistance. Consequently, we developed a high-throughput method for phenotyping bacteria under antimicrobial exposure at the scale of individual bacterial cells. Imaging conditions were optimised on an Opera Phenix confocal microscope (Perkin-Elmer) and novel analysis pipelines were established for both Gram-negative bacilli and Gram-positive cocci. The potential of this approach was illustrated using isolates of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus. HCI enabled the detection and assessment of subtle morphological characteristics, undetectable through conventional phenotypical methods, that could reproducibly distinguish between bacteria exposed to different classes of antimicrobials with distinct modes of action (MOA). In addition, distinctive responses were observed between susceptible and resistant isolates. By phenotyping single bacterial cells, we observed intra-population differences, which may be critical in identifying persistence or emerging resistance during antimicrobial treatment. The work presented here outlines a comprehensive method for investigating morphological changes at scale in bacterial populations under specific perturbation.

ImportanceHigh-content imaging (HCI) is a microscopy technique that permits the screening of multiple cells simultaneously in high resolution to detect subtle morphological and phenotypic variation. The power of this methodology is that is can generate large datasets comprised of multiple parameters taken from individual cells subjected to range of different conditions. We aimed to develop novel methods for using HCI to study bacterial cells exposed to a range of different antibiotic classes. Using an Opera Phenix confocal microscope (Perkin-Elmer) and novel analysis pipelines we created a method to study the morphological characteristics of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus when exposed to antibacterial drugs with differing modes of action. By imaging individual bacterial cells at high resolution and scale, we observed intra-population differences associated with different antibiotics. The outlined methods are highly relevant for how we begin to better understand and combat antimicrobial resistance.


title: Characterizing scaling laws in gut microbial dynamics from time series data: caution is warranted

abstract: Many studies have revealed that both host and environmental factors can impact the gut microbial compositions, implying that the gut microbiota is considerably dynamic1-5. In their Article, Ji et al.6 performed comprehensive analysis of multiple high-resolution time series data of human and mouse gut microbiota. They found that both human and mouse gut microbiota dynamics can be characterized by several robust scaling laws describing short- and long-term changes in gut microbiota abundances, distributions of species residence and return times, and the correlation between the mean and the temporal variance of species abundances. They claimed that those scaling laws characterize both short- and long-term dynamics of gut microbiota. However, we are concerned that their interpretation is quite misleading, because all the scaling laws can be reproduced by the shuffled time series with completely randomized time stamps of the microbiome samples.


title: Hypoxia-inducible factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis

abstract:
Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1 (HIF1) was previously proposed to support virus replication through its metabolic regulatory function. HIF1 protein levels rise in response to HCMV infection in non-hypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1 in HCMV replication by generating primary human cells with HIF1 knocked out using CRISPR/Cas9. When HIF1 was absent, we found that HCMV replication was enhanced, showing that HIF1 suppresses viral replication. We used untargeted metabolomics to determine if HIF1 regulates metabolite concentrations in HCMV infected cells. We discovered that in HCMV-infected cells, HIF1 suppresses intracellular and extracellular concentrations of kynurenine. HIF1 also suppressed the expression of the indoleamine 2,3-dioxygenase 1 (IDO1) rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication while activating AhR with an exogenous ligand increases HCMV replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1 reduces HCMV replication by regulating metabolism and metabolite signaling.

ImportanceViruses, like human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1 (HIF1) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1 suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1-dependent mechanism. Our findings describe a functional connection between HIF1, IDO1, and AhR that allows HIF1 to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.


title: Identification of Nocardia species using Autof MS 1000 and Bruker MALDI-TOF MS systems: A comparison

abstract: Nocardia is an important cause of clinically invasive disease, but for most clinical laboratories, identification of these isolates to the species level is challenging. Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for identification of most bacterial and fungal isolates. In this multicenter study, we evaluated the identification of Nocardia isolates using Autof MS1000 and Bruker Biotyper. A total of 86 non-duplicate Nocardia isolates from 7 hospital laboratories were evaluated. Further, we carried out sequence analysis of 16S rRNA, gyrB, secA1, hsp65, and rpoB genes as a reference method for Nocardia species identification. The 86 isolates were directly spotted on the target plate and plate protein extraction was performed. Data were analyzed by SPSS 19.0. In total, 72 (83.7%) strains (score [≥] 9.0) and 70 (81.4%) strains (score [≥] 2.0) were correctly identified by the Autof MS1000 and Bruker Biotyper systems, respectively, at the species level. There was no significant difference (P > 0.05) between the two systems using the same protein extraction method. In conclusion, the Autof MS 1000 and Bruker MALDI-TOF systems showed no difference in identification of Nocardia spp. to the species level and could meet the most important clinical requirement for species identification.


title: Screening by deep sequencing reveals mediators of miRNA tailing in C. elegans

abstract: microRNAs are frequently modified by addition of untemplated nucleotides to the 3 end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using C. elegans. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1/CDE-1/PUP-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2 - here named GLD-2-related 2 (GLDR-2) - is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median=20.7h), as observed in other systems. Although we observe that the proportion of tailed species increases over time after biogenesis, disrupting tailing does not alter microRNA decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

category: molecular biology


title: Allosteric MAPKAPK2 Inhibitors Improve Plaque Stability in Advanced Atherosclerosis

abstract: Atherosclerotic vascular disease resulting from unstable plaques is the leading cause of morbidity and mortality in subjects with type 2 diabetes (T2D), and thus a major therapeutic goal is to discover T2D drugs that can also promote atherosclerotic plaque stability. Genetic or pharmacologic inhibition of mitogen-activated protein kinase-activated protein kinase-2 (MK2) in obese mice improves glucose homeostasis and enhances insulin sensitivity. We developed two novel orally active small-molecule inhibitors of MK2, TBX-1 and TBX-2, and tested their effects on metabolism and atherosclerosis in high-fat Western diet (WD)-fed Ldlr-/- mice. Ldlr-/- mice were first fed the WD to allow atherosclerotic lesions to become established, and the mice were then treated with TBX-1 or TBX-2. Both compounds improved glucose metabolism and lowered plasma cholesterol and triglyceride, without an effect on body weight. Most importantly, the compounds decreased lesion area, lessened plaque necrosis, and increased fibrous cap thickness in the aortic root lesions of the mice. Thus, in a preclinical model of high-fat feeding and established atherosclerosis, MK2 inhibitors improved metabolism and also enhanced atherosclerotic plaque stability, suggesting potential for further clinical development to address the epidemic of T2D associated with atherosclerotic vascular disease.


title: Deficiency of gluconeogenic enzyme PCK1 promotes non-alcoholic steatohepatitis progression by activation of PI3K/AKT/PDGF axis

abstract:
Background and AimsNonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. However, the pathomechanisms underlying NASH are incompletely explored. Phosphoenolpyruvate carboxykinase 1 (PCK1) catalyzes the first rate-limiting step of gluconeogenesis. This study was designed to determine the role of PCK1 in regulating NASH progression.

MethodsLiver metabolism, hepatic steatosis, and fibrosis were evaluated at 24 weeks in liver-specific Pck1-knockout mice fed with NASH diet or chow diet. Gain- or loss-of-function approaches were used to explore the underlying mechanism in vitro. AKT and RhoA inhibitors were evaluated for NASH treatment in vivo.

ResultsHepatic PCK1 was downregulated in patients with NASH and mouse models of NASH. Mice with liver Pck1 deficiency displayed hepatic lipid disorder and liver injury fed with normal diet, while showed aggravated fibrosis and inflammation when fed NASH diet. Mechanistically, PCK1 deficiency upregulated genes involved in fatty acid transport and lipid droplet formation. Moreover, metabolomics analysis showed the accumulation of glycerol 3-phosphate, the substrate of triglyceride synthesis. Furthermore, the loss of hepatic PCK1 could activate the RhoA/PI3K/AKT pathway, which leads to increased secretion of PDGF-AA and promotes the activation of hepatic stellate cells. Accordingly, treatment with RhoA and AKT inhibitors alleviated NASH progression in the presence of Pck1 deletion in vivo.

ConclusionsPCK1 deficiency plays a key role in the development of hepatic steatosis and fibrosis by facilitating the RhoA/PI3K/AKT/PDGF-AA axis. These findings provide a novel insight into therapeutic approaches for the treatment of NASH.


title: Deficiency of gluconeogenic enzyme PCK1 promotes murine NASH progression and fibrosis by activation PI3K/AKT/PDGF axis


title: The biogeographic history of eelpouts and related fishes: linking phylogeny, environmental change, and patterns of dispersal in a globally distributed fish group

abstract: Modern genetic data sets present unprecedented opportunities to understand the evolutionary origins of taxonomic groups comprising hundreds to thousands of species. When the timing of key events are known, it is also possible to investigate biogeographic history in the context of major phenomena (e.g., continental drift). In this study, we investigated the biogeographic history of the suborder Zoarcoidei, a globally distributed fish group that includes species inhabiting both poles and multiple taxa that produce antifreeze proteins to survive chronic subfreezing temperatures. We first generated a multi-locus, time-calibrated phylogeny for the group. We then used biogeographic modeling to reconstruct ancestral ranges across the tree and quantify the type and frequency of biogeographic events (e.g., founder, dispersal). With these results, we considered how the cooling of the Southern and Arctic Oceans, which reached their present-day subfreezing temperatures 10-15 million years ago (Mya) and 2-3 Mya, respectively, may have shaped the evolutionary history of Zoarcoidei, with an emphasis on the most speciose and widely distributed family, eelpouts (family Zoarcidae). Our phylogenetic results clarified standing issues in the Zoarcoidei taxonomy and showed that the group began to diversify in the Oligocene [~]31-32 Mya, with the center of origin for all families in north temperate waters. Within-area speciation was the most common biogeographic event in the groups history (80% of all events) followed by dispersal (20%). Finally, we found mixed evidence for polar ocean cooling underpinning Zoarcoidei diversification, with support limited to eelpout speciation in the Southern Ocean over the last 10 million years.

category: evolutionary biology


title: High-content single-cell combinatorial indexing

abstract: Single-cell genomics assays have emerged as a dominant platform for interrogating complex biological systems. Methods to capture various properties at the single-cell level typically suffer a tradeoff between cell count and information content, which is defined by the number of unique and usable reads acquired per cell. We and others have described workflows that utilize single-cell combinatorial indexing (sci)1, leveraging transposase-based library construction2 to assess a variety of genomic properties in high throughput; however, these techniques often produce sparse coverage for the property of interest. Here, we describe a novel adaptor-switching strategy,  s3, capable of producing one-to-two order-of-magnitude improvements in usable reads obtained per cell for chromatin accessibility (s3-ATAC), whole genome sequencing (s3-WGS), and whole genome plus chromatin conformation (s3-GCC), while retaining the same high-throughput capabilities of predecessor  sci technologies. We apply s3 to produce high-coverage single-cell ATAC-seq profiles of mouse brain and human cortex tissue; and whole genome and chromatin contact maps for two low-passage patient-derived cell lines from a primary pancreatic tumor.

category: genetics


title: Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease

abstract: The main protease (3CL Mpro) from SARS-CoV-2, the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the -ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other -ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation changes induced by inhibitors we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.


title: Plant-derived benzoxazinoids act as antibiotics and shape bacterial communities

abstract: Plants synthesize and release specialized metabolites into their environment that can serve as chemical cues for other organisms. Metabolites that are released from the roots are important factors in determining which microorganisms will colonize the root and become part of the plant rhizosphere. Root exudates can be converted by soil microorganisms, which can result in the formation of toxic compounds. How individual members of the plant rhizosphere respond to individual compounds and how the differential response of individual microorganisms contributes to the response of a microbial community remains an open question. Here, we investigated the impact of derivatives of benzoxazinoids, a class of plant root exudates released by important crops such as wheat and maize, on a collection of 180 root-associated bacteria. Phenoxazine, derived in soil from benzoxazinoids, inhibited the growth of root-associated bacteria in vitro in an isolate-specific manner, with sensitive and resistant isolates present in most of the studied clades. Using synthetic communities, we show that community stability is a consequence of the resilience of its individual members, with communities assembled from tolerant isolates being overall more tolerant to benzoxazinoids. However, the performance of an isolate in a community context was not correlated with its individual performance but appeared to be shaped by interactions between isolates. These interactions were independent of the overall community composition and were strain-specific, with interactions between different representatives of the same bacterial genera accounting for differential community composition.

category: plant biology


title: Host preference and invasiveness of commensals in the Lotus and Arabidopsis root microbiota

abstract: Healthy plants are colonized by microorganisms from the surrounding environment, which form stable communities and provide beneficial services to the host. Culture-independent profiling of the bacterial root microbiota shows that different plant species are colonized by distinct bacterial communities, even if they share the same habitat. It is, however, not known to what extent the host actively selects these communities and whether commensal bacteria are adapted to a specific plant species. Here, we report a sequence-indexed culture collection from roots and nodules of the model legume Lotus japonicus that contains representatives from the majority of species found in culture-independent profiles. Using taxonomically paired synthetic communities from L. japonicus and the crucifer Arabidopsis thaliana in a multi-species gnotobiotic system, we detect clear signatures of host preference among commensal bacteria in a community context, but not in mono-associations. Sequential inoculation of either host reveals strong priority effects during the assembly of the root microbiota, where established communities are resilient to invasion by late-comers. However, we found that host preference by commensal bacteria confers a competitive advantage in their native host. We reveal that host preference is prevalent in commensal bacteria from diverse taxonomic groups and that this trait is directly linked to their invasiveness into standing root-associated communities.


title: Natural variation in GmRAV confers ecological adaptation through photoperiod control of flowering time and maturity in soybean

abstract: Photoperiod strictly controlled vegetative and reproductive growth stages in soybean. A soybean GmRAV transcription factor containing both AP2 and B3 domains was shown to be a key component of this process. We identified six polymorphisms in GmRAV promoter that showed significant association with flowering time and maturity of soybean in one or multiple environments. Soybean varieties with minor polymorphism exhibited longer growth period contributing to soybean adaptation to lower latitudes. The cis-acting element GT1CONSENSUS motif of GmRAV promoter controlled the growth period and shortened R5-R7 by reducing the expression level of GmRAV in soybean. Three GmRAV-overexpressing (GmRAV-ox) transgenic lines displayed later flowering time and maturity, shorter height and fewer numbers of leaves compared with control plants, and transgenic inhibition of GmRAV (GmRAV-i) soybean displayed earlier flowering time and maturity, and increased plant heights. 163 GmRAV-target genes were determined to be putatively directly bound and transcriptionally regulated by GmRAV by combining the results from the DAP-seq and RNA-seq analyses. Two GmRAV binding motifs [C(A/G/T)A(C)ACAA(G/T)A(C/T)A(G/T)] and [C(T/A)A(C/T) C(T/G)CTG] were identified. GmRAV acting downstream of E3E4 delayed soybean growth period by repressing GmFT5a transcriptional activity to guaranteed both vegetative and reproductive phase long enough to allow necessary energy reserved to be accumulated.


title: Expression of a fungal lectin in Arabidopsis enhances plant growth and resistance towards microbial pathogens and plant-parasitic nematode

abstract: Coprinopsis cinerea lectin 2 (CCL2) is a fucoside-binding lectin from the basidiomycete C. cinerea that is toxic to the bacterivorous nematode Caenorhabditis elegans as well as animal-parasitic and fungivorous nematodes. We expressed CCL2 in Arabidopsis to assess its protective potential towards plant-parasitic nematodes. Our results demonstrate that expression of CCL2 enhances host resistance against the cyst nematode Heterodera schachtii. Surprisingly, CCL2-expressing plants were also more resistant to fungal pathogens including Botrytis cinerea, and the phytopathogenic bacterium Pseudomonas syringae. In addition, CCL2 expression positively affected plant growth indicating that CCL2 has the potential to improve two important agricultural parameters namely biomass production and general disease resistance. The mechanism of the CCL2-mediated enhancement of plant disease resistance depended on fucoside-binding by CCL2 as transgenic plants expressing a mutant version of CCL2 (Y92A), compromised in fucoside-binding, exhibited wild type disease susceptibility. The protective effect of CCL2 did not seem to be direct as the lectin showed no growth-inhibition towards B. cinerea in in vitro assays. We detected, however, a significantly enhanced transcriptional induction of plant defense genes in CCL2- but not CCL2-Y92A-expressing lines in response to infection with B. cinerea compared to wild type plants. This study demonstrates a potential of fungal defense lectins in plant protection beyond their use as toxins.


title: The Th1 cell regulatory circuitry is largely conserved between human and mouse

abstract: Gene expression programmes controlled by lineage-determining transcription factors are often conserved between species. However, infectious diseases have exerted profound evolutionary pressure, and therefore the genes regulated by immune-specific transcription factors might be expected to exhibit greater divergence due to exposure to species-specific pathogens. T-bet (Tbx21) is the immune-specific lineage-defining transcription factor for T helper type I (Th1) immunity, which is fundamental for the immune response to intracellular pathogens but also underlies inflammatory diseases. We therefore compared T-bet genomic targets between mouse and human CD4+ T cells and correlated T-bet binding patterns with species-specific gene expression. Remarkably, we show that the vast majority of T-bet regulated genes are conserved between mouse and human, either via preservation of a binding site or via an alternative binding site associated with transposon-linked insertion. We also identified genes that are specifically targeted by T-bet in humans or mice and which exhibited species-specific expression. These results provide a genome-wide cross-species comparison of T-bet target gene regulation that will enable more accurate translation of genetic targets and therapeutics from pre-clinical models of inflammatory disease into human clinical trials.


title: Identification of Fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration

abstract:
The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no FDA-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 years of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo, and that it inhibits activation of the NLRP3-ASC inflammasome in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other anti-depressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies triangulate to link fluoxetine as a potential drug repurposing candidate for a major unmet medical need that causes blindness in millions of people in the United States and across the world.

Significance StatementDry age-related macular degeneration (AMD) affects the vision of millions of people worldwide. There is currently no FDA-approved treatment for dry AMD. The inflammasome components NLRP3 and ASC have been implicated in the pathogenesis of dry AMD. We report that fluoxetine, which is approved for the treatment of clinical depression, directly binds the NLRP3 protein and prevents the assembly and activation of the NLRP3-ASC inflammasome. As a result, it also blocks the degeneration of retinal pigmented epithelium (RPE) cells in an animal model of dry AMD. Furthermore, we demonstrate through an analysis of health insurance databases that use of this FDA-approved anti-depressant drug is associated with reduced incidence of dry AMD. These studies identify that fluoxetine is a potential repurposing candidate for AMD, a prevalent cause of blindness.


title: A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase

abstract: Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) was known to response to two types of termination signals, while the mechanism underlying such termination especially the specific elements of the polymerase involved in is still unclear, due to the lack of a termination complex structure. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation S43Y that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNA polymerase (RdRp) of T7 RNAP without affecting the major DNA-dependent RNA polymerase (DdRp) activity of the enzyme, indicating the relationship between transcription termination and RdRp activity, and suggesting a model in which the stem-loop terminator induces the RdRp activity which competes with the ongoing DdRp activity to cause transcription termination. The T7 RNAP S43Y mutant as an enzymatic reagent for in vitro transcription reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.


title: Moderate increases in channel discharge are positively related to ecosystem respiration in forested Ozark streams

abstract: The natural flow regime is considered the "master variable" in lotic systems, controlling structure and function at organismal, population, community, and ecosystem levels. We sought to estimate forested headwater stream metabolism across two dominant flow regimes (Runoff and Groundwater) in northern Arkansas and evaluate potential differences in, and drivers of, gross primary production, ecosystem respiration, and net ecosystem metabolism. Flow regimes differed in intermittency, substrate heterogeneity, hyporheic connectivity, and dominant water source (subsurface runoff vs. groundwater), which we expected to result in differences in primary production and respiration. Average daily gross primary production (GPP) and ecosystem respiration (ER) estimated from field data collected from May 2015-June 2016 tended to be greater in Groundwater streams. Respiration was positively related to discharge (R2= 0.98 p< 0.0001) and net metabolism became more heterotrophic with increasing average annual discharge across sites (R2= 0.94, p= 0.0008). Characterizing ecosystem-level responses to differences in flow can reveal mechanisms governing stream metabolism and, in turn, provide information regarding trophic state and energy inputs as efforts continue to determine global trends in aquatic carbon sources and fates.

category: ecology


title: Why do some primate mothers carry their infant's corpse? A cross-species comparative study

abstract: Non-human primates respond to the death of a conspecific in diverse ways, some which may present phylogenetic continuity with human thanatological behaviours. Of these responses, infant corpse carrying by mothers (ICC) is the most-frequently reported. Despite its prevalence, quantitative analyses of this behaviour are scarce and inconclusive. We compiled a database of 409 published cases across 50 different primate species of mothers responses to their infants deaths to test hypotheses proposed to explain between- and within-species variation in corpse carrying. Using Bayesian phylogenetic regressions, we preliminarily identified three factors as possible predictors of ICC occurrence. However, using an information-theoretic approach, no combination of these predictors performed better than the null model, offering no support for any of the hypotheses we tested. In contrast, for those cases where infants corpses were carried, infant age affected ICC duration, with longer ICC observed for younger infants. This result may provide support for hypotheses that suggest that ICC is a by-product of a strong mother-infant bond. The results are discussed in the context of the evolution of emotion and their implications for evolutionary thanatology are considered.

category: animal behavior and cognition


title: Cooperative Communication with Humans Evolved to Emerge Early in Dogs

abstract:
While we know that dogs evolved from wolves through a process of domestication, it remains unclear how this process may have affected dog cognitive development. Here we tested dog (N=44) and wolf (N=37) puppies, 5-18 weeks old, on a battery of temperament and cognition tasks. Dog puppies were more attracted to humans, read human gestures more skillfully and made more eye contact with humans than wolf puppies. The two species were similarly attracted to objects and performed similarly on nonsocial measures of memory and inhibitory control. These results demonstrate the role of domestication in enhancing the cooperative communication skills of dogs through selection on attraction to humans, which altered developmental pathways.

One Sentence Summary

Domestication altered dogs developmental pathways to enhance their cooperative communication with humans.


title: Ureterorenoscopic (URS) lithotripsy and balloon dilation cause acute kidney injury and distal renal tubule damage

abstract: Ureterorenoscopy (URS) is believed to be a safe and effective procedure to treat ureteral stone or ureteral stricture. Rapidly increased intrarenal pressure during URS may have a negative impact on the kidney, but the effect on renal functions is not well known. The aim of this study is to evaluate whether URS balloon dilation or lithotripsy would cause acute kidney injury (AKI), which was evaluated by urine neutrophil gelatinase associated lipocalin (NGAL) and renal tubular damage, which was evaluated by urine  glutathione S-transferase (GST) and {pi}GST. This prospective study consisted of 207 patients with mean age 53.8 years old between September 2012 and June 2013. Four groups were included: the ureteral stricture group (group 1), the ureteral stone group (group 2), and two control groups. URS induced increased uNGAL on both Days 1 and 14 in both groups, and only elevated u-{pi}GST levels were noted on Day 14 after URSL. On Day 14, the difference between low-grade and high-grade hydronephrosis was significant in group 1 (p < 0.001) rather than group 2 (p = 0.150). By multivariate logistic regression analysis, age, baseline eGFR, and stone size > 1.0 cm were associated with complete recovery of hydronephrosis after URS on Day 14. Ureteral stone patients with preserved renal function suffered more AKI (uNGAL) than those with impaired renal function. However, URS-related AKI had no significant difference between stone [≤] 1 cm and > 1 cm subgroups. Besides, urine GST and {pi}GST were both significantly higher in stone > 1 cm subgroup when compared to [≤] 1 cm subgroup. In conclusion, URS laser lithotripsy and balloon dilatation all resulted in AKI and renal tubular damage on Day 14 though post-URS double-J (DBJ) stenting was performed in every patient.


title: Nucleus accumbens neurons encode initiation and vigor of reward approach behavior

abstract:
The nucleus accumbens (NAc) is considered an interface between motivation and action, with NAc neurons playing an important role in promoting reward approach. However, the encoding by NAc neurons that contribute to this role remains unknown. Here, we trained male rats to find rewards in an 8-arm radial maze. The activity of 62 neurons, mostly in the shell of the NAc, were recorded while rats ran towards each reward place. General linear model (GLM) analysis showed that variables related to the vigor of the locomotor approach, like speed and acceleration, and the fraction of the approach run completed were the best predictors of the firing rate for most NAc neurons. Nearly 23% of the recorded neurons, here named locomotion-off cells, were inhibited during the entire approach run, suggesting that reduction in firing of these neurons promotes initiation of locomotor approach. Another 24% of the neurons presented a peak of activity during acceleration followed by a valley during deceleration (peak-valley cells). Together, these neurons accounted for most of the speed and acceleration encoding identified in the GLM analysis. Cross-correlations between firing and speed indicated that the spikes of peak-valley cells were followed by increases in speed, suggesting that the activity of these neurons drives acceleration. In contrast, a further 19% of neurons presented a valley during acceleration followed by a peak just prior to or after reaching reward (valley-peak cells). These findings suggest that these three classes of NAc neurons control the initiation and vigor of the locomotor approach to reward.

Significance StatementDeciphering the mechanisms by which the NAc controls the vigor of motivated behavior is critical to better understand and treat psychiatric conditions in which motivation is dysregulated. Manipulations of the NAc profoundly impair subjects ability to spontaneously approach reward-associated locations, preventing them from exerting effort to obtain reward. Here, we identify for the first time specific activity of NAc neurons in relation to spontaneous approach behavior. We discover three classes of neurons that could control initiation of movement and the speed vs. time trajectory during locomotor approach. These results suggest a prominent but heretofore unknown role for the NAc in regulating the kinematics of reward approach locomotion.

category: neuroscience


title: NMDARs expressed in Granule Cells are involved in cerebellar plasticity and motor learning

abstract:
Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic NMDA receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers nitric oxide signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-Purkinje cell (PF-PC) synapses. Consistent with this finding, the selective deletion of GCs NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

Significance StatementLearning depends on synaptic plasticity. The signaling mechanisms that control induction of plasticity determine the learning rules at the specific synapse involved. Moreover, the relationship between the activity patterns of synaptic inputs and the type, direction, and level of plasticity induced may evolve during development. Here, we establish a key link between NMDA receptor activation presynaptic to cerebellar Purkinje cells, downstream signaling mechanisms, and the ability of adult animals to learn a cerebellar motor task.