title: Strong SARS-CoV-2 N-specific CD8+ T immunity induced by engineered extracellular vesicles associates with protection from lethal infection in mice.

abstract: SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut- based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.


title: Impact of Nosema ceranae invasion on sucrose solution consumption, midgut epithelial cell structure, and lifespan of Apis cerana cerana workers

abstract: Nosema ceranae is an intracellular fungal parasite for honeybees, leading to chronic disease named bee nosemosis with worldwide distribution. Asian honeybee (Apis cerana) is the original host for N. ceranae, but the impact of N. ceranae infection on A. cerana physiology is largely unknown. In this current work, workers of Apis cerana cerana, a subspecies of Asian honeybee, were artificially inoculated with N. ceranae spores and reared under lab conditions, followed by detection of fungal spore load as well as host sucrose solution consumption, midgut epithelial cell structure, and lifespan. The result of spore counting suggested that the spore load in the host midgut decreased significantly during 1 dpi-2 dpi, whereas that displayed an elevated trend among 2 dpi-13 dpi. The sucrose solution consumption of workers in N. ceranae-inoculated groups among 1 dpi-20 dpi was always higher than that of workers in un-inoculated groups; additionally, the difference of sucrose solution consumption between these two groups at 4 dpi, 5 dpi, and 13 dpi was of significance. Based on microscopic observation of paraffin sections, darkly stained parasites were clearly detected in the midgut epithelial cells of N. ceranae-inoculated workers at 7 dpi-10 dpi, whereas no parasite was observed in those of un-inoculated workers. In addition, the boundaries of un-inoculated host epithelial cells were intact and the darkly stained nucleus were clear, while the boundaries of midgut epithelial cells of N. ceranae-inoculated workers were blurred, the nucleus were almost disappeared, and the nucleic acid substances were diffused. Moreover, the survival rates of workers in both N. ceranae-inoculated groups and un-inoculated groups at 1 dpi-5 dpi were pretty high and then started to decrease at 5 dpi; the survival rate of workers in N. ceranae-inoculated groups was always lower than that in un-inoculated groups, with significant difference between these two groups during 11 dpi-20 dpi. These results together indicate that the quantity of fungal spores continuously elevated with the microsporidian multiplication, causing energetic stress for workers and host cell structure damage, which further negatively affected the host lifespan. Our findings offer a solid basis not only for exploring the molecular mechanism underlying N. ceranae infection but also for investigating the interaction between N. ceranae and eastern honeybee.

category: molecular biology


title: Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

abstract: The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 [A] resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 [A]. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.


title: FMRP inhibits translation elongation independent of mRNA G-quadruplexes

abstract: Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome, the leading form of inherited intellectual disability and the most common monogenic cause of autism spectrum disorders. FMRP is an RNA-binding protein that controls neuronal mRNA localization and translation. Notably, FMRP is thought to inhibit translation elongation after being recruited to target transcripts via binding RNA G-quadruplexes (G4s) within the coding sequence. Here we directly tested this model and report that FMRP inhibits translation elongation independent of mRNA G4s. Furthermore, we found that the RGG box motif together with its natural C-terminal domain forms a non-canonical RNA-binding domain (ncRBD) that binds reporter mRNA and all four polymeric RNA sequences. The ncRBD is essential for FMRP to inhibit translation. Transcripts that are bound by FMRP through the ncRBD co-sediment with heavy polysomes, which is consistent with stalling elongating ribosomes and a subsequent accumulation of slowed polysomes. Together, this work shifts our understanding of how FMRP inhibits translation elongation and supports a model where repression is driven by local FMRP and mRNA concentrations rather than target mRNA sequence.


title: Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

abstract: It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5' UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5' UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.


title: Monomodular Pseudomonas aeruginosa phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain

abstract:
Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy for introducing a more precise approach in the way we apply antimicrobial therapy. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had been previously identified as an antimicrobial agent candidate, able to interact with the Gram-negative surface and disrupt it. Further evidence is hereby provided on this matter, based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 [A] away from the catalytic site and located at the opposite side of the lysin molecule. This substrate binding region was conserved among phylogenetically related lysins lacking an additional cell wall binding domain, but not among those containing such a module. Two glutamic acids were identified as relevant for the peptidoglycan degradation activity, although Pae87 antimicrobial activity was seemingly unrelated to it. In contrast, an antimicrobial peptide-like region within Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and have antibacterial activity by itself. Therefore, we propose an antimicrobial mechanism for Pae87 in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell wall function, either with or without the participation of Pae87 catalytic activity.

SynopsisThe structure of the monomodular Pseudomonas aeruginosa bacteriophage JG004 lysin Pae87 is presented and investigated in relationship with its function repurposed as an antimicrobial agent. The structure with its peptidoglycan ligand revealed a possible cell wall binding region. A C-terminal antimicrobial peptide-like region is shown to be important for disrupting the bacterial cell wall.


title: Recognition of HIV-1 capsid licenses innate immune response to viral infection

abstract: Cyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1- encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.


title: Uncertainty-guided learning with scaled prediction errors in the basal ganglia

abstract:
To accurately predict rewards associated with states or actions, the variability of observations has to be taken into account. In particular, when the observations are noisy, the individual rewards should have less influence on tracking of average reward, and the estimate of the mean reward should be updated to a smaller extent after each observation. However, it is not known how the magnitude of the observation noise might be tracked and used to control prediction updates in the brain reward system. Here, we introduce a new model that uses simple, tractable learning rules that track the mean and standard deviation of reward, and leverages prediction errors scaled by uncertainty as the central feedback signal. We provide a normative analysis, comparing the performance of the new model with that of conventional models in a value tracking task. We find that the new model has an advantage over conventional models when tested across various levels of observation noise. Further, we propose a possible biological implementation of the model in the basal ganglia circuit. The scaled prediction error feedback signal is consistent with experimental findings concerning dopamine prediction error scaling relative to reward magnitude, and the update rules are found to be consistent with many features of striatal plasticity. Our results span across the levels of implementation, algorithm, and computation, and might have important implications for understanding the dopaminergic prediction error signal and its relation to adaptive and effective learning.

Author SummaryThe basal ganglia system is a collection of subcortical nuclei in the mammalian brain. This system and its dopaminergic inputs are associated with learning from rewards. Here, dopamine is thought to signal errors in reward prediction. The structure and function of the basal ganglia system are not fully understood yet--for example, the basal ganglia are split into two antagonistic pathways, but the reason for this split and the role of the two pathways are unknown. Further, it has been found that under some circumstances, rewards of different sizes lead to dopamine responses of similar size, which cannot be explained with the reward prediction error theory. Here, we propose a new model of learning in the basal ganglia--the scaled prediction error model. According to our model, both reward average and reward uncertainty are tracked and represented in the two basal ganglia pathways. The learned reward uncertainty is then used to scale dopaminergic reward prediction errors, which effectively renders learning adaptive to reward noise. We show that such learning is more robust than learning from unscaled prediction errors and that it explains several physiological features of the basal ganglia system.

category: neuroscience


title: TCF7L2 acts as a molecular switch in midbrain to control mammal vocalization through a transcriptional repression mechanism

abstract: Vocalization is an essential medium for sexual and social signaling in birds and mammals. Periaqueductal gray (PAG) a conserved midbrain structure is believed to be responsible for innate vocalizations, but its molecular regulation remains largely unknown. Here, through a mouse forward genetic screening we identified one of the key Wnt/ {beta}-catenin effectors TCF7L2/TCF4 controls ultrasonic vocalization (USV) production and syllable complexity during maternal deprivation and sexual encounter. Expression of TCF7L2 in PAG excitatory neurons is necessary for the complex trait, while TCF7L2 loss reduces neuronal gene expressions and synaptic transmission in PAG. TCF7L2-mediated vocal {beta}-catenin-binding domain but dependent of its DNA binding ability. Patient mutations associated with severe speech delay disrupt the transcriptional repression effect of TCF7L2, while mice carrying those mutations display severe USV impairments. Therefore, we conclude that TCF7L2 orchestrates gene expression in midbrain to control vocal production through a transcriptional repression mechanism.


title: Closed-loop recruitment of striatal parvalbumin interneurons prevents the onset of compulsive behaviors

abstract: A prominent electrophysiological feature of compulsive behaviours is striatal hyperactivity; but its underlying dysfunctional cellular mechanisms still need to be characterized. Within the striatum, parvalbumin-positive interneurons (PVI) exert a powerful feedforward inhibition essential for the regulation of striatal activity. To investigate the potential implication of striatal PVI in aberrant repetitive behaviors, we used the Sapap3 mutant mice which exhibit compulsive-like behaviours characterized by excessive self-grooming. When striatal PVI in the centromedial striatum of Sapap3 mice were we optogenetically activated, we first showed that the number of compulsive-like events were greatly reduced. To investigate further the critical time-window when striatal PVI needed to be recruited for the behavioural regulation of compulsive-like grooming, we then designed a novel closed-loop stimulation pipeline. We identified a transient 1-4 Hz oscillations in the orbitofrontal cortex that temporally predicted grooming onsets. Exploiting this delta band signal as a biomarker, we were able to provide on-demand stimulation of striatal PVI shortly before predicted grooming events. This targeted closed-loop optogenetics approach greatly reduced grooming events and demonstrated that the recruitment of striatal PVI regulated the initiations of compulsive-like behaviours.


title: The hippocampus as a sorter and reverberatory integrator of sensory inputs

abstract: In entorhinal-hippocampal networks, the trisynaptic pathway, including the CA3 recurrent circuit, processes episodes of context and space1-3. Recurrent connectivity can generate reverberatory activity4-6, an intrinsic activity pattern of neurons that occurs after sensory inputs have ceased. However, the role of reverberatory activity in memory encoding remains incompletely understood. Here we demonstrate that in mice, synchrony between conditioned stimulus (CS) and unconditioned stimulus (US)-responsible cells occurs during the reverberatory phase, lasting for approximately 15 s, but not during CS and US inputs, in the CA1 and the reverberation is crucial for the linking of CS and US in the encoding of delay-type cued-fear memory. Retrieval-responsive cells developed primarily during the reverberatory phase. Mutant mice lacking N-methyl-D-aspartate receptors (NRs) in CA3 showed a cued-fear memory impairment and a decrease in synchronized reverberatory activities between CS- and US-responsive CA1 cells. Optogenetic CA3 silencing at the reverberatory phase during learning impaired cued-fear memory. Our findings suggest that reverberation recruits future retrieval-responsive cells via synchrony between CS- and US-responsive cells. The hippocampus uses reverberatory activity to link CS and US inputs, and avoid crosstalk during sensory inputs.


title: The dorsal hippocampus' role in context-based timing in rodents

abstract: To act proactively, we must predict when future events will occur. Individuals generate temporal predictions using cues that indicate an event will happen after a certain duration elapses. Neural models of timing focus on how the brain represents these cue-duration associations. However, these models often overlook the fact that situational factors frequently modulate temporal expectations. For example, in realistic environments, the intervals associated with different cues will often covary due to a common underlying cause. According to the  common cause hypothesis, observers anticipate this covariance such that, when one cues interval changes, temporal expectations for other cues shift in the same direction. Furthermore, as conditions will often differ across environments, the same cue can mean different things in different contexts. Therefore, updates to temporal expectations should be context-specific. Behavioral work supports these predictions, yet their underlying neural mechanisms are unclear. Here, we asked whether the dorsal hippocampus mediates context-based timing, given its broad role in context-conditioning. Specifically, we trained rats with either hippocampal or sham lesions that two cues predicted reward after either a short or long duration elapsed (e.g., tone-8s / light-16s). Then, we moved rats to a new context and extended the long-cues interval (e.g., light-32s). This caused rats to respond later to the short cue, despite never being trained to do so. Importantly, when returned to the initial training context, sham rats shifted back toward both cues original intervals. In contrast, lesion rats continued to respond at the long cues newer interval. Surprisingly, they still showed contextual modulation for the short cue, responding earlier like shams. These data suggest the hippocampus only mediates context-based timing if a cue is explicitly paired and/or rewarded across distinct contexts. Furthermore, as lesions did not impact timing measures at baseline or acquisiton for the long cues new interval, our data suggests that the hippocampus only modulates timing when context is relevant.


title: Anti-thrombotic treatment enhances antibiotic efficiency in a humanized model of meningococcemia

abstract: Meningococcal infections remain particularly difficult to treat. Despite antibiotic therapy, the state of the patients often rapidly deteriorates. Early clinical studies suggest that meningococci acquire a form of resistance to antibiotic treatments during infections. Taking advantage of a humanized animal model of infection, we confirm that adherent bacteria become highly resistant to antibiotic treatments as early as 3-6 hours post infection, although fully sensitive in vitro. Within this time frame, meningococci adhere to the endothelium via their type IV pili, proliferate and eventually fill the vessel lumen. Using intravital imaging, we show that rapidly upon infection blood flow is dramatically decreased, thus limiting antibiotic access to infected vessels. Concomitantly, fibrin is deposited inside infected vessels in proximity to bacterial aggregates. Pharmacologically impairing thrombin generation by inhibiting Factor X activity not only improves blood flow in infected vessels, but also enhances the efficacy of the antibiotic treatment. Our results indicate that the combined administration of anticoagulants together with antibiotics might represent a therapeutic approach to treat meningococcal sepsis more efficiently.

category: pathology


title: Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1

abstract: Polo-Like-Kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosomes pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa) and zebrafish embryos in parallel to phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation and that a conserved C-terminal PLK1 binding site between humans and zebrafish is needed for PCM maintenance through mediating PLK1-dependent substrate phosphorylation events. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexins C-terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to anchor PLK1 moderating its potential to phosphorylate PCM substrates required for PCM maintenance and function.

category: cell biology


title: WNK kinases sense molecular crowding and rescue cell volume via phase separation

abstract: When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.


title: Oil Red O based method for Exosome Labelling and Detection

abstract:
With the realization of the role of exosomes in diseases especially cancer, exosome research is gaining popularity in biomedical sciences. To understand exosome biology, their labelling and tracking studies are important. New and improved methods of exosome labelling for detection and tracking of exosomes need to be developed to harness their therapeutic and diagnostic potential. In this paper, we report a novel, simple and effective method of labelling and detecting exosomes using Oil red O (ORO) which is a dye commonly used for lipid staining. Using ORO is a cost effective and easy approach with intense red colouration of stained exosomes. Further, the issues faced with commonly used lipophilic dyes for exosomes labelling such as long term persistence of dyes, aggregation and micelle formation of dyes, difficulty to distinguish dye particles from labelled exosomes and detection of large aggregates of dye or dye-exosome are not seen with ORO dye. This method shows good labelling efficiency of exosomes with very sensitive detection and real-time tracking of the cellular uptake of exosomes.

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title: Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion

abstract: Cis and trans-interactions in cadherins are the foundations of multicellularity. While the trans-interaction mediate cell-cell adhesion, the cis-interaction is postulated as strengthening to trans by clustering. The well-accepted model in cadherin-adhesion is that the  trans precedes cis via a diffusion-trap kinetic model. Here we report that cadherin-23, a non-classical cadherin with an extended extracellular region, undergoes clustering in solution via lateral interactions independent of trans and phase separate as liquid droplets. In cellulo using fluorescence-recovery after the photobleaching, we noticed a significantly slow-diffusion of cadherin-23 at the intercellular junctions, indicating the diffusion of a cluster. The cis-clustering accelerates the cell-cell adhesion and, thus, kinetically controls cell-adhesion via  cis precedes trans model. Though the connection of cis-clustering with the rapid adhesion is yet to explore, M2-macrophages that predominantly express cadherin-23 undergo fast attachments to circulatory tumor cells during metastasis.


title: Hypoxia Dynamically Regulates DBC1 Ubiquitination and Stability by SIAH2 and OTUD5 in Breast Cancer Progression

abstract: DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is dynamically regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted the competitive binding of SIAH2 with OTUD5 to DBC1, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. Siah2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of Siah2/DBC1. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.


title: A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates

abstract:
We present a multiomic cell atlas of human lung development that combines single cell RNA and ATAC sequencing, high throughput spatial transcriptomics and single cell imaging. Coupling single cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial and erythrocyte/leukocyte compartments from 5-22 post conception weeks. We identify new cell states in all compartments. These include developmental-specific secretory progenitors that resemble cells in adult fibrotic lungs and a new subtype of neuroendocrine cell related to human small cell lung cancer; observations which strengthen the connections between development and disease/regeneration. Our datasets are available for the community to download and interact with through our web interface (https://fetal-lung.cellgeni.sanger.ac.uk). Finally, to illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signalling and transcription factor hierarchies which we test using organoid models.

HighlightsO_LISpatiotemporal atlas of human lung development from 5-22 post conception weeks identifies 147 cell types/states.
C_LIO_LITracking the developmental origins of multiple cell compartments, including new progenitor states.
C_LIO_LIFunctional diversity of fibroblasts in distinct anatomical signalling niches.
C_LIO_LIResource applied to interrogate and experimentally test the transcription factor code controlling neuroendocrine cell heterogeneity and the origins of small cell lung cancer.
C_LI

category: developmental biology


title: Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

abstract:
Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of {beta}-arrestin ({beta}arr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for {beta}arrs, agonist-stimulation first drives the translocation of {beta}arrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of {beta}arr translocation to endosomes although {beta}arrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like {beta}arr1 conformation, rescues endosomal translocation of {beta}arr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in {beta}arr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of {beta}arr1 with {beta}2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of {beta}arr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery.

SignificanceThe interaction of G protein-coupled receptors (GPCRs) with {beta}-arrestins ({beta}arrs) is a critical step in their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and {beta}arrs have been utilized as biosensors and regulators of functional outcomes, allosteric targeting of receptor-transducer complexes to encode gain of function has not been documented so far. Here, we discover that a conformation-specific synthetic intrabody recognizing GPCR-bound {beta}arr1 can allosterically enhance endosomal trafficking of {beta}arr1 and agonist-induced ERK1/2 MAP kinase activation. This intrabody promotes an active-like {beta}arr1 conformation and enhances the interaction of {beta}2-adaptin with {beta}arr1. Our findings establish a conceptual framework to allosterically modulate protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling responses.

category: biochemistry


title: HP1β Chromo Shadow Domain facilitates H2A ubiquitination for BRCA1 recruitment at DNA double-strand breaks

abstract: Efficient DNA double strand break (DSB) repair by homologous recombination (HR), as orchestrated by histone and non-histone proteins, is critical to genome stability, replication, transcription, and cancer avoidance. Here we report that Heterochromatin Protein1 beta (HP1{beta}) acts as a key component of the HR DNA resection step by regulating BRCA1 enrichment at DNA damage sites, a function largely dependent on the HP1{beta} chromo shadow domain (CSD). HP1{beta} itself is enriched at DSBs within gene-rich regions through a CSD interaction with Chromatin Assembly Factor 1 (CAF1) and HP1 {beta} depletion impairs subsequent BRCA1 enrichment. An added interaction of the HP1 {beta} CSD with the Polycomb Repressor Complex 1 ubiquitinase component RING1A facilitates BRCA1 recruitment by increasing H2A lysine 118-119 ubiquitination, a marker for BRCA1 recruitment. Our findings reveal that HP1{beta} interactions, mediated through its CSD with RING1A, promote H2A ubiquitination and facilitate BRCA1 recruitment at DNA damage sites, a critical step in DSB repair by the HR pathway. These collective results unveil how HP1{beta} is recruited to DSBs in gene-rich regions and how HP1{beta} subsequently promotes BRCA1 recruitment to further HR DNA damage repair by stimulating CtIP-dependent resection.


title: Privacy-Preserving Federated Neural Network Learning for Disease-Associated Cell Classification

abstract: Training accurate and robust machine learning models requires a large amount of data that is usually scattered across data-silos. Sharing or centralizing the data of different healthcare institutions is, however, unfeasible or prohibitively difficult due to privacy regulations. In this work, we address this problem by using a novel privacy-preserving federated learning-based approach, PriCell, for complex machine learning models such as convolutional neural networks. PriCell relies on multiparty homomorphic encryption and enables the collaborative training of encrypted neural networks with multiple healthcare institutions. We preserve the confidentiality of each institutions input data, of any intermediate values, and of the trained model parameters. We efficiently replicate the training of a published state-of-the-art convolutional neural network architecture in a decentralized and privacy-preserving manner. Our solution achieves an accuracy comparable to the one obtained with the centralized solution, with an improvement of at least one-order-of-magnitude in execution time with respect to prior secure solutions. Our work guarantees patient privacy and ensures data utility for efficient multi-center studies involving complex healthcare data.


title: Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis

abstract: High-resolution respirometry (HRR) can assess PBMC bioenergetics, but no standardized medium for PBMC preparation and HRR analysis exist. Here, we study the effect of four different media (MiR05, PBS, RPMI, Plasmax) on quantification, size, and HRR analysis (Oxygraph-O2k) of intact PBMCs. Remarkably, PBMC quantification was 21% higher in MiR05 than PBS and Plasmax, and 28% higher than in RPMI, causing O2 flux underestimation during HRR due to inherent adjustments. Moreover, smaller cell size of PBMCs and aggregation was observed in MiR05. We suggest optimization of HRR with a standardized, plasma-like medium for future HRR analysis of intact PBMCs.


title: Telomere elongation in the gut extends zebrafish lifespan

abstract: Telomere shortening is a hallmark of aging and is counteracted by telomerase. The gut is one of the earliest organs to exhibit short telomeres and tissue dysfunction during normal zebrafish aging. This is recapitulated in prematurely aged telomerase mutants (tert-/-). Here, we show that gut-specific telomerase activity in tert-/- zebrafish prevents premature aging. Induction of telomerase rescues gut senescence and low cell proliferation to wild-type levels, while restoring gut tissue integrity, inflammation, and age-dependent gut microbiota dysbiosis. Remarkably, averting gut dysfunction results in a systemic beneficial impact. Gut-specific telomerase activity rescues premature aging markers in remote organs, such as the reproductive (testes) and hematopoietic (kidney marrow) systems. Functionally, it also rescues age-dependent loss of male fertility and testes atrophy. Finally, we show that gut-specific telomerase activity increases the lifespan of telomerase mutants. Our work demonstrates that delaying telomere shortening in the gut is sufficient to systemically counteract aging in zebrafish.


title: Fatty acids produced by the gut microbiota dampen host inflammatory responses by modulating intestinal SUMOylation

abstract: The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. These metabolites regulate intestinal cell activities by modulating either gene transcription or post-translational modifications of gut proteins. The effect of gut commensal bacteria on SUMOylation, an essential ubiquitin-like modification in intestinal physiology, remains however unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in different intestinal cell lines in a pH-dependent manner. We demonstrate that these metabolites induce an oxidative stress which inactivates intestinal deSUMOylases and promotes the hyperSUMOylation of chromatin-bound proteins. In order to determine the impact of these modifications on intestinal physiology, we focused on the NF-{kappa}B signaling pathway, a key player in inflammation known to be regulated by SUMOylation. We demonstrated that the hyperSUMOylation induced by SCFAs/BCFAs inhibits the activation of the NF-{kappa}B pathway in intestinal cells by blocking the degradation of the inhibitory factor I{kappa}B in response to TNF. This results in a decrease in pro-inflammatory cytokines expression, such as IL8 or CCL20, as well as a decrease in intestinal epithelial permeability in response to TNF. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses by the gut microbiota.


title: Ccdc38 is required for sperm flagellum biogenesis and male fertility in mouse

abstract:
Sperm flagellum is essential for male fertility, defects in flagellum biogenesis are associated with male infertility. Deficiency of CCDC42 is associated with malformation of the mouse sperm flagella. Here, we find that the testis-specific expressed protein CCDC38 (coiled-coil domain containing 38) interacts with CCDC42 and localizes on manchette and sperm tail during spermiogenesis. Inactivation of CCDC38 in male mice results in distorted manchette, multiple morphological abnormalities of the flagella (MMAF) of spermatozoa, and eventually male sterility. Furthermore, we find that CCDC38 interacts with intra-flagellar transport protein 88 (IFT88) as well as the outer dense fibrous 2 (ODF2), and its depletion reduces the transportation of ODF2 to flagellum. Altogether, our results uncover the essential role of CCDC38 during sperm flagellum biogenesis, and suggesting the defects of these genes might be associated with male infertility in human being.

Summary statementWe demonstrated that CCDC38, localizes on manchette and sperm tail, is crucial for male fertility.


title: unc-37/Groucho and lsy-22/AES repress Wnt target genes in C. elegans asymmetric cell divisions

abstract: Asymmetric cell division (ACD) is a fundamental mechanism of cell fate specification and adult tissue homeostasis. In C. elegans, the Wnt/{beta}-catenin asymmetry (W{beta}A) pathway regulates ACDs throughout embryonic and larval development. Under control of Wnt ligand-induced polarity, the transcription factor POP-1/TCF functions with the coactivator SYS-1/{beta}-catenin to activate gene expression in the signaled cell or, in absence of the coactivator, to repress Wnt target genes in the unsignaled daughter cell. To date, investigation of Groucho function in W{beta}A is lacking, and the function of LSY-22/AES has only been evaluated in C. elegans neurons. Further, conflicting evidence shows TCF utilizing Groucho-mediated repression may be either aided or repressed by AES addition. Here we demonstrate a genetic interaction between Groucho corepressors and POP-1/TCF in the distal tip cells (DTCs), seam cells (SCs) and embryonic endoderm development. In the DTCs, signaled cell fate increases after individual and double Groucho loss of function, representing the first demonstration of Groucho function in wildtype W{beta}A ACDs. Further, W{beta}A target gene misexpression occurs more frequently than DTC fate changes, suggesting derepression generates an intermediate cell fate. In the SCs, loss of UNC-37/Groucho or LSY-22/AES in a POP-1/TCF hypomorphic background enhances SC expansion and target gene misregulation. Moreover, while POP-1/TCF depletion in lsy-22/AES nulls yielded an expected increase in SCs we observed a surprising SC decrease in unc-37/Groucho nulls subjected to POP-1/TCF depletion. This phenotype correlates with UNC-37/Groucho regulation of pop-1/tcf expression since POP-1/TCF levels are increased in unc-37/Groucho null SCs. Lastly, Groucho functions in embryonic endoderm development since we observe ectopic endoderm transgene expression in unc-37/Groucho and lsy-22/AES knockdown in a HDA-1 background. Together, these data indicate Groucho-mediated modulation of cell fate via regulation of POP-1/TCF repression is widespread in W{beta}A ACDs and suggests a novel role of LSY-22/AES as a bona fide TCF repressor.


title: TrpA1 is a shear stress mechanosensing channel regulating intestinal homeostasis in Drosophila

abstract: Adult stem cells are essential for maintaining normal tissue homeostasis and supporting tissue repair. Although genetic and biochemical programs controlling adult stem cell behavior have been extensively investigated, how mechanosensing regulates stem cells and tissue homeostasis is not well understood. Here, we show that shear stress can activate enteroendocrine cells, but not other gut epithelial cell types, to regulate intestine stem cell-mediated gut homeostasis. This shear stress sensing is mediated by transient receptor potential A1 (TrpA1), a Ca2+-permeable ion channel that expressed only in enteroendocrine cells among all gut epithelial cells. Genetic depletion of TrpA1 or modification of its shear stress sensing function causes reduced intestine stem cell proliferation and intestine growth. We further show that among the TrpA1 splice variants, only select isoforms are activated by shear stress. Altogether, our results suggest the naturally occurring mechanical force such as fluid passing generated shear stress regulates intestinal stem cell-mediated tissue growth by activating enteroendocrine cells, and Drosophila TrpA1 as a new shear stress sensor.


title: Crystal structures of bacterial Small Multidrug Resistance transporter EmrE in complex with structurally diverse substrates

abstract: Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 [A] structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.


title: LinkExplorer: Predicting, explaining and exploring links in large biomedical knowledge graphs

abstract:
SummaryMachine learning algorithms for link prediction can be valuable tools for hypothesis generation. However, many current algorithms are black boxes or lack good user interfaces that could facilitate insight into why predictions are made. We present LinkExplorer, a software suite for predicting, explaining and exploring links in large biomedical knowledge graphs. LinkExplorer integrates our novel, rule-based link prediction engine SAFRAN, which was recently shown to outcompete other explainable algorithms and established black box algorithms. Here, we demonstrate highly competitive evaluation results of our algorithm on multiple large biomedical knowledge graphs, and release a web interface that allows for interactive and intuitive exploration of predicted links and their explanations.

Availability and ImplementationA publicly hosted instance, source code and further documentation can be found at https://github.com/OpenBioLink/Explorer.

Contactmatthias.samwald@meduniwien.ac.at

Supplementary informationSupplementary data are available at Bioinformatics online.

category: bioinformatics


title: MicrogliaST: a web server for microglia spatiotemportal pattern analysis in normal and disordered brains

abstract: Brain is the most complex organ of living organisms, as the celebrated cells in the brain, microglia play an indispensable role in the brains immune microenvironment. Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative diseases and malignant of the central nervous system. However, little is known about the dynamic characteristics of microglia during development or disease conditions. Recently, the single-cell RNA sequencing technologies have become possible to characterize the heterogeneity of immune system in brain. But it posed computational challenges on integrating and utilizing the massive published datasets to dissect the spatiotemporal characterization of microglia. Here, we present microgliaST (bio-bigdata.hrbmu.edu.cn/MST), a database consisting of single-cell microglia transcriptomes across multiple brain regions and developmental periods. Based on high-quality microglia markers collected from published papers, we annotated and constructed human and mouse transcriptomic profiles of 273,374 microglias, comprising 12 regions, 12 periods and 3 conditions (normal, disease, treatment). In addition, MicrogliaST provides multiple analytical tools to elucidate the landscape of microglia under disorder conditions, conduct personalized difference analysis and spatiotemporal dynamic analysis. More importantly, microgliaST paves an ingenious way to the study of brain environment, and also provides insights into clinical therapy assessments.


title: Mode selection mechanism in traveling and standing waves revealed by Min wave reconstituted in artificial cells

abstract:
Reaction-diffusion coupling (RDc) generates spatiotemporal patterns, including two dynamic wave modes: traveling and standing waves. Although mode selection plays a significant role in the spatiotemporal organization of living cell molecules, the mechanism for selecting each wave mode remains elusive. Here, we investigated a wave mode selection mechanism using Min waves reconstituted in artificial cells, emerged by the RDc of MinD and MinE. Our experiments and theoretical analysis revealed that the balance of membrane binding and dissociation from the membrane of MinD determines the mode selection of the Min wave. We successfully demonstrated that the transition of the wave modes can be regulated by controlling this balance and found hysteresis characteristics in the wave mode transition. These findings highlight a novel role of the balance between activators and inhibitors as a determinant of the mode selection of waves by RDc and depict a novel mechanism in intracellular spatiotemporal pattern formations.

TeaserActivator-inhibitor balance determines whether a nonlinear wave in live cells becomes a traveling wave or standing wave.

category: biophysics


title: Actin-microtubule dynamic composite forms responsive active matter with memory

abstract: Active cytoskeletal materials in vitro demonstrate self-organising properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in the living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possessing adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite, where self-assembling microtubules and actin filaments collectively self-organise due to the activity of microtubules-percolating molecular motors. We demonstrate that microtubules spatially organise actin filaments that in turn guide microtubules. The two networks align in an ordered fashion using this feedback loop. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli suggesting possible autoregulatory behaviour in changing mechanochemical environment. We thus establish artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.


title: PD1+ T-cells correlate with Nerve Fiber Density as a prognostic biomarker in patients with resected perihilar cholangiocarcinoma.

abstract:
Background & AimsPerihilar cholangiocarcinoma (pCCA) is a rare hepatobiliary malignancy. Nerve fiber invasion (NFI) shows cancer invading the nerve and is considered an aggressive feature. Nerve fiber density (NFD) consists of small nerve fibers without cancer invasion and is divided into high NFD (high numbers of small nerve fibers) or low NFD (low numbers of small nerve fibers). We aim to explore differences in immune cell populations and survival.

MethodsWe applied multiplex immunofluorescence (mIF) on 47 pCCA surgically resected patients and investigated the immune cell composition in the tumor microenvironment (TME) of different nerve fiber phenotypes (NFI, high and low NFD). Extensive group comparison was carried out and the association with overall survival (OS) was assessed.

ResultsThe NFI ROI was measured with highest CD68+ macrophage levels among 3 ROIs (NFI compared to tumor free p= 0.016 and to tumor p=0.034). Further, for NFI patients the density of co-inhibitory markers CD8+PD1+ and CD68+PD1+ were more abundant in the tumor rather than NFI ROI (p= 0.004 and p= 0.0029 respectively). Comparison between patients with NFD and NFI groups, the signals of co-expression of CD8+PD1+ as well as CD68+PD1+ were significantly higher in the high NFD group (p= 0.027 and p= 0.044, respectively). The OS for high NFD patients was 92 months median OS (95% CI:41-142), for low NFD patients 20 months ((95% CI: 4-36) and for NFI group of patients 19 months (95% CI 7-33). The OS for high NFD patients was significantly better compared to low NFD (p= 0.046) and NFI (p= 0.032).

ConclusionsPD1+ T-cells correlate with high NFD as a prognostic biomarker, the biological pathway behind this needs to be investigated.

Lay summaryNerve fibers play a dual role in the tumor microenvironment in pCCA. In our previous study, we showed that the presence of high numbers of small nerve fibers is associated with a better overall survival. In addition, we found that in high NFD patients PD1+ T-cells are significantly overexpressed. Therefore, we present high NFD as a promising prognostic biomarker.

Graphical abstract

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category: cancer biology


title: Thalamo-Prefrontal Connectivity Correlates with Early Command-Following After Severe Traumatic Brain injury

abstract: Recovery of consciousness after traumatic brain injury (TBI) is heterogeneous and difficult to predict. Structures such as the thalamus and prefrontal cortex are thought to be important in facilitating consciousness. We sought to investigate whether the integrity of thalamo-prefrontal circuits, assessed via diffusion tensor imaging (DTI), was associated with the return of goal-directed behavior after severe TBI. We classified a cohort of severe TBI patients (N = 25, 20 males) into Early and Late/Never outcome groups based on their ability to follow commands within 30 days post-injury. We assessed connectivity between whole thalamus, and mediodorsal thalamus (MD), to prefrontal cortex (PFC) subregions including dorsolateral PFC (dlPFC), medial PFC (mPFC), anterior cingulate (ACC), and orbitofrontal (OFC) cortices. We found that the integrity of thalamic projections to PFC subregions (L OFC, L and R ACC, and R mPFC) was significantly associated with Early command-following. This association persisted when the analysis was restricted to prefrontal-mediodorsal (MD) thalamus connectivity. In contrast, dlPFC connectivity to thalamus was not significantly associated with command-following. Using the integrity of thalamo-prefrontal connections, we created a linear regression model that demonstrated 72% accuracy in predicting command-following after a leave-one-out analysis. Together, these data support a role for thalamo-prefrontal connectivity in the return of goal-directed behavior following TBI.


title: Sub-second striatal dopamine dynamics assessed by simultaneous fast-scan cyclic voltammetry and fluorescence biosensor

abstract: Fast-scan cyclic voltammetry (FSCV) is an electrochemical method used to detect dopamine on a subsecond time scale. Recordings using FSCV in freely behaving animals revolutionized the study of behaviors associated with motivation and learning. Despite this advance, FSCV cannot distinguish between catecholamines, which limits its use to brain regions where dopamine is the predominant neurotransmitter. It has also been difficult to detect dopamine in vivo in some striatal subregions with FSCV. Recently, fluorescent biosensors for dopamine were developed, allowing for discrimination between catecholamines. However, the performance of these biosensors relative to FSCV has not been determined. Thus, we compared fluorescent photometry responses of the dopamine biosensor, dLight, with FSCV. We also used dLight photometry to assess changes in tonic and phasic dopamine, which has not been possible with FSCV. Finally, we examined dopamine dynamics during Pavlovian conditioning in striatal subregions, including the dorsolateral striatum where dopamine measurements are challenging with FSCV.


title: Functional dissection of neural connectivity in generalized anxiety disorder

abstract: Differences in the correlated activity of networked brain regions have been reported in individuals with generalized anxiety disorder (GAD) but an overreliance on the null-hypothesis significance testing (NHST) framework limits the identification and characterization of disorder-relevant relationships. In this preregistered study, we applied a Bayesian statistical framework as well as NHST to the analysis of resting-state fMRI scans from females with GAD and demographically matched healthy comparison females. Eleven a-priori hypotheses about functional correlativity (FC) were evaluated using Bayesian (multilevel model) and frequentist (t-test) inference. Reduced FC between the ventromedial prefrontal cortex (vmPFC) and the posterior-mid insula (PMI) was confirmed by both statistical approaches. FC between the vmPFC-anterior insula, the amygdala-PMI, and the amygdala-dorsolateral prefrontal cortex (dlPFC) region pairs did not survive multiple comparison correction using the frequentist approach. However, the Bayesian model provided evidence for these region pairs having decreased FC in the GAD group. Leveraging Bayesian modeling, we demonstrate decreased FC of the vmPFC, insula, amygdala, and dlPFC in females with GAD. Exploiting the Bayesian framework revealed FC abnormalities between region pairs excluded by the frequentist analysis, as well as other previously undescribed regions, demonstrating the benefits of applying this statistical approach to resting state FC data.


title: Withdrawal from repeated nicotine vapor exposure increases somatic signs of physical dependence, anxiety-like behavior, and brain reward thresholds in adult male rats.

abstract:
In recent years, there has been a dramatic increase in nicotine vapor consumption via electronic nicotine delivery systems (i.e., e-cigarettes), particularly in adolescents. While recent work has focused on the health effects of nicotine vapor exposure, its effects on the brain and behavior remain unclear. In this study, we assessed the effects that cessation from repeated nicotine vapor exposure had on behavioral and neuronal measures of withdrawal. For Experiment 1, fifty-six adult male rats were tested for plasma cotinine levels, somatic withdrawal signs, and anxiety-like behavior in the elevated plus maze, immediately following precipitated withdrawal from repeated exposure to 12 or 24 mg/mL nicotine vapor. In Experiment 2, twelve adult male rats were tested for intracranial self-stimulation (ICSS) across 14 days of exposure to 24 mg/mL nicotine vapor and across the 14 days immediately following nicotine exposure. Results revealed that plasma cotinine, somatic signs, anxiety-like behavior, and ICSS stimulation thresholds were all observed to be elevated during withdrawal in the 24 mg/mL nicotine group, when compared to vehicle controls (50/50 vegetable glycerin/propylene glycol). The data suggest that cessation from repeated nicotine vapor exposure using our preclinical model leads to nicotine dependence and withdrawal, and demonstrates that the vapor system described in these experiments is a viable pre-clinical model of e-cigarette use in humans. Further characterization of the mechanisms driving nicotine vapor abuse and dependence is needed to improve policies and educational campaigns related to e-cigarette use.

HighlightsO_LIA rodent model of nicotine e-cigarette vapor use was utilized to assess effects of cessation from repeated nicotine vapor exposure on behavioral and neuronal measures of drug withdrawal.
C_LIO_LICessation of repeated nicotine vapor exposure resulted in increased plasma cotinine levels, somatic withdrawal signs, and anxiety-like behavior.
C_LIO_LICessation of repeated nicotine vapor exposure resulted in elevations of ICSS reward threshold.
C_LIO_LIElectrode implantations for ICSS were mapped by location and threshold to a standardized reference atlas of the rat brain to facilitate comparisons with the published literature.
C_LI


title: RNA-sequencing of human post-mortem hypothalamus and nucleus accumbens identifies expression profiles associated with obesity.

abstract: Obesity, the accumulation of body fat to excess, may cause serious negative health effects, including increased risk of heart disease, type 2 diabetes, stroke and certain cancers. The biology of obesity is complex and not well understood, involving both environmental and genetic factors and affecting metabolic and endocrine mechanisms in tissues of the gut, adipose, and brain. Previous RNA sequencing studies have identified transcripts associated with obesity and body mass index in blood and fat, often using animal models, but RNA sequencing studies in human brain tissue related to obesity have not been previously undertaken. We conducted both large and small RNA sequencing of hypothalamus (207 samples) and nucleus accumbens (276 samples) from individuals defined as consistently obese (124 samples), consistently normal weight as controls (148 samples) or selected without respect to BMI and falling within neither case nor control definition (211 samples), based on longitudinal BMI measures. The samples were provided by three cohort studies with brain donation programs; the Framingham Heart Study (FHS), the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP). For each brain region and large/small RNA sequencing set, differential expression of obesity, BMI, brain region and sex was performed. Analyses were done transcriptome-wide as well as with a priori defined sets of obesity or BMI-associated mRNAs and microRNAs (miRNAs). There are sixteen mRNAs and five microRNAs that are differentially expressed (adjusted p < 0.05) by obesity or BMI in these tissues, several of which were validated with qPCR data. The results include many that are BMI-associated, such as APOBR and CES1, as well as many associated with the immune system and some with addiction, such as the gene sets "cytokine signaling in immune system" and "opioid signaling". In spite of the relatively large number of samples, our study was likely under-powered to detect other transcripts or miRNA with relevant but smaller effects.

category: genomics


title: Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior in response to glaucomatous insult

abstract: In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF{beta}2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF{beta}2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF{beta}2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHA retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with longer process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF{beta}2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONHA-encapsulated hydrogel to investigate ONHA behavior in response to glaucomatous insult.

category: bioengineering


title: A quantitative analysis of the similarities and differences of the HIV/FIV gag genome

abstract: 1.Among viruses, human immunodeficiency virus (HIV) presents the greatest challenge to humans. Here, we retrieved genome sequences from NCBI and were then run through LALIGN bioinformatics software to compute the E value, bit score, Waterman eggert score, and percent identity, which are four important indicators of how similar the sequences are. The E value was 3.1 x 10^-9, the percent identity was 54.4 percent, and the bit score was 51.9. It was also sensed that bases 1600 to 1990 in HIV and bases 800 to 910 in FIV have a higher than normal similarity. This reflects that while the DNA sequences of the gag region of both the HIV and FIV genomes are rather similar, it is unlikely that this similarity is due to random chance; therefore, there are a noticeable number of differences. A better understanding of the level of similarity and differences in the gag region of the genome sequence would facilitate our understanding of structural and cellular behavioral differences between FIV and HIV, and in the long term, it will provide new insights into the differences observed in previous studies or even facilitate the development of an effective HIV treatment.


title: Validating amino acid variants in proteogenomics using sequence coverage by multiple reads

abstract:
Mass spectrometry-based proteome analysis usually implies matching mass spectra of proteolytic peptides to amino acid sequences predicted from nucleic acid sequences. At the same time, due to the stochastic nature of the method when it comes to proteome-wide analysis, in which only a fraction of peptides are selected for sequencing, the completeness of protein sequence identification is undermined. Likewise, the reliability of peptide variant identification in proteogenomic studies is suffering. We propose a way to interpret shotgun proteomics results, specifically in data-dependent acquisition mode, as protein sequence coverage by multiple reads, just as it is done in the field of nucleic acid sequencing for the calling of single nucleotide variants. Multiple reads for each position in a sequence could be provided by overlapping distinct peptides, thus, confirming the presence of certain amino acid residues in the overlapping stretch with much lower false discovery rate than conventional 1%. The source of overlapping distinct peptides are, first, miscleaved tryptic peptides in combination with their properly cleaved counterparts, and, second, peptides generated by several proteases with different specificities after the same specimen is subject to parallel digestion and analyzed separately. We illustrate this approach using publicly available multiprotease proteomic datasets and our own data generated for HEK-293 cell line digests obtained using trypsin, LysC and GluC proteases. From 5000 to 8000 protein groups are identified for each digest corresponding to up to 30% of the whole proteome coverage. Most of this coverage was provided by a single read, while up to 7% of the observed protein sequences were covered two-fold and more. The proteogenomic analysis of HEK-293 cell line revealed 36 peptide variants associated with SNP, seven of which were supported by multiple reads. The efficiency of the multiple reads approach depends strongly on the depth of proteome analysis, the digesting features such as the level of miscleavages, and will increase with the number of different proteases used in parallel proteome digestion.

Graphical abstract

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title: Validated method for automated glioma diagnosis from GFAP immunohistological images: a complete pipeline

abstract:
Background and objectiveGlial fibrillar acid protein is a common marker for brain tumor because of its particular rearrangement during tumor development. It is commonly used in manually histological glioma detection and grading. An automatic pipeline for tumor diagnosis based on GFAP is proposed in the present manuscript for detecting and grading canine brain glioma in stages III and IV.

MethodsThe study was performed on canine brain tumor stages III and IV as well as healthy tissue immunohistochemically stained for gliofibrillar astroglial protein. Four stereological indexes were developed using the area of the image as reference unit: density of glioma protein, density of neuropil, density of astrocytes and the glioma nuclei number density. Images of the slides were subset for image analysis (n=1415) and indexed. The stereological indexes of each subset constituted an array of data describing the tumor phase of the subset. A 5% of these arrays were used as training set for decision tree classification with PCA. The other arrays were further classified in a supervised approach. ANOVA and PCA analysis were applied to the indexes.

ResultsThe final pipeline is able to detect brain tumor and to grade it automatically. Added to it, the role the neuropil during tumor development has been quantified for the first time. While astroglial cells tend to disappear, glioma cells invade all the tumor area almost to a saturation in stage III before reducing the density in stage IV. The density of the neuropil is reduced during the tumour growth.

ConclusionsThe method validated ere allows the automated diagnosis and grading of glioma in dogs. This method opens the research of the role of the neuropil in tumor development.

Graphical abstract

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title: Enhanced competitive protein exchange at the nano-bio interface enables ultra-deep coverage of the human plasma proteome

abstract: We have developed a scalable system that leverages protein-nano interactions to overcome current limitations of deep plasma proteomics in large cohorts. Introducing proprietary engineered nanoparticles (NPs) into a biofluid such as blood plasma leads to the formation of a selective and reproducible protein corona at the particle-protein interface, driven by the relationship between protein-NP affinity and protein abundance. Here we demonstrate the importance of tuning the protein to NP-surface ratio (P/NP), which determines the competition between proteins for binding. We demonstrate how optimized P/NP ratio affects protein corona composition, ultimately enhancing performance of a fully automated NP-based deep proteomic workflow (Proteograph). By limiting the available binding surface of NPs and increasing the binding competition, we identify 1.2 - 1.7x more proteins with only 1% false discovery rate on the surface of each NP, and up to 3x compared to a standard neat plasma proteomics workflow. Moreover, increased competition means proteins are more consistently identified and quantified across replicates, yielding precise quantification and improved coverage of the plasma proteome when using multiple physicochemically distinct NPs. In summary, by optimizing NPs and assay conditions, we capture a larger and more diverse set of proteins, enabling deep proteomic studies at scale.


title: Artificial Metabolic Networks: enabling neural computation with metabolic networks

abstract: Metabolic networks have largely been exploited as mechanistic tools to predict the behavior of microorganisms with a defined genotype in different environments. However, flux predictions by constraint-based modeling approaches are limited in quality unless labor intensive experiments including the measurement of media intake fluxes, are performed. Using machine learning instead of an optimization of biomass flux - on which most existing constraint-based methods are based - provides ways to improve flux and growth rate predictions. In this paper, we show how Recurrent Neural Networks can surrogate constraint-based modeling and make metabolic networks suitable for backpropagation and consequently be used as an architecture for machine learning. We refer to our hybrid - mechanistic and neural network - models as Artificial Metabolic Networks (AMN). We showcase AMN and illustrate its performance with an experimental dataset of Escherichia coli growth rates in 73 different media compositions. We reach a regression coefficient of R2=0.78 on cross-validation sets. We expect AMNs to provide easier discovery of metabolic insights and prompt new biotechnological applications.

category: systems biology


title: OptiDiff: structural variation detection from single optical mapping reads

abstract: Detecting structural variation (SV) in eukaryotic genomes is of broad interest due to its often dramatic phenotypic effects, but remains a major, costly challenge based on DNA sequencing data. A cost-effective alternative in detecting large-scale SV has become available with advances in optical mapping technology. However, the algorithmic approaches to identifying SVs from optical mapping data are limited. Here, we propose a novel, open-source SV detection tool, OptiDiff, which employs a single molecule based approach to detect and classify homozygous and heterozygous SVs at coverages as low as 20x, showing better performance than the state of the art.


title: Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X

abstract: Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. sensory response), and also mediate intracellular signaling cascades normally of longer time scales (e.g., Ca2+-dependent neuritogenesis). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate that the recently developed GCaMP-X outperforms GCaMP in long-term probe expression and/or chronic Ca2+ imaging. GCaMP-X shows much improved compatibility with neurons and thus more reliable than GCaMP as demonstrated in vivo by acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations over an extended time frame. Chronic Ca2+ imaging data ([≥]1 month) are acquired from the same set of cultured cortical neurons, unveiling that spontaneous/local Ca2+ activities would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length or soma size, the major metrics of oscillatory Ca2+, including rate, amplitude, synchrony among different neurons or organelles have also been examined along with the developmental stages. Both neuritogenesis and Ca2+ signals are dysregulated by GCaMP in virus-infected or transgenic neurons, in direct contrast to GCaMP-X without any noticeable side-effect. Such in vitro data altogether consolidate the unique importance of oscillatory Ca2+ to activity-dependent neuritogenesis, as one major factor responsible for the distinctions between GCaMP vs GCaMP-X in vivo. For the first time with GCaMP-X of long-term expression in neurons, spontaneous and sensory-evoked Ca2+ activities are imaged and evaluated both in vitro and in vivo, providing new opportunities to monitor neural development or other chronic processes concurrently with Ca2+ dynamics.


title: Long-term two-photon imaging of spinal cord in freely behaving mice

abstract: The spinal cord is critical to the perception of peripheral information under sensory-guided motor behaviors in health and disease. However, the cellular activity underlie spinal cord function in freely behaving animals is not clear. Here, we developed a new method for imaging the spinal cord at cellular and subcellular resolution over weeks under naturalistic conditions. The method involves an improved surgery to reduce spinal movement, and the installation of a miniaturized two-photon microscope to obtain high-resolution imaging in moving mice. In vivo calcium imaging demonstrated that dorsal horn neurons show a sensorimotor program-dependent synchronization and heterogeneity under distinct cutaneous stimuli in behaving mice. The long-term imaging of sensory neurons revealed that in the spinal cord, healthy mice demonstrated stereotyped responses. However, in a neuropathic pain model, plasticity changes and neuronal sensitization were observed. We provide a practical method to study the function of spinal cord on sensory perception and disorders in freely behaving mice.


title: Differential recognition and cytokine induction by the peptidorhamnomannan from Sporothrix brasiliensis and S. schenckii

abstract: Sporotrichosis is a deep mycosis caused by dimorphic species of the genus Sporothrix, with differences in pathogenicity between S. schenckii and S. brasiliensis species. Recently, it was discovered that the cell wall peptidorhamnomannan (PRM) of Sporothrix spp. is a pathogen associated molecular pattern (PAMP). Interestingly, S. brasiliensis PRM has additional unknown rhamnose residues. We hypothesize that the structural differences of Sporothrix spp PRMs impact the hosts immune response and may explain the severity of sporotrichosis caused by S. brasiliensis. Here we demonstrate that S. brasiliensis yeasts and its PRM (S.b PRM) induced a strong inflammatory response in human PBMCs, with high production of TNF-, IL-6 and IL-1{beta} and induction of T-helper cytokines IFN-{gamma}, IL-17 and IL-22. In contrast, S. schenckii yeasts and its PRM induced higher concentrations of interleukin-1 receptor antagonist (IL-1Ra), which resulted in low production of T-helper cytokines such as IL-17 and IL-22. CR3 and dectin-1 were required for cytokine induction by both PRMs, while TLR2 and TLR4 were required for the response of S.s PRM and S.b PRM, respectively. IL-1{beta} and IL-1 production induced by S. brasiliensis yeasts and S.b PRM were dependent on inflammasome and caspase-1 activation. S. schenckii and S.s PRM were able to induce IL-1{beta} independent of ROS. In conclusion, these findings improve our understanding of the pathogenesis of Sporothrix spp. by reporting differences of immunological responses induced by S. schenckii and S. brasiliensis. The study also opens the gateway for novel treatment strategies targeting local inflammation and tissue destruction induced by S. brasiliensis infection through IL-1 inhibition.

category: immunology


title: Integrated cyto-physiological and proteomic analyses reveal new insight into CMS mechanism in a novel upland cotton CMS line LD6A

abstract: Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we elucidated molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed for physiological and proteomic changes during the development process. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in CMS and its maintainer line. Transmission electron micrographs of anther tapetum showed that inner ridge of CMS mitochondria was relatively indistinct than that of LD6B with narrower membranous space at tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.


title: Mechanics of human embryo compaction

abstract: The shaping of the human embryo begins with compaction, during which cells come into close contact and form a tighter structure1,2. Assisted reproductive technology (ART) studies suggest that human embryos fail compaction primarily because of defective adhesion3,4. Based on our current understanding of animal morphogenesis5,6, other morphogenetic engines, such as cell contractility, could be involved in shaping the human embryo. However, the molecular, cellular and physical mechanisms driving human embryo morphogenesis remain uncharacterized. Using micropipette aspiration on human embryos donated to research, we have mapped cell surface tensions during compaction. This reveals a 4-fold increase of tension at the cellmedium interface while cell-cell contacts keep a steady tension. Comparison between human and mouse reveals qualitatively similar but quantitively different mechanical strategies, with human embryos being mechanically least efficient. Inhibition of cell contractility and cell-cell adhesion in human embryos reveal that only contractility controls the surface tension responsible for compaction. Interestingly, if both cellular processes are required for compaction, they exhibit distinct mechanical signatures when faulty. Analyzing the mechanical signature of naturally failing embryos, we find evidence that non-compacting embryos or partially compacting embryos with excluded cells have defective contractility. Together, our study reveals that an evolutionarily conserved increase in cell contractility is required to generate the forces driving the first morphogenetic movement shaping the human body.


title: Multiplex PCR reveals unique trends in pathogen and parasitoid infestations of alfalfa leafcutting brood cells.

abstract: The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. Megachile rotundata is a solitary bee that forms brood cocoons where its larvae can develop. During the developmental stages of growth, broods can be preyed upon by multiple different fungal and bacterial pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollinator services provided by Megachile rotundata and reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of the most common bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. Co-infection of single brood cells by multiple Ascosphaera species was confirmed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species including multiple new predators and pathogenetic species not previously documented in associated with M. rotundata brood cells.

category: ecology


title: Examining historical rates of leafcutting bee cell pathogens to establish baseline infectivity rates for alfalfa seed growers

abstract: The alfalfa leafcutting bee (Megachile rotundata) is one of the primary pollinators for the alfalfa seed industry. The alfalfa leafcutting bee is a solitary cavity nesting bee. Female Megachile rotundata bees will construct and provision individual brood cells lined with cut leaves (cocoon) and will gather nectar and pollen to place within the constructed cocoon. The female bee will lay a single egg within the constructed cocoon and leave the egg to undergo larval stage development and pupation into the adult stage. During this time multiple pathogens and parasitoids can prey on the developing larvae, resulting in the loss of the future adult bee. A major concern for commercial alfalfa seed growers is the presence of invertebrate pests and fugal pathogens. In the present study, we used historical data from the Parma Cocoon Diagnostic Laboratory to determine baseline rates of pathogen and parasite infection of Megachile rotundata cells and used this analysis to determine historical infection rates and cutoffs for management practices. Additionally, using a Faxitron (X-ray) analysis for Megachile rotundata cell obtained in 2020, we compared the presence of chalkbrood, pathogens, and parasitoids in samples collected from both growers stocks and newly purchased Canada bees. The results of the investigation demonstrate historical averages of the presence of chalkbrood, pathogens, and parasitoids. We also show a significant increase in chalkbrood and predators in 2007-2011 and a significant difference in chalkbrood and predators between bee samples obtained from Canada and grower stocks.


title: A Tm4sf1-Marked Subpopulation of Endothelial Stem/Progenitor Cells Identified by Lung Single-Cell Omics of Pulmonary Arterial Hypertension

abstract:
RationaleThe identification and role of endothelial progenitor cells (EPCs) in pulmonary arterial hypertension (PAH) remains controversial. Single-cell omics analysis can shed light on EPCs and their potential contribution to PAH pathobiology.

ObjectivesWe aim to identify EPCs in rat lungs and assess their relevance to preclinical and human PAH.

MethodsDifferential expression, gene set enrichment, cell-cell communication, and trajectory reconstruction analyses were performed on lung endothelial cells from single-cell RNA-seq of Sugen-hypoxia, monocrotaline, and control rats. Relevance to human PAH was assessed in multiple independent blood and lung transcriptomic datasets.

Measurements and Main ResultsA subpopulation of endothelial cells (EA2) marked by Tm4sf1, a gene strongly implicated in cancer, harbored a distinct transcriptomic signature including Bmpr2 downregulation that was enriched for pathways such as inflammation and angiogenesis. Cell-to-cell communication networks specific to EA2 were activated in PAH such as CXCL12 signaling. Trajectory analysis demonstrated EA2 has a stem/progenitor cell phenotype. Analysis of independent datasets revealed Tm4sf1 is a marker for hematopoietic stem cells and is upregulated in PAH peripheral blood, particularly in patients with worse WHO functional class. EA2 signature genes including Procr and Sulf1 were found to be differentially regulated in the lungs of PAH patients and in PAH models in vitro, such as BMPR2 knockdown.

ConclusionsOur study uncovered a novel Tm4sf1-marked stem/progenitor subpopulation of rat lung endothelial cells and demonstrated its relevance to preclinical and human PAH. Future experimental studies are warranted to further elucidate the pathogenic role and therapeutic potential of targeting EA2 and Tm4sf1 in PAH.


title: Synthesis of cell-laden alginate microgels with tunable compositions based on microfluidic pico-injection technique

abstract: We report a microfluidic pico-injection-based approach for reliably generating monodisperse cell-laden alginate microgels whose composition can be tuned in situ through modulation of the cross-linker concentration. Separating the gelation from emulsification allows for a better control over the microgel size with a microfluidic drop-maker, and an instant adjustment of the microgel composition with a pico-injector.


title: A unique Thy-1-negative immuno-fibroblast population emerges as a key determinant of fibrotic outcomes to biomaterials

abstract:
Microporous annealed particle (MAP) hydrogels are an exciting new development in biomaterial design. They regulate innate and acquired immunity which has been linked to their ability to evade normal host-material fibrosis. Yet, resident stromal fibroblasts, not immune cells, are the arbiters of the extracellular matrix assembly that characterizes fibrosis. In other idiopathic fibrotic disorders, a fibroblast subpopulation defined by its loss of cell surface Thy-1 expression is strongly correlated with degree of fibrosis. We have previously shown that Thy-1 is a critical v{beta}3 integrin regulator that enables normal fibroblast mechanosensing and here, leveraging non-fibrosing MAP gels, we demonstrate that Thy-1-/- mice mount a robust response to MAP gels that remarkably resembles a classical foreign body response. We further find that within the naive, Thy-1+ fibroblast population exists a distinct and cryptic SMA+ Thy-1- population that emerges in response to IL-1{beta} and TNF. Employing single-cell RNA sequencing, we find that IL-1{beta}/TNF-induced Thy-1- fibroblasts actually consist of two distinct subpopulations, both of which are strongly pro-inflammatory. These findings illustrate the emergence of a unique pro-inflammatory, pro-fibrotic fibroblast subpopulation that is central to material-associated fibrosis likely through amplifying local inflammatory signaling.

Significance StatementDespite decades of research, implanted biomaterials are still significantly hampered by the foreign body response and fibrotic encapsulation.

Advancements in material design and immunomodulation have made positive impacts, yet, a fuller mechanistic understanding of aberrant ECM remodeling in the biomaterial microenvironment could improve approaches in biomaterial-host interactions. Here, we leverage anti-fibrotic MAP hydrogels and demonstrate that their ability to evade fibrosis is linked to fibroblast Thy-1 (CD90) surface expression. Thy-1-/- mice exhibit elevated NF{kappa}B signaling and elevated fibrosis in response to MAP gel implantation. Interestingly, pro-inflammatory cytokines elicit a Thy-1-/SMA+ myofibroblast subpopulation. Single-cell RNA-Seq more fully identifies an  immunofibroblast subpopulation defined by Thy-1 loss and a pro-inflammatory/fibrotic cytokine, chemokine, and cytokine receptor expression profile, suggesting a self-perpetuating pro-fibrotic subpopulation.


title: PCR-free, label-free detection of sequence-specific DNA with single-molecule sensitivity using in vitro N-hybrid system in microfluidic drops

abstract: We propose a novel method that can detect DNA with high specificity at the single-molecule level by employing the in vitro N-hybrid strategy realized in sub-picoliter microfluidic drops. It detects target DNA based on the specific interactions of the target-encoded proteins with their partner molecules, and achieves single-molecule sensitivity via signal-transduction and signal-amplification during gene-expression processes in a sub-picoliter droplet, therefore effectively avoiding complicated procedures in labeling-based methods or biases and artifacts in PCR-based methods.


title: High-throughput direct screening of restriction endonuclease using microfluidic fluorescence-activated drop sorter based on SOS response in E. coli

abstract: A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route for expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient fashion. Briefly, we construct a host bacterial cell to link the RE genotype to the phenotype of {beta}-galactosidase expression based on the bacterial SOS response, and use a high-throughput microfluidic platform to isolate, detect and sort the REs. We employ this strategy to screen for the XbaI gene from constructed libraries of varied sizes. In single round of sorting, a 30-fold target enrichment was obtained within 1 h. The direct screening approach we propose shows potential for efficient search of desirable REs in natural samples compared to the conventional RE-screening method, and is amenable to being adapted to high-throughput screening of other genotoxic targets.


title: Quantitative Assessment of 3D Printed Blood Vessels Produced with J750™ Digital Anatomy™ for Suture Simulation

abstract:
Blood vessels anastomosis is one of the most challenging and delicate tasks to learn in many surgical specialties, especially for vascular and abdominal surgeons. Such a critical skill implies a learning curve that goes beyond technical execution. The surgeon needs to gain proficiency in adapting gestures and the amount of force expressed according to the type of tissue he/she is dealing with. In this context, surgical simulation is gaining a pivotal role in the training of surgeons, but currently available simulators can provide only standard or simplified anatomies, without the chance of presenting specific pathological conditions and rare cases.

3D printing technology, allowing the manufacturing of extremely complex geometries, find a perfect application in the production of realistic replica of patient-specific anatomies. According to available technologies and materials, morphological aspects can be easily handled, while the reproduction of tissues mechanical properties still poses major problems, especially when dealing with soft tissues.

The present work focuses on blood vessels, with the aim of identifying - by means of both qualitative and quantitative tests - materials combinations able to best mimic the behavior of the biological tissue during anastomoses, by means of J750 Digital Anatomy technology and commercial photopolymers from Stratasys. Puncture tests and stitch traction tests are used to quantify the performance of the various formulations. Surgical simulations involving anastomoses are performed on selected clinical cases by surgeons to validate the results.

A total of 37 experimental materials were tested and 2 formulations were identified as the most promising solutions to be used for anastomoses simulation. Clinical applicative tests, specifically selected to challenge the new materials, raised additional issues on the performance of the materials to be considered for future developments.


title: Single-cell network biology characterizes cell-type gene regulation for drug repurposing and phenotype prediction in Alzheimer's disease

abstract: Dysregulation of gene expression in Alzheimers disease (AD) remains elusive, especially at the cell type level. Gene regulatory network, a key molecular mechanism linking transcription factors (TFs) and regulatory elements to govern target gene expression, can change across cell types in the human brain and thus serve as a model for studying gene dysregulation in AD. However, it is still challenging to understand how cell type networks work abnormally under AD. To address this, we integrated single-cell multi-omics data and predicted the gene regulatory networks in AD and control for four major cell types, excitatory and inhibitory neurons, microglia and oligodendrocytes. Importantly, we applied network biology approaches to analyze the changes of network characteristics across these cell types, and between AD and control. For instance, many hub TFs target different genes between AD and control (rewiring). Also, these networks show strong hierarchical structures in which top TFs (master regulators) are largely common across cell types, whereas different TFs operate at the middle levels in some cell types (e.g., microglia). The regulatory logics of enriched network motifs (e.g., feed-forward loops) further uncover cell-type-specific TF-TF cooperativities in gene regulation. The cell type networks are highly modular. Several network modules with cell-type-specific expression changes in AD pathology are enriched with AD-risk genes and putative targets of approved and pending AD drugs, suggesting possible cell-type genomic medicine in AD. Finally, using the cell type gene regulatory networks, we developed machine learning models to classify and prioritize additional AD genes. We found that top prioritized genes predict clinical phenotypes (e.g., cognitive impairment). Overall, this single-cell network biology analysis provides a comprehensive map linking genes, regulatory networks, cell types and drug targets and reveals mechanisms on cell-type gene dyregulation in AD.


title: Lemaitre's law and the spatial distributions of the cone photoreceptors in the retinas of vertebrates

abstract: By applying the principle of maximum entropy, we demonstrate the universality of the spatial distributions of the cone photoreceptors in the retinas of vertebrates. We obtain Lemaitres law as a special case of our formalism.