title: Structural and temporal basis for agonism in the α4β2 nicotinic acetylcholine receptor

abstract: Despite decades of study, the structural mechanisms underpinning agonist efficacy in pentameric ligand-gated ion channels remain poorly understood. Here, a combination of extensive equilibrium and dynamical-nonequilibrium molecular dynamics simulations was used to obtain a detailed description of the structural and dynamic changes induced within the human 4{beta}2 nicotinic acetylcholine receptor by a full and a partial agonist, namely acetylcholine and nicotine, and map how these rearrangements propagate within this receptor. These simulations reveal how the agonists modulate the patterns associated with intra and inter-domain communication and the evolution of the agonist-specific structural rearrangements. For the first time, we show that full and partial agonists, although generally using similar routes for through-receptor signal transmission, induce different amplitudes of conformational rearrangements in key functional motifs, thus impacting the rates of signal propagation within the protein. The largest agonist-induced conformational differences are located in the Cys loop, loops C and 1-{beta}1 in the 4 subunit, loops F and {beta}1-{beta}2 in the {beta}2 subunit and in the extracellular selectivity filter.


title: GANscan: continuous scanning microscopy using deep learning deblurring

abstract: Most whole slide imaging (WSI) systems today rely on the "stop-and-stare" approach, where, at each field of view, the scanning stage is brought to a complete stop before the camera snaps a picture. This procedure ensures that each image is free of motion blur, which comes at the expense of long acquisition times. In order to speed up the acquisition process, especially for large scanning areas, such as pathology slides, we developed an acquisition method in which the data is acquired continuously while the stage is moving at high speeds. Using generative adversarial networks (GANs), we demonstrate this ultra-fast imaging approach, referred to as GANscan, which restores sharp images from motion blurred videos. GANscan allows us to complete image acquisitions at 30x the throughput of stop-and-stare systems. This method is implemented on a Zeiss Axio Observer Z1 microscope in brightfield mode, requires no specialized hardware, and accomplishes successful reconstructions at stage speeds of up to 5,000 m/s. We validate the proposed method by imaging H&E stained tissue sections. Our method not only retrieves crisp images from fast, continuous scans, but also corrects any defocusing that occurs during scanning. Using a consumer GPU, the inference runs at <20ms/ image.

category: bioengineering


title: Thermodynamic limitations of metabolic strategies for PHB production from formate and fructose in Cupriavidus necator

abstract:
The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields of different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that metabolic strategies for PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified reactions of the core metabolism associated with low and high PHB yield. The findings of this study aid in understanding metabolic adaptation. Furthemore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria.

HighlightsO_LIElementary flux modes of C. necator for PHB synthesis from fructose and formate.
C_LIO_LIMetabolite sampling identified common reactions among EFMs with low driving force.
C_LIO_LIPHB from formate shows low driving forces for aconitase, citrate lyase, NADPH synthesis.
C_LIO_LIPhosphoketolase and ATP citrate lyase increased driving forces and PHB production.
C_LI


title: Omicron and Alpha P680H block SARS-CoV2 spike protein from accessing cholinergic inflammatory pathway via α9-nAChR mitigating the risk of MIS-C

abstract: Sequence homology between neurotoxins and the site encompassing the furin cleavage site 680SPRRAR685 in the spike protein (S) of CoV2 suggested that this site could interact with nicotinic acetylcholine receptors (nAChRs). Molecular dynamics simulations confirm robust structural similarity between wild-type (WT) CoV2 and the binding motif of -conotoxin to 9 nAChR, which is known to modulate IL-1{beta} in immune cells. We show that the structural integrity of this binding motif is eliminated by Alpha P681H mutation, reemerged in Delta variant P681R, and disappeared again with Omicron N679H/P681H. Interactions between the toxin-mimic CoV2 motif and 9-nAChR are expected to trigger the release of pro-inflammatory cytokines an effect that is mollified by Alpha and Omicron. Clinical features of this interaction site are relevant because, contrary to most regions in the S protein, the furin binding site does not appear to trigger an immune response prior to cleavage, indicating that the cholinergic pathway should be activated in the respiratory tract and nasal mucosa where 9-nAChR co-localizes with the virus. The correlation of changes on this motif by the different variants closely matches the reported cases of Multisystem Inflammatory Syndrome in Children by the CDC, and predicts significant mitigation of MIS-C with the Omicron variant. Our findings strongly motivate further study of this cholinergic pathway as one source of the cytokine storm triggered by CoV2.

category: biophysics


title: Effect of an amyloidogenic SARS-COV-2 protein fragment on α-synuclein monomers and fibrils

abstract: Using molecular dynamic simulations we study whether amyloidogenic regions in viral proteins can initiate and modulate formation of -synuclein aggregates, thought to be the disease-causing agent in Parkinsons Disease. As an example we choose the nine-residue fragment SFYVYSRVK (SK9), located on the C-terminal of the Envelope protein of SARS-COV-2. We probe how the presence of SK9 affects the conformational ensemble of -synuclein monomers and the stability of two resolved fibril polymorphs. We find that the viral protein fragment SK9 may alter -synuclein amyloid formation by shifting the ensemble toward aggregation-prone and preferentially rod-like fibril seeding conformations. However, SK9 has only little effect of the stability of pre-existing or newly-formed fibrils.


title: Frequency dependent growth of bacteria in living materials

abstract: The fusion of living bacteria and man-made materials represents a new frontier in medical and biosynthetic technology. However, the principles of bacterial signal processing inside three dimensional and fluctuating environments of synthetic materials remain elusive. Here, we study bacterial growth in a three-dimensional hydrogel. We find that bacteria expressing an antibiotic resistance module can take advantage of ambient kinetic disturbances to improve growth while encapsulated. We show that these changes in bacterial growth are specific to disturbance frequency and hydrogel density. This remarkable specificity is consistent with stochastic resonance theory, which we leverage to explain how bacteria can integrate spatial and temporal information to control growth. This research provides a quantitative foundation for the control of living materials and a systematic framework towards understanding bacterial information processing in three-dimensional environments.


title: Topologically frustrated dynamics in an uncharged Tetra-PEG gel

abstract: Based on our previous discovery of non-diffusive topologically frustrated dynamics in a charged system where electrostatic interactions between the charged guest and the charged host gel play a role (Nature Communications, 2018, 9, 2248; Physical Review Letters, 2021, 126, 057802), we have investigated the onset of this effect in an uncharged gel matrix. Using sodium (polystyrene sulfonate) as the guest macromolecule and the ideal tetra-PEG gel, we find the emergence of the non-diffusive topologically frustrated dynamical state with a hierarchy of segmental dynamics represented by a stretched exponential of exponent {beta} around 1/3. Our results demonstrate the universal behavior of the topologically frustrated dynamical state.


title: Biophysical fitness landscape of the SARS-CoV-2 Delta variant receptor binding domain

abstract: Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased number of deaths. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single mutations as well as the double mutation on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than receptor binding, is the main biophysical parameter determining the fitness landscape of the Delta variant RBD and is determined by the L452R mutation.


title: First passage time analysis of spatial mutation patterns reveals evolutionary dynamics of pre-existing resistance in colorectal cancer

abstract: The footprint left by early cancer dynamics on the spatial arrangement of tumour cells is poorly understood, and yet could encode information about how therapy resistant sub-clones grew within the expanding tumour. Novel methods of quantifying spatial tumour data at the cellular scale are required to link evolutionary dynamics to the resulting spatial architecture of the tumour. Here, we propose a framework using first passage times of random walks to quantify the complex spatial patterns of tumour cell population mixing. First, using a toy model of cell mixing we demonstrate how first passage time statistics can distinguish between different pattern structures. We then apply our method to simulated patterns of wild-type and mutated tumour cell population mixing, generated using an agent-based model of expanding tumours, to explore how first passage times reflect mutant cell replicative advantage, time of emergence and strength of cell pushing. Finally, we analyse experimentally measured patterns of genetic point mutations in human colorectal cancer, and estimate parameters of early sub-clonal dynamics using our spatial computational model. We uncover a wide range of mutant cell replicative advantages and timings, with the majority of sampled tumours consistent with boundary driven growth or short-range cell pushing. By analysing multiple sub-sampled regions in a small number of samples, we explore how the distribution of inferred dynamics could inform about the initial mutational event. Our results demonstrate the efficacy of first passage time analysis as a new methodology for quantifying cell mixing patterns in vivo, and suggest that patterns of sub-clonal mixing can provide insights into early cancer dynamics.

category: cancer biology


title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

abstract: Despite its pivotal roles in biology, how the transcriptional activity of c-MYC is attuned quantitatively remain poorly defined. Here, we show that heat shock factor 1 (HSF1), the master transcriptional regulator of the heat-shock, or proteotoxic stress, response, acts as a key modifier of the c-MYC-mediated transcription. HSF1 deficiency diminishes c-MYC DNA binding and dampens its transcriptional activity genome-widely. Mechanistically, c-MYC, MAX, and HSF1 assemble into a transcription factor complex on genomic DNAs and, surprisingly, the DNA binding of HSF1 is dispensable. Instead, HSF1 physically recruits the histone acetyltransferase GCN5, thereby promoting histone acetylation and augmenting c-MYC transcriptional activity. Thus, our studies reveal that HSF1 specifically potentiates the c-MYC-mediated transcription, distinct from its role in the canonical heat-shock response. Importantly, this mechanism of action engenders two distinct c-MYC activation states, primary and advanced, which may be important to accommodate diverse physiological and pathological conditions.


title: Galaxy-SynBioCAD: Automated Pipeline for Synthetic Biology Design and Engineering

abstract: We introduce the Galaxy-SynBioCAD portal, the first toolshed for synthetic biology, metabolic engineering, and industrial biotechnology. The tools and workflows currently shared on the portal enables one to build libraries of strains producing desired chemical targets covering an end-to-end metabolic pathway design and engineering process from the selection of strains and targets, the design of DNA parts to be assembled, to the generation of scripts driving liquid handlers for plasmid assembly and strain transformations. Standard formats like SBML and SBOL are used throughout to enforce the compatibility of the tools. In a study carried out at four different sites, we illustrate the link between pathway design and engineering with the building of a library of E. coli lycopene-producing strains. We also benchmarked our workflows on literature and expert validated pathways. Overall, we find an 83% success rate in retrieving the validated pathways among the top 10 pathways generated by the workflows.

category: synthetic biology


title: Rank Selection Genetic Algorithm optimises robust parameter estimation for systems biology models

abstract: The ability to reliably predict and infer cellular responses to environmental exposures would offer a major advance in the investigation of immune regulation in health and disease. One possible approach is the use of in silico modelling. Design of such a mathematical kinetic model would be based on existing knowledge of a biological system and utilise a partial data set to parameterise. However, the process of parameter estimation, key for the accuracy of the model, is difficult to conduct by hand, and thus a computational alternative is necessary. We report the utility of Genetic Algorithm with Rank Selection (GARS) as a parameter estimation tool on multiple biological models, including heat shock, signal transduction via ERK, circadian rhythm and NF{kappa}B systems, where it showed strong accuracy and superiority to the Extended Kalman Filter method, Algebraic Difference Equations, and MATLAB fminsearch approaches. GARS parameter estimation is a valuable tool for biological data because it reliably infers system behaviour from partial data sets, allowing for the prediction of cellular responses to environmental exposures.

category: systems biology


title: Detection of functional activity in brain white matter using fiber architecture informed synchrony mapping

abstract: A general linear model is widely used for analyzing fMRI data, in which the blood oxygenation-level dependent (BOLD) signals in gray matter (GM) evoked in response to neural stimulation are modeled by convolving the time course of the expected neural activity with a canonical hemodynamic response function (HRF) obtained a priori. The maps of brain activity produced reflect the magnitude of local BOLD responses. However, detecting BOLD signals in white matter (WM) is more challenging as the BOLD signals are weaker and the HRF is different, and may vary more across the brain. Here we propose a model-free approach to detect changes in BOLD signals in WM by measuring task-evoked increases of BOLD signal synchrony in WM fibers. The proposed approach relies on a simple assumption that, in response to a functional task, BOLD signals in relevant fibers are modulated by stimulus-evoked neural activity and thereby show greater synchrony than when measured in a resting state, even if their magnitudes do not change significantly. This approach is implemented in two technical stages. First, for each voxel a fiber-architecture-informed spatial window is created with orientation distribution functions constructed from diffusion imaging data. This provides the basis for defining neighborhoods in WM that share similar local fiber architectures. Second, a modified principal component analysis (PCA) is used to estimate the synchrony of BOLD signals in each spatial window. The proposed approach is validated using a 3T fMRI dataset from the Human Connectome Project (HCP) at a group level. The results demonstrate that neural activity can be reliably detected as increases in fMRI signal synchrony within WM fibers that are engaged in a task with high sensitivities and reproducibility.

category: neuroscience


title: Somatostatin Neurons in the Bed Nucleus of the Stria Terminalis Play a Sex-Dependent Role in Binge Drinking

abstract:
Alcohol use disorder (AUD) is characterized by alcohol use coupled with chronic relapse and involves brain regions including the bed nucleus of the stria terminalis (BNST). Here, we explore whether a subpopulation of BNST neurons, somatostatin (SST) expressing GABAergic neurons, play a role in an animal model of binge-like alcohol consumption, the Drinking in the Dark (DID) model. Chemogenetic activation of BNST SST neurons reduced binge alcohol consumption in female but not male SST-Cre mice, while inhibition of these neurons in the same mice had no effect. In addition, chemogenetic activation of these neurons did not cause apparent changes in models of anxiety-like behavior in either sex. Basal SST cell counts and intrinsic excitability of SST neurons were compared to attempt to understand sex differences in DREADD-induced changes in drinking, and while males had a greater number of BNST SST neurons, this effect went away when normalizing for total BNST volume. Together, these results suggest SST neurons in the BNST should be further explored as a potential neuronal subtype modulated by AUD, and for their therapeutic potential.

HIGHLIGHTSO_LIChemogenetic activation of BNST SST neurons reduces binge drinking in female but not male mice
C_LIO_LIChemogenetic activation of these neurons has no effect on anxiety-like behavior in either sex
C_LIO_LIElectrophysiology revealed no clear sex differences in intrinsic excitability BNST SST neurons between males and females
C_LIO_LIImaging revealed males had greater overall BNST SST cell numbers than females, but this effect could be explained by normalizing for total BNST volume
C_LI


title: Temporal continuity shapes visual responses of macaque face patch neurons

abstract: Macaque inferior temporal cortex neurons respond selectively to complex visual images, with recent work showing that they are also entrained reliably by the evolving content of natural movies. To what extent does visual continuity itself shape the responses of high-level visual neurons? We addressed this question by measuring how cells in face-selective regions of the macaque temporal cortex were affected by the manipulation of a movies temporal structure. Sampling the movie at 1s intervals, we measured neural responses to randomized, brief stimuli of different lengths, ranging from 800 ms dynamic movie snippets to 100 ms static frames. We found that the disruption of temporal continuity strongly altered neural response profiles, particularly in the early onset response period of the randomized stimulus. The results suggest that models of visual system function based on discrete and randomized visual presentations may not translate well to the brains natural modes of operation.


title: A novel analgesic pathway from parvocellular oxytocin neurons to the periaqueductal gray

abstract:
The hypothalamic neuropeptide, oxytocin (OT), exerts prominent analgesic effects via central and peripheral action. Here we discovered a novel subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells being targeted by OT projections are GABAergic in nature. Both optogenetically-evoked axonal OT release in the vlPAG as well as chemogenetic activation of OTR vlPAG neurons results in a long-lasting overall increase of vlPAG neuronal activity. This then leads to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia. Finally, we describe a novel OT[->]vlPAG[->]spinal cord circuit that seems critical for analgesia in the context of both inflammatory and neuropathic pain.

Highlights- We generated a new transgenic knock-in rat line (OTR-IRES-Cre)
- A distinct parvOT neuronal population projects to vlPAG but not the SON or spinal cord
- OT excites vlPAG OTR neurons which indirectly inhibit SC WDR neurons
- This novel parvOT[->]vlPAG[->]SC pathway alleviates nociception but not the affective component of pain


title: Regional and cell type-specific afferent and efferent projections of the mouse claustrum

abstract: The claustrum (CLA) is a conspicuous subcortical structure interconnected with cortical and subcortical regions. However, its regional anatomy and cell-type-specific connections in the mouse remain largely undetermined. Here, we accurately delineated the boundary of the mouse CLA and quantitatively investigated its inputs and outputs brain-wide using anterograde and retrograde viral tracing and fully reconstructed single claustral principal neurons. At a population level, the CLA reciprocally connects with all isocortical modules. It also receives inputs from at least 35 subcortical structures but sends projections back to only a few of them. We found that cell types projecting to the CLA are differentiated by cortical areas and layers. We classified single CLA principal neurons into at least 9 cell types that innervate the diverse sets of functionally linked cortical targets. Axons of interneurons within the CLA arborize along almost its entire anteroposterior extent. Together, this detailed wiring diagram of the cell-type-specific connections of the mouse CLA lays a foundation for studying its functions.


title: Theta-phase connectivity between medial prefrontal and posterior areas underlies novel instructions implementation

abstract:
Implementing novel instructions is a complex and uniquely human cognitive ability, that requires the rapid and flexible conversion of symbolic content into a format that enables the execution of the instructed behavior. Preparing to implement novel instructions, as opposed to their mere maintenance, involves the activation of the instructed motor plans, and the binding of the action information to the specific context in which this should be executed. Recent evidence and prominent computational models suggest that this efficient configuration of the system might involve a central role of frontal theta oscillations in establishing top-down long-range synchronization between distant and task-relevant brain areas. In the present EEG study (human subjects, 30 females, 4 males), we demonstrate that proactively preparing for the implementation of novels instructions, as opposed to their maintenance, involves a strengthened degree of connectivity in the theta frequency range between medial prefrontal and motor/visual areas. Moreover, we replicated previous results showing oscillatory features associated specifically with implementation demands, and extended on them demonstrating the role of theta oscillations in mediating the effect of task demands on behavioral performance. Taken together, these findings support our hypothesis that the modulation of connectivity patterns between frontal and task-relevant posterior brain areas is a core factor in the emergence of a behavior-guiding format from novel instructions.

Significance statementEveryday life requires the use and manipulation of currently available information to guide behavior and reach specific goals. In the present study we investigate how the same instructed content elicits different neural activity depending on the task being performed. We show that medial prefrontal theta oscillations are larger when novel instructions have to be implemented, rather than simply maintained. Crucially, connectivity between medial prefrontal cortex and posterior areas is strengthened during instructions implementation, suggesting that theta oscillations play a role in setting up a dynamic and flexible network of task-relevant regions optimized for the execution of the instructed behavior.


title: Odorant receptor orthologues in conifer-feeding beetles display conserved responses to ecologically relevant odors

abstract: Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer-feeding bark beetles Ips typographus L. ( Ityp) and Dendroctonus ponderosae Hopkins ( Dpon) (Scolytinae), and the pine weevil Hylobius abietis L. ( Habi; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. Results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2-phenylethanol (2-PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2-PE, suggesting a shared evolutionary history of 2-PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer-feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm non-host plants, and 2-PE being important for intraspecific communication and/or playing a putative role in beetle-microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs.


title: Antisense modulation of IL7R splicing to control sIL7R expression in human CD4 T cells

abstract: The interleukin 7 receptor (IL7R) is strongly associated with increased risk to develop multiple sclerosis (MS), an autoimmune disease of the central nervous system, and this association is likely driven by upregulation of the soluble isoform of IL7R (sIL7R). Expression of sIL7R is determined by exclusion of the alternative exon 6 from IL7R transcripts, and our previous work revealed that the MS risk allele of the SNP rs6897932 within this exon enhances the expression of sIL7R by promoting exclusion of exon 6. sIL7R potentiates the activity of IL7, leading to enhanced expansion of T cells and increased disability in the Experimental Autoimmune Encephalomyelitis (EAE) murine model of MS. This role in modulating T cell-driven immunity positions sIL7R as an attractive therapeutic target whose expression could be reduced for treatment of MS or increased for treatment of cancers. In this study we identified novel antisense oligonucleotides (ASOs) that effectively control the inclusion (anti-sIL7R ASOs) or exclusion (pro-sIL7R ASOs) of this exon in a dose-dependent fashion. These ASOs provided excellent control of exon 6 splicing and sIL7R secretion in human primary CD4+ T cells. Supporting their potential for therapeutic targeting, we showed that lead anti-sIL7R ASOs correct the enhanced exon 6 exclusion imposed by the MS risk allele of rs6897932, whereas lead pro-sIL7R ASOs phenocopy it. The data presented here form the foundation for future pre-clinical studies that will test the therapeutic potential of these ASOs in MS and immuno-oncology.

category: immunology


title: SARS-CoV-2 peptide vaccine elicits T-cell responses in mice but does not protect against infection or disease

abstract: There is significant interest in T-cell mediated immunity against SARS-CoV-2. Both vaccination and infection have been observed to elicit durable T-cell responses against the virus. The classical role of CD4+ T-cell responses in coordinating humoral immunity is well understood but it is less clear to what degree, if any, T-cell responses play a direct protective role against infection In this study we vaccinated BALB/c mice with peptides derived from the SARS-CoV-2 proteome designed to either elicit T-cell responses or B-cell responses against linear epitopes. These peptides were administered in combination with either of two adjuvants, poly(I:C) and the STING agonist BI-1387466. Both adjuvants consistently elicited responses against the same peptides, preferentially from the group selected for predicted T-cell immunogenicity. The magnitude of T-cell responses was, however, significantly higher with BI-1387466 compared with poly(I:C). Neither adjuvant group, however, provided any protection against infection with the murine adapted virus SARS-CoV-2-MA10 or from disease following infection.


title: Fate-resolved gene regulatory signatures of individual B lymphocytes in the early stages of Epstein-Barr Virus infection

abstract: Epstein-Barr Virus (EBV) infection of B lymphocytes elicits diverse host responses via complex, well-adapted transcriptional control dynamics. Consequently, this host-pathogen interaction provides a powerful system to explore fundamental cellular processes that contribute to consensus fate decisions including cell cycle arrest, apoptosis, proliferation, and differentiation. Here we capture these responses and fates with matched single-cell transcriptomics and chromatin accessibility, from which we construct a genome-wide multistate model of early infection dynamics. Notably, our model captures a previously uncharacterized EBV+ analog of a multipotent activated precursor state that can yield early memory B cells. We also find that a marked global reduction in host chromatin accessibility occurs during the first stages of infection in subpopulations of EBV+ cells that display senescent and pre-apoptotic hallmarks induced by innate antiviral sensing and proliferation-linked DNA damage. However, cells in proliferative infection trajectories exhibit greater accessibility at select host sites linked to B cell activation and survival genes as well as key regions within the viral genome. To further investigate such loci, we implement a bioinformatic workflow (crisp-ATAC) to identify phenotype-resolved regulatory signatures. This customizable method applies user-specified logical criteria to produce genome-wide single-cell ATAC-and ChIP-seq range intersections that are used as inputs for cis-linkage prediction and ontology tools. The resulting tri-modal data yield exquisitely detailed hierarchical perspectives of the transforming regulatory landscape during critical stages of an oncogenic viral infection that simulates antigen-induced B cell activation and differentiation. We anticipate these resources will guide investigations of gene regulatory modules controlling EBV-host dynamics, B cell effector fates, and lymphomagenesis. To demonstrate the utility of this resource, this work concludes with the discovery of EBV infection dynamics in FCRL4+ / TBX21+ Tissue-Like Memory B cells, an unconventional subset with notable associations to numerous immune disorders.


title: The extracellular sialidase NEU3 induces neutrophil priming

abstract: Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have four sialidases, but their biological functions are unclear. In this report, we show that incubation of human neutrophils with the human sialidase NEU3, but not NEU1, NEU2 or NEU4, inducess human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed neutrophil markers CD66, CD11B, and CD18 to localize to the cell cortex, and decreases the localization of the unprimed neutrophil markers CD43 and CD62L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.


title: A novel lipase with dual localisation in Trypanosoma brucei

abstract: Phospholipases are esterases involved in lipid catabolism. In pathogenic micro-organisms (bacteria, fungi, parasites) they often play a critical role in virulence and pathogenicity. A few phospholipases (PL) have been characterised so far at the gene and protein level in unicellular parasites including African trypanosomes (AT). They could play a role in different processes such as host-pathogen interaction, antigenic variation, intermediary metabolism. By mining the genome database of AT we found putative new phospholipase candidate genes and here we provided biochemical evidence that one of these has lypolytic activity. This protein has a unique non-canonical glycosome targeting signal responsible for its dual localisation in the cytosol and the peroxysomes-related organelles named glycosomes. We also show that this new phospholipase is excreted by these pathogens and that antibodies directed against this protein are generated during an experimental infection with T. brucei gambiense, a subspecies responsible for infection in humans. This feature makes this protein a possible tool for diagnosis.

category: microbiology


title: Ivermectin does not protect against SARS-CoV-2 infection in the Syrian hamster model

abstract: Ivermectin, an FDA-approved antiparasitic drug, has been reported to have in vitro activity against SARS-CoV-2. An increasing off-label use of Ivermectin for COVID-19 has been reported. We here assessed the effect of Ivermectin in Syrian hamsters infected with the SARS-CoV-2 Beta (B.1.351) variant. Infected animals received a clinically relevant dose of Ivermectin (0.4 mg/kg subcutaneously dosed) once daily for four consecutive days after which the effect was quantified. Ivermectin monotherapy did not reduce lung viral load and even significantly worsened the SARS-CoV-2-induced lung pathology. Additionally, it did not potentiate the activity of Molnupiravir (Lagevrio) when combined with this drug. This study contributes to the growing body of evidence that Ivermectin does not result in a beneficial effect in the treatment of COVID-19. These findings are important given the increasing, dangerous off-label use of Ivermectin for the treatment of COVID-19.


title: Molnupiravir (MK-4482) is efficacious against Omicron and other SARS-CoV-2 variants in the Syrian hamster COVID-19 model

abstract:
The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC) containing a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here we assessed the efficacy of MK-4482 against the earlier Alpha, Beta and Delta VOCs and Omicron in the Syrian hamster COVID-19 model. Omicron replication and associated lung disease in vehicle treated hamsters was reduced compared to the earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of Alpha, Beta and Delta VOC infected hamsters. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious virus titers compared to viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.

One Sentence SummaryMK-4482 inhibits replication of multiple SARS-CoV-2 variants of concern, including Omicron, in the Syrian hamster COVID-19 model


title: Metamorphism of catalytic domain controls transposition in Tn3 family transposases

abstract: Transposons account for a remarkable diversity of mobile genetic elements that play the role of genome architects in all domains of life. Tn3 is a family of widespread and among first identified bacterial transposons notorious for their contribution to dissemination of antibiotic resistance. Transposition within this family is mediated by a large TnpA transposase facilitating both transposition and target immunity. The structural framework for understanding the mechanism of the TnpA transposition is however absent. Here, we describe the cryo-EM structures of TnpA from Tn4430 in apo form and paired with transposon ends. We show that TnpA has a unique architecture and exhibits a family-specific regulation mechanism involving metamorphic refolding of the RNase H-like catalytic domain. The TnpA structure, constrained by a double dimerization interface, creates a unique topology that suggests a specific role for target DNA in transpososome assembly, activation, and target immunity.

category: molecular biology


title: Melanin Nanoparticles as an Alternative to Natural Melanin in Retinal Pigment Epithelium Cells and Their Therapeutic Effects against Age-Related Macular Degeneration

abstract:
Melanin is a natural pigment that is widely distributed in many parts of the human body, such as the skin and retinal pigment epithelium (RPE) in eyes. In contrast to skin melanin, which is being constantly synthesized by the epidermal melanocytes, melanin in the RPE does not regenerate. Melanin is known to function as a potential radical scavenger and photoprotective agent. However, the protective effects of melanin against oxidative stress decline with increasing age. This phenomenon has been significantly correlated with the pathogenesis of age-related macular degeneration (AMD). To increase the potential antioxidant and photoprotective characteristics of the melanin, we designed a new strategy by replenishment of melanin using PEGylated-synthetic melanin nanoparticles (MNPs) in the RPE for the treatment of AMD. We performed experiments using AMD-like cellular and mouse models and demonstrated that MNPs are safe, biocompatible, and selectively target reactive oxygen species (ROS) with powerful antioxidant properties. MNPs can traffic and accumulate in the RPE and are exclusively located in cytosol, but not the nucleus and mitochondria of the cells, for up to three months after a single-dose intravitreal (IVT) injection. Our findings demonstrate, for the first time, that MNPs are able to substitute for natural melanin in the RPE of eyes and suggest the potential efficacy of MNPs as a natural radical scavenger against oxidative stress in ROS-related diseases, such as AMD.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/481315v2_ufig1.gif" ALT="Figure 1">
View larger version (26K):
org.highwire.dtl.DTLVardef@18929e6org.highwire.dtl.DTLVardef@1dc929org.highwire.dtl.DTLVardef@181c8c2org.highwire.dtl.DTLVardef@1a36eab_HPS_FORMAT_FIGEXP  M_FIG C_FIG Intracellular trafficking showing that PEGylated-synthetic melanin nanoparticles (MNPs) reach their target and are stable in the retinal pigment epithelial cells for up to 3 months via a single-dose intravitreal (IVT) injection.


title: Single-cell analysis of skeletal muscle macrophages reveals age- associated functional subpopulations

abstract: Tissue-resident macrophages represent a group of highly responsive innate immune cells that acquire diverse functions by polarizing towards distinct subgroups. The subgroups of macrophages that reside in skeletal muscle (SKM) and their changes during aging are poorly characterized. By single-cell transcriptomic analysis, we found that mouse SKM macrophages primarily comprise two large populations, "healing" LYVE1+ and "proinflammatory" LYVE1-macrophages. SKM macrophages were further classified into four functional subgroups based on the expression levels of another cell-surface marker, MHCII: LYVE1+/MHCII-lo (similar to alternatively activated M2), LYVE1-/MHCII-hi (similar to classically activated M1), and two new subgroups, LYVE1+/MHCII-hi and LYVE1-/MHCII-lo. Notably, the new subgroup LYVE1+/MHCII-hi had traits of both M2 and M1 macrophages, while the other new subgroup, LYVE1-/MHCII-lo, expressed high levels of mRNAs encoding cytotoxicity proteins. Flow cytometric analysis validated the presence of the four macrophage subgroups in SKM. In old SKM, LYVE1-macrophages were more abundant than LYVE1+ macrophages. Furthermore, complementary unsupervised classification revealed the emergence of specific macrophage subclusters expressing abundant proinflammatory markers, including S100a8 and S100a9 in aged SKM. In sum, our study has identified dynamically polarized mouse SKM macrophages and further uncovered the contribution of specific macrophage subpopulations to the proinflammatory status in old SKM.

category: cell biology


title: Condensin II is required for efficient Spindle Assembly Checkpoint activation in Drosophila male meiosis

abstract: Reductional nuclear division in meiosis is essential for diploid life. A fundamental event in meiosis is chromatin condensation, through mechanisms not yet fully understood. Current data suggest that Condensins are key players in building and sustaining mitotic and meiotic chromosome structure. In Drosophila, Condensin II appears to be dispensable for faithful mitosis in somatic tissues yet essential in the germline. Previous work has demonstrated that in Drosophila male meiosis, Condensin II is required for the segregation of homologous chromosomes into distinct territories during prophase I, possibly through the resolution of chromosomal intertwines. Here we show that in addition to this well-established function in meiotic chromatin assembly, Condensin II is required for robust Spindle Assembly Checkpoint (SAC) signaling in male meiosis. In the absence of Condensin II, spermatocytes undergo faster meiotic divisions and display reduced ability to prolong meiosis in the presence of spindle poisons. This is attributed to the inability to recruit a key SAC component (Mad1) to the kinetochore. Importantly, we demonstrate that the absence of a robust SAC response in Condensin II mutants, and consequent accelerated meiosis, is a strong contributor to the meiotic defects associated with these mutants. We show that artificial prolongation of meiotic divisions, using conditions that delay anaphase onset in a SAC-independent manner, is sufficient to rescue segregation defects and aneuploidy associated with Condensin II mutations. We therefore conclude that Condensin II can be dispensable for the resolution of topological problems and chromosome condensation if cells are able to prolong meiosis. Yet, the newly found role of this complex in the robustness of the SAC reduces meiotic timing leading to severe chromosome segregation defects.


title: 222-nm far UVC exposure results in DNA damage and transcriptional changes to mammalian cells.

abstract: Ultraviolet (UV) germicidal tools have recently gained attention as a disinfection strategy against the COVID-19 pandemic but the safety profile arising from their exposure have been controversial and impeded larger scale implementation. We compare the emerging 222-nm far UVC and 277-nm UVC LED disinfection modules with the traditional UVC mercury lamp emitting at 254 nm to understand their effects on human retinal cell line ARPE-19 and HEK-A keratinocytes. Cells illuminated with 222-nm far UVC survived while those treated with 254-nm and 277-nm wavelengths underwent apoptosis via JNK/ATF2 pathway. However, cells exposed to 222-nm far UVC presented the highest degree of DNA damage as evidenced by yH2AX staining. Globally, these cells presented transcriptional changes in cell cycle and senescence pathways. Thus, the introduction of 222-nm far UVC lamps for disinfection purposes should be carefully considered and designed with the inherent dangers involved.


title: Vitronectin mediates survival of human WJ-MSCs under inflammatory temperature stress via cell cycle arrest.

abstract:
Due to their anti-inflammatory and immunomodulatory capabilities, mesenchymal stem cells (MSCs) are being widely used in cell-based therapies for the treatment of a wide spectrum of inflammatory disorders. Despite their promises, substantial cell loss post transplantation leads to compromised therapeutic benefits in clinical trials, which remains a challenge to overcome. Inflammatory microenvironment comprises the presence of pro-inflammatory cytokines, elevated temperature, etc., which could hamper MSC viability following transplantation. Thus, identifying the underlying molecular factors controlling survival mechanism under such stress conditions, thereby, improving MSC survival becomes important for optimising MSC-based therapy. Also, since MSCs from different origins have significantly varied biology, choosing the appropriate MSC source could be crucial in determining the fate of transplanted MSCs in stressful milieu.

As extracellular matrix (ECM) components can mediate cell survival signals, in the present study, we have evaluated the role of ECM matricellular protein, vitronectin (VTN), in the survival of human Whartons Jelly MSCs (WJ-MSCs) under the condition of inflammatory temperature stress. On exposure to 40{degrees}C, WJ-MSCs underwent cell cycle arrest with no significant change in viability status, along with an induction in VTN expression both at mRNA and protein levels. Interestingly, inhibition of pro-survival signalling pathways, ERK or PI3K, at 40{degrees}C led to further upregulation in VTN expression without any significant impact on viability or cell cycle arrest status. However, on knocking down VTN in WJ-MSCs at 40{degrees}C, decrease in viable population along with reversal of cell cycle arrest were noted. Moreover, inhibition of pro-survival pathways ERK or PI3K, in VTN knocked down WJ-MSCs at 40{degrees}C, led to a dramatic reduction in the viable population accompanied with reversal in cell cycle arrest.

Altogether, our findings highlighted the protective role of VTN in mediating survival of WJ-MSCs under inflammatory temperature stress via adopting cell cycle arrest mechanism.


title: Using dilution grazing assays to measure size fractionated phytoplankton mortality rates across the freshwater-to-marine continuum

abstract: There is a need for a unified grazing method that can be used across the freshwater-to-marine continuum. To accomplish this, this research utilized dilution grazing assays across the freshwater-to-marine continuum and across the oligotrophic-to-hypereutrophic gradient by measuring size fractions of dilution-based mortality. This was investigated by using 0.7 {micro}m and 0.2 {micro}m prefiltered water and major ion solutions (MIS) as diluent media for use in the Landry-Hassett grazing bioassays run in lake, river, estuarine (riverine and lagoonal), and oceanic shelf systems. Because MIS does not include vitamins that would be in prefiltered natural water, vitamin effects on grazing rate determination were also investigated for the MIS bioassays. Results show that the dilution grazing method can be broadly applied across the freshwater-to-marine continuum and across trophic gradients.

category: ecology


title: PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression

abstract: Autosomal dominant polycystic kidney disease (ADPKD), among the most common human genetic conditions and a frequent etiology of kidney failure, is primarily caused by heterozygous PKD1 mutations. Kidney cyst formation ensues when the PKD1 dosage falls below a critical threshold. However, no framework exists to harness the remaining allele or reverse PKD1 decline. Here, we show that mRNAs produced by the noninactivated PKD1 allele are cis-repressed via their 3-UTR miR-17 binding element. Eliminating this motif (Pkd1{Delta}17) improves mRNA stability, raises Polycystin-1 levels, and alleviates cyst growth in cellular, ex vivo, and mouse PKD models. Remarkably, Pkd2 is also autoinhibited via its 3-UTR miR-17 motif, and Pkd2{Delta}17-induced Polycystin-2 derepression partly compensates and retards cyst growth in Pkd1-mutant models. Moreover, acutely blocking Pkd1/2 cis-inhibition, including after cyst onset, attenuates murine PKD. Finally, PKD1{Delta}17 or PKD2{Delta}17 alleles revert cyst-pathogenic sequala in patient-derived primary ADPKD cultures. Thus, evading 3-UTR cis-interference and enhancing PKD1/2 mRNA translation is a potentially mutation-agnostic ADPKD-arresting approach.

category: genetics


title: High nucleotide similarity of three Copia lineage LTR retrotransposons among plant genomes

abstract:
Transposable elements (TEs) are mobile genetic elements found in the majority of eukaryotic genomes. Because of their mobility in the host genome, TEs can deeply impact the structure and evolution of chromosomes and can induce mutations affecting coding genes. In response to these potential threats, host genomes use various processes to repress the TE expression, leading to an arm-race between TEs for their persistence and host genomes for their protection. In plants, the major group of TEs is the Long Terminal Repeats retrotransposons (LTR-RT). They are classified into superfamilies (Gypsy, Copia) and sub-classified into lineages according to similarities, structures and presence of coding domains. Among the different ways LTR-RTs can proliferate, horizontal transfer (HT), defined as the nonsexual transmission of nuclear and plastid genetic material between species, is a process allowing LTR-RTs to invade a new genome. Although this phenomenon was considered rare in eukaryotic organisms, recent studies demonstrate numerous potential transfers of LTR-RTs, suggesting that HT may be more frequent than initially estimated.

This study aims to determine which LTR-RT lineages are shared with high similarity among 69 reference genomes that represent the major groups of green plants. We first identified and classified 88,450 LTR-RTs and determined 143 cases of high similarities between pairs of genomes. Most of them involved three Copia lineages (Oryco/Ivana, Retrofit/Ale and Tork/Tar/Ikeros) and very few of them included the Gypsy superfamily. Interestingly, a detailed analysis of three high similarities involving the Tork/Tar/Ikeros group of lineages indicates a patchy distribution of the elements and phylogenetic incongruities, indicating they originated from potential HTs. Overall, our results demonstrate that three specific lineages of Copia share outstanding similarity between very distant species and may probably be involved in horizontal transfer mechanisms.

category: genomics


title: Convergence of monosynaptic inputs from neurons in the brainstem and forebrain on parabrachial neurons that project to the paraventricular nucleus of the thalamus

abstract: The paraventricular nucleus of the thalamus (PVT) projects to areas of the forebrain involved in behavior. Homeostatic challenges and salient cues activate the PVT and evidence shows that the PVT regulates appetitive and aversive responses. The brainstem is a source of afferents to the PVT and the present study was done to determine if the lateral parabrachial nucleus (LPB) is a relay for inputs to the PVT. Retrograde tracing experiments with cholera toxin B (CTB) demonstrate that the LPB contains more PVT projecting neurons than other regions of the brainstem including the catecholamine cell groups. The hypothesis that the LPB is a relay for brainstem signals to the PVT was assessed using an intersectional monosynaptic rabies tracing approach. Sources of inputs included the reticular formation, periaqueductal gray (PAG), nucleus cuneiformis, superior and inferior colliculi, and the LPB. Distinctive clusters of input cells to LPB-PVT projecting neurons were also found in the dorsolateral bed nucleus of the stria terminalis (BSTDL) and the lateral central nucleus of the amygdala (CeL). Anterograde viral tracing demonstrates that LPB-PVT neurons densely innervate all of the PVT in addition to providing collateral innervation to the preoptic area, lateral hypothalamus, zona incerta and PAG but not the BSTDL and CeL. The paper discusses the anatomical evidence that suggests that the PVT is part of a network of interconnected neurons involved in arousal, homeostasis, and the regulation of behavioral states with forebrain regions potentially providing descending modulation or gating of signals relayed from the LPB to the PVT.

category: animal behavior and cognition


title: Neuromuscular embodiment of feedback control elements in Drosophila flight

abstract: While insects like Drosophila are flying, aerodynamic instabilities require that they make millisecond-timescale adjustments to their wing motion to stay aloft and on course. These stabilization reflexes can be modeled as a proportional-integral (PI) controller; however, it is unclear how such control might be instantiated in insects at the level of muscles and neurons. Here, we show that the b1 and b2 motor units--prominent components of the flys steering muscles system--modulate specific elements of the PI controller: the angular displacement (integral, I) and angular velocity (proportional, P), respectively. Moreover, these effects are observed only during the stabilization of pitch. Our results provide evidence for an organizational principle in which each muscle contributes to a specific functional role in flight control, a finding that highlights the power of using top-down behavioral modeling to guide bottom-up cellular manipulation studies.


title: Distinct glycerophospholipids potentiate Gsα-activated adenylyl cyclase activity

abstract: Nine mammalian adenylyl cyclases (AC) are pseudoheterodimers with two hexahelical membrane domains which are isoform-specifically conserved. Previously we proposed that these membrane domains are orphan receptors (10.7554/eLife.13098; 10.1016/j.cellsig.2020.109538). Lipids extracted from fetal bovine serum at pH 1 inhibited several mAC activities. Guided by a lipidomic analysis we tested glycerophospholipids as potential ligands. Contrary to expectations we surprisingly discovered that 1-stearoyl-2-docosahexaenoyl-phosphatidic acid (SDPA) potentiated Gs-activated activity of human AC isoform 3 seven-fold. The specificity of fatty acyl esters at glycerol positions 1 and 2 was rather stringent. 1-Stearoyl-2-docosahexaenoyl-phosphatidylserine and 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine significantly potentiated several Gs-activated mAC isoforms to different extents. SDPA appears not interact with forskolin activation of AC isoform 3. SDPA enhanced Gs-activated AC activities in membranes from mouse brain cortex. The action of SDPA was reversible. Unexpectedly, SDPA did not affect cAMP generation in HEK293 cells stimulated by isoproterenol, PGE2 and adenosine, virtually excluding a role as an extracellular ligand and, instead, suggesting an intracellular role. In summary, we discovered a new dimension of intracellular AC regulation by chemically defined glycerophospholipids.

category: biochemistry


title: mRNA Treatment Rescues Niemann-Pick Disease Type C1 in Patient Fibroblasts

abstract: Messenger RNA (mRNA) holds great potential as a disease-modifying treatment for a wide array of monogenic disorders. Niemann-Pick disease type C1 (NP-C1) is an ultra-rare monogenic disease that arises due to loss-of-function mutations in the NPC1 gene, resulting in the entrapment of unesterified cholesterol in the lysosomes of affected cells and a subsequent reduction in their capacity for cholesterol esterification. This causes severe damage to various organs including the brain, liver, and spleen. In this work, we describe the use of NPC1-encoded mRNA to rescue the protein insufficiency and pathogenic phenotype caused by biallelic NPC1 mutations in cultured fibroblasts derived from an NP-C1 patient. We first evaluated engineering strategies for the generation of potent mRNAs capable of eliciting high protein expression across multiple cell types. We observed that "GC3" codon optimization, coupled with N1-methylpseudouridine base modification, yielded an mRNA that was approximately a thousand-fold more potent than wildtype, unmodified mRNA in a luciferase reporter assay, and consistently superior to other mRNA variants. Our data suggest that the improved expression associated with this design strategy was due in large part to the increased secondary structure of the designed mRNAs. Both codon optimization and base modification appear to contribute to increased secondary structure. Applying these principles to the engineering of NPC1-encoded mRNA, we observed a normalization in NPC1 protein levels after mRNA treatment, as well as a rescue of the mutant phenotype. Specifically, mRNA treatment restored the cholesterol esterification capacity of patient cells to wildtype levels, and induced a significant reduction in both unesterified cholesterol levels (>57% reduction compared to Lipofectamine-treated control in a cholesterol esterification assay) and lysosome size (157 m2 reduction compared to lipofectamine-treated control). These findings show that engineered mRNA can correct the deficit caused by NPC1 mutations. More broadly, they also serve to further validate the potential of this technology to correct diseases associated with loss-of-function mutations in genes coding for large, complex, intracellular proteins.


title: SEQUIN: rapid and reproducible analysis of RNA-seq data in R/Shiny

abstract: SEQUIN is a new web application (app) that allows fast and intuitive RNA-sequencing data analysis for organisms, tissues, and single cells. Integrated app functions enable uploading datasets, quality control, gene set enrichment, data visualization, and differential gene expression analysis. We also present the iPSC Profiler, a practical tool that helps to measure pluripotency and cell differentiation. Freely available to the public, SEQUIN empowers scientists to investigate transcriptome data firsthand with cutting edge statistical methods.

category: bioinformatics


title: Spatiotemporal growth pattern during plant nutation implies fast dynamics for cell wall mechanics and chemistry: a multiscale study in Averrhoa carambola

abstract:
Nutation is the most striking and ubiquitous example of the rhythmic nature of plant development. Although there is a consensus that this wide oscillatory motion is driven by growth, its internal mechanisms have not been fully elucidated yet. In this work, we study the specific case of nutation in compound leaves in the archetypal Averrhoa carambola plant. We quantify the macroscopic growth kinematics with time lapse imaging, image analysis and kinematics modeling. We further characterize the mechanical and chemical properties of the cell wall with atomic force microscopy and immunolabelling. Our data first reveal that the differential growth driving nutation is localized and peaks where the average growth drops. We then show this specific spatiotemporal growth profile is compatible with local contraction events. At the cell wall level, differential growth is further colocalized with an asymmetry of the cell wall elastic modulus, and with an asymmetric distribution of homogalacturonans (HG). Our results not only back up the hypothesis of HG being involved in plant growth, but also build up on it by suggesting a dynamic nature for this process.

Significance StatementNutation is an oscillatory motion displayed by many organs of growing plants. Most works on nutation focus on its relation to external stimuli attempting to explain its origins. By contrast, its internal physiological mechanisms remain to be fully explored. Here we propose an experimental and multiscale characterization of undisturbed nutation. We determine the macroscopic growth profile and show it is compatible with cell expansion but also local contractions in the tissues. At the microscopic level, we reveal that both the rigidity and composition of the cell wall are asymmetrically distributed where nutation occurs. The combination of results on both scales brings contributions to the understanding of interplay between global movement, local growth, cell wall mechanics and cell wall biochemistry.


title: The structural context of PTMs at a proteome wide scale

abstract: The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry (MS)-based proteomics has identified and quantified tens of thousands of post-translational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, unstructured regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis which revealed examples of spatial co-regulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.


title: A Neural Model of Task Compositionality with Natural Language Instructions

abstract: AO_SCPLOWBSTRACTC_SCPLOWWe present neural models of one of humans most astonishing cognitive feats: the ability to interpret linguistic instructions in order to perform novel tasks with just a few practice trials. Models are trained on a set of commonly studied psychophysical tasks, and receive linguistic instructions embedded by transformer architectures pre-trained on natural language processing. Our best performing models can perform an unknown task with a performance of 80% correct on average based solely on linguistic instructions (i.e. 0-shot learning), and 90% after 3 learning updates. We found that the resulting neural representations capture the semantic structure of interrelated tasks even for novel tasks, allowing for the composition of practiced skills in unseen settings. Finally, we also demonstrate how this model can generate a linguistic description of a task it has identified using motor feedback, which, when communicated to another network, leads to near perfect performance (95%). To our knowledge, this is the first experimentally testable model of how language can structure sensorimotor representations to allow for task compositionality.


title: Functional definition of thyroid hormone response elements based on a synthetic STARR-seq screen

abstract: When bound to thyroid hormone, the nuclear receptor TR1 activates the transcription of a number of genes in many cell types. It mainly acts by binding DNA as an heterodimer with RXRs at specific response elements related to the DR4 consensus sequence. However, the number of DR4-like elements in the genome exceed by far the number of occupied sites, indicating that minor variations in nucleotides composition deeply influence the DNA-binding capacity and transactivation activity of TR1. An improved protocol of synthetic STARR-seq was used to quantitatively assess the transcriptional activity of thousands of synthetic sites in parallel. This functional screen highlights a strong correlation between the affinity of the heterodimers for DNA and their capacity to mediate the thyroid hormone response.


title: Recombination smooths the time-signal disrupted by latency in within-host HIV phylogenies

abstract: Within-host HIV evolution involves latency and re-activation of integrated provirus that has the potential to disrupt the temporal signal induced by the evolutionary race between host immune responses and viral evolution. Yet, within-host HIV phylogenies tend to show clear, ladder-like trees structured by the time of sampling. Recombination complicates this dynamic by allowing latent HIV viruses to re-integrate as fragments in the genomes of contemporary virus populations. That is, recombination violates the fundamental assumption made by the phylogenetic methods typically used to study within-host HIV sequence data that evolutionary history can be represented by a single bifurcating tree. In this paper we develop a coalescent-based simulator of within-host HIV evolution that includes, latency, recombination, and population dynamics that allows us to study the relationship between the true, complex genealogy of within-host HIV, encoded as an Ancestral Recombination Graph (ARG), and the observed phylogenetic tree. We show how recombination recovers the disruption of the temporal signal of within-host HIV evolution caused by latency by mixing fragments of ancestral, latent genomes into the contemporary population through recombination. In effect, recombination averages over extant heterogeneity, whether it stems from mixed time-signals or population bottlenecks. Further, we establish that the signals of latency and recombination can be observed in phylogenetic trees despite being an incorrect representation of the true evolutionary history. Using an Approximate Bayesian Computation method, we develop a set of statistical probes to tune our simulation model to nine longitudinally-sampled within-host HIV phylogenies, finding evidence for recombination rates at the lower end of published estimates and relatively small latent pool sizes ranging from about 1000 to 2500 cells.

category: evolutionary biology


title: Phylogenetic Network Dissimilarity Measures That Take Branch Lengths Into Account

abstract: Dissimilarity measures for phylogenetic trees have long been used for analyzing inferred trees and understanding the performance of phylogenetic methods. Given their importance, a wide array of such measures have been developed, some of which are based on the tree topologies alone, and others that also take branch lengths into account. Similarly, a number of dissimilarity measures of phylogenetic networks have been developed in the last two decades. However, to the best of our knowledge, all these measures are based solely on the topologies of phylogenetic networks and ignore branch lengths. In this paper, we propose two phylogenetic network dissimilarity measures that take both topology and branch lengths into account. We demonstrate the behavior of these two measures on pairs of related networks. Furthermore, we show how these measures can be used to cluster a set of phylogenetic networks obtained by an inference method, illustrating this application on the posterior sample of phylogenetic networks. Both measures are implemented in the publicly available software package PhyloNet.


title: Early identification of cooperative fragments for protein-protein interaction stabilization

abstract: Modulating protein-protein interactions (PPIs) is an effective approach to drug discovery, with several drugs in the clinic that inhibit PPIs. The orthogonal approach of PPI stabilization has developed slowly, a function of the complicated dynamics of multi-component protein complexes. In contrast to PPI inhibition, where ligand affinity is the driving parameter for efficacy, cooperativity is frequently the directing variable for PPI stabilization. Here we show how STD NMR allows for early-stage detection of cooperativity using the hub protein 14-3-3, a focused library of fragments and several 14-3-3 partner proteins. Further, we validate that the observed enhancement in STD signal is a function of cooperativity of the ternary 14-3-3 complex, using mutagenesis and X-ray crystallography. Additionally, we assess the differential cooperativity of three fragments in a panel of 14-3-3 interaction partners. Finally, we demonstrate how selective 14-3-3 complex formation is a function of cooperativity effects


title: Fuzzy Supertertiary Interactions within PSD-95 Enable Ligand Binding

abstract: The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by PDZ3, SH3 and GuK domains, regulate PSD-95 activity. Here we combined Discrete Molecular Dynamics and single molecule FRET to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand.


title: Sporulation activated via σW protects Bacillus from a Tse1 peptidoglycan hydrolase T6SS effector

abstract: Bacterial communities constantly interact with various community members employing diverse offensive and defensive tools to reach coexistence. The extracellular matrix and sporulation are defensive mechanisms used by Bacillus cells when they interact with Pseudomonas strains expressing a type VI secretion system (T6SS). Here, we define Tse1 as the main toxin mobilized by the Pseudomonas T6SS that triggers sporulation in Bacillus. We characterize Tse1 as a peptidoglycan hydrolase that indirectly alters the dynamics and functionality of the Bacillus cell membrane. We also delineate the response of Bacillus cells to Tse1, which through the coordinated actions of the extracellular sigma factor {sigma}W and the cytoplasmic histidine kinases KinA and KinB, culminates in activation of the sporulation cascade. We propose that this cellular developmental response is conserved in Bacilli to defend against the toxicity of T6SS-mobilized Tse1 effector.


title: Neutral sphingomyelinase 1 regulates cellular fitness at the level of ER stress and cell cycle

abstract: Neutral sphingomyelinase 1 (nSMase1) belongs to the sphingomyelinase enzyme family that hydrolyzes sphingomyelin to produce signaling active lipid ceramide and phosphorylcholine. The molecular characterization and biological function of nSMase1 remain poorly studied. Here, we report that nSMase1 (gene name: SMPD2) knockdown reduces LAMP1 at the mRNA levels and is required for initiating a full-potential unfolded protein response under ER stress. Additionally, SMPD2 KD dramatically reduces the global protein translation rate. We further show that SMPD2 KD cells are arrested in the G1 phase of the cell cycle and that two important cell cycle regulating processes - PI3K/Akt pathway and Wnt signaling pathway are altered. Taken together, we propose a role for nSMase1 in buffering ER stress and modulating cellular fitness via cell cycle regulation.


title: Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

abstract: Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.


title: Complex effects of chytrid parasites on the growth of the cyanobacterium Planktothrix rubescens across interacting temperature and light gradients

abstract: Chytrids are important drivers of aquatic ecosystems as phytoplankton parasites. The interaction between these parasites and their hosts are shaped by abiotic factors such as temperature and light. Here, we performed a full-factorial experiment to study how temperature and light interact to affect the dynamics of the bloom-forming toxic cyanobacterium Planktothrix rubescens and its chytrid parasite. We used a dynamic host-parasite model to explore how temperature and light affect long term dynamics. At low temperatures, chytrids do not survive. Higher light and temperature levels stimulated both phytoplankton and chytrid growth, with complex effects on their dynamics. Model exploration indicates that increasing temperature and light shifts equilibrium outcomes from P. rubescens persisting alone to stable coexistence and then to limit cycles. This provides an alternative biological explanation for why P. rubescens is mainly found in the relatively cold and dark lake metalimnion - it may enable avoidance of its parasite. Our study emphasizes the importance of investigating how abiotic factors interact with biotic interactions to drive complex outcomes.


title: QTL analysis reveals an oligogenic architecture of a rapidly adapting trait during the European invasion of common ragweed

abstract: Biological invasions offer a unique opportunity to investigate evolution over contemporary time-scales. Rapid adaptation to local climates during range expansion can be a major determinant of invasion success, yet fundamental questions remain about its genetic basis. This study sought to investigate the genetic basis of climate adaptation in invasive common ragweed (Ambrosia artemisiifolia). Flowering time adaptation is key to this annual species invasion success, so much so that it has evolved repeated latitudinal clines in size and phenology across its native and introduced ranges despite high gene flow among populations. Here, we produced a high-density linkage map (4,493 SNPs) and paired this with phenotypic data from an F2 mapping population (n=336) to identify one major and two minor quantitative trait loci (QTL) underlying flowering time and height differentiation in this species. Within each QTL interval, several candidate flowering time genes were also identified. Notably, the major flowering time QTL detected in this study was found to overlap with a previously identified haploblock (putative inversion). Multiple genetic maps of this region identified evidence of suppressed recombination in specific genotypes, consistent with inversions. These discoveries support the expectation that a concentrated genetic architecture with fewer, larger and more tightly-linked alleles should underlie rapid local adaptation during invasion, particularly when divergently-adapting populations experience high-levels of gene flow.


title: PanGraph: scalable bacterial pan-genome graph construction

abstract: The genomic diversity of microbes is commonly parameterized as population genetic polymorphisms relative to a reference genome of a well-characterized, but arbitrary, isolate. Reference genomes contain a fraction of the microbial pangenome, the set of genes observed within all isolates of a given species, and are thus blind to both the dynamics of the accessory genome, as well as variation within gene order and copy number. With the wide-spread usage of long-read sequencing, the number of high-quality, complete genome assemblies has increased dramatically. Traditional computational approaches towards whole-genome analysis either scale poorly, or treat genomes as dissociated bags of genes, and thus are not suited for this new era. Here, we present PanGraph, a Julia based library and command line interface for aligning whole genomes into a graph, wherein each genome is represented as an undirected path along vertices, which in turn, encapsulate homologous multiple sequence alignments. The resultant data structure succinctly summarizes population-level nucleotide and structural polymorphisms and can be exported into a several common formats for either downstream analysis or immediate visualization.


title: Three membrane fusion pore families determine the pathways of pore dilation

abstract:
During exocytosis secretory vesicles fuse with a target membrane and release neurotransmitters, hormones or other bioactive molecules through a membrane fusion pore. The initially small pore may subsequently dilate for full contents release, as commonly observed in amperometric traces. The size, shape and evolution of the pore is critical to the course of contents release, but exact fusion pore solutions accounting for membrane tension and bending energy constraints have not been available. Here we obtained exact solutions for fusion pores between two membranes. We find three families: a narrow pore, a wide pore and an intermediate tether-like pore. For high tensions these are close to the catenoidal and tether solutions recently reported for freely hinged membrane boundaries. We suggest membrane fusion initially generates a stable narrow pore, and the dilation pathway is a transition to the stable wide pore family. The unstable intermediate pore is the transition state that sets the energy barrier for this dilation pathway. Pore dilation is mechanosensitive, as the energy barrier is lowered by increased membrane tension. Finally, we study fusion pores in nanodiscs, powerful systems for the study of individual pores. We show that nanodiscs stabilize fusion pores by locking them into the narrow pore family.

SignificanceDuring neurotransmission, hormone release and other fundamental processes, secretory vesicles fuse their membranes with target membranes to release contents through an initially small membrane fusion pore that subsequently dilates. Dilation is assisted by proteins such as SNAREs and synaptotagmin. While macroscopic soap film shapes are well characterized, finding exact solutions for microscopic cellular membrane surfaces is made more complex by bending energy constraints. Here, computational analysis revealed three families of fusion pores between two membranes. Our work suggests membrane fusion generates a member of the narrow pore family, and pore dilation is a transition to the wide pore family. The energy barrier that SNAREs or synaptotagmin must surmount to achieve dilation is set by a third unstable intermediate pore family.


title: Three membrane fusion pore families determine the pathway to pore dilation


title: ATPase activity of DFCP1 controls selective autophagy

abstract: Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 had a minor effect on bulk autophagic flux, DFCP1 was required to maintain the autophagic flux of p62 under both fed and starved conditions, and this was dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localized to forming omegasomes, these omegasomes failed to constrict. Consequently, the release of nascent autophagosomes from omegasomes was markedly delayed. DFCP1 was found to associate with ubiquitinated proteins, and degradation of ubiquitinated cargoes such as protein aggregates, mitochondria and micronuclei was strongly inhibited when DFCP1 was knocked out or mutated. Thus, DFCP1 mediates ATPase-driven constriction of omegasomes to release autophagosomes for selective autophagy.


title: Actin turnover protects the cytokinetic contractile ring from structural instability

abstract:
In common with other actomyosin contractile cellular machineries, actin turnover is required for normal function of the cytokinetic contractile ring. Cofilin is an actin-binding protein contributing to turnover by severing actin filaments, required for cytokinesis by many organisms. In fission yeast cofilin mutants, contractile rings suffer bridging instabilities in which actin bundles peel away from the plasma membrane into straight bridges. The origin of this behaviour is unclear. Here we used molecularly explicit simulations of the fission yeast contractile ring to examine the role of cofilin. Simulations reproduced the experimentally observed cycles of bridging and reassembly during constriction, each lasting [~] 6 min, and the tendency for bridging to occur in ring segments with low myosin II Myo2 density. The lack of cofilin severing produced [~] 2-fold longer filaments and, consequently, [~] 2-fold higher ring tensions. Simulations identified bridging as originating in the boosted ring tension, which increased centripetal forces that detached actin from Myo2 that anchored actin to the membrane. Thus, cofilin serves a critical role in cytokinesis by protecting the contractile ring from bridging, the principal structural threat.

Summary statementMolecularly explicit simulations showed that cofilin-mediated actin severing protects the fission yeast cytokinetic contractile ring from instabilities in which actin peels away into straight bridges.


title: Spinning Disk Multifocal Microscopy for Dynamic Arbitrarily Shaped Surface Imaging at Centimetre Scale and Micrometre Resolution

abstract: The large-scale fluorescence microscopy has enabled the observation of dynamic physiological activities at the single cellular level across the mouse cortex, such as distributed neuronal population representations. However, video-rate high-resolution microscopy at sophisticated biological surfaces in nature keeps a challenging task for the tradeoff between the speed, resolution, and field of view. Here we propose Spinning Disk Multifocal Microscopy (SDiM) for arbitrarily shaped surfaces, which enables imaging at centimeter field-of-view, micrometer resolution and up to 30 frames per second across the depth range of 450 {micro}m. We apply this technique in various microscopic systems, including customized macroscopic systems and the Real-time Ultra-large-Scale imaging at High resolution macroscopy (RUSH), in both the reflective mode and the fluorescence mode, and in the study of cortex-wide single-neuron imaging and immune cell tracking. SDiM provides an opportunity for studying the cortex-wide multi-scale cellular interactions in biological tissues.

category: bioengineering


title: Cooperativity of myosin II motors in the non-regulated and regulated thin filaments investigated with high-speed AFM

abstract:
Skeletal myosins II are non-processive molecular motors, that work in ensembles to produce muscle contraction while binding to the actin filament. Although the molecular properties of myosin II are well known, there is still debate about the collective work of the motors: is there cooperativity between myosin motors while binding to the actin filaments? In this study, we used high-speed AFM to evaluate this issue. We observed that the initial binding of small arrays of myosin heads to the non-regulated actin filaments did not affect the cooperative probability of subsequent bindings to neighboring sites and did not lead to an increase in the fractional occupancy of the actin binding sites. These results suggest that myosin motors are independent force generators when connected in small arrays, and that the binding of one myosin does not alter the kinetics of other myosins. In contrast, the probability of binding of myosin heads to regulated thin filaments under activating conditions (at high Ca2+ concentration and with 2 M ATP) was increased with the initial binding of one myosin, leading to a larger occupancy of neighboring available binding sites. The result suggests that myosin cooperativity is defined by the activation status of the thin filaments.

eLife digestMuscle contraction is the result of large ensembles of the molecular motor myosin II working in coordination while attached to actin. Myosin II produces the power stroke, responsible for force generation. In this paper, we used High-Speed Atomic Force Microscopy (HS-AFM) to determine the potential cooperativity between myosin motors bound to non-regulated and regulated thin filaments. Based on the direct visualization of myosin-actin interaction, probability of myosin binding, and the myosin fractional occupancy of binding sites along non-regulated and regulated actin filaments, our results show no cooperative effects over [~]100 nm of the actin filament length. In contrast, there is myosin cooperativity within the activated thin filament, that induces a high affinity of myosin heads to the filaments. Our results support the independent behaviour of myosin heads while attached to actin filaments, but a cooperative behavior when attached to regulated thin filaments.

category: biophysics


title: Trends in Arabidopsis Research post genome sequencing- A Scientometric study

abstract: 1.Arabidopsis thaliana, a model plant, is intensively researched because of the intrinsic advantages associated with its life cycle, genetics, and other characteristics. In the present study, we analysed the publication data of research work done on A. thaliana during 2001-2020, a period when the whole genome sequence of the plant was available to the researchers. The current meta-analysis showed that 31965 research papers were published globally on Arabidopsis during the study period. The USA topped the countries with the maximum share of publications (28.97%), followed by China, Japan, and West European countries. The analysis showed a temporal shift in the number of publications from different countries and their research focus. After 2013, the research output from China was higher than that from the USA, and there was a shift in focus from developmental biology to stress-related topics. Plant Physiology journal carried the most research work (2490 publications) on Arabidopsis, which was followed by Plant Cell (2376), the Plant Journal (2260) and the Journal of Experimental Botany (1121). However, there was a progressive decline in the publication in these journals, and a shift was evident in favour of open access journals like Frontiers in Plant Science. The publication and citation numbers also show ongoing Arabidopsis researchs relevance to plant sciences, particularly curiosity-driven and discovery-based science. This article delves into the patterns in the prominence of research areas, ideas and foci for the future Arabidopsis research roadmap.

category: plant biology


title: Quantifying the phenotypic information in mRNA abundance

abstract: Quantifying the dependency between mRNA abundance and downstream cellular phenotypes is a fundamental open problem in biology. Advances in multimodal single cell measurement technologies provide an opportunity to apply new computational frameworks to dissect the contribution of individual genes and gene combinations to a given phenotype. Using an information theory approach, we analyzed multimodal data of the expression of 83 genes in the Ca2+ signaling network and the dynamic Ca2+ response in the same cell. We found that the overall expression levels of these 83 genes explain approximately 60% of Ca2+ signal entropy. The average contribution of each single gene was 16%, revealing a large degree of redundancy between genes. Using different heuristics we estimated the dependency between the size of a gene set and its information content, revealing that on average a set of 53 genes contains 90% of the information about Ca2+ signaling within the cellular transcriptional state. Our results provide the first direct quantification of information content about complex cellular phenotype that exists in mRNA abundance measurements.


title: Single-cell analysis reveals the range of transcriptional states of circulating human neutrophils

abstract: Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. To date, neutrophils have been characterized based on discrete parameters including cell-surface markers, buoyancy, maturation status, or tissue localization. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two endpoints defined by either relative transcriptional inactivity (Nh2) or high expression of type I interferon-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.


title: Functional stemness-related genes revealed by single-cell profiling of naïve and stimulated human CD34+ cells from CB and mPB

abstract: Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. However, the underlying mechanisms of the stemness differences because of the cell sources and the culture stimulation are not fully understood. Here, we applied single-cell RNA-seq (scRNA-seq) to analyze the naive and stimulated human CD34+ cells from cord blood (CB) and mobilized peripheral blood (mPB). We collected over 16,000 single-cell data to construct a comprehensive trajectory inference map and characterized the HSCs population on the hierarchy top, which is under quiescent state. Then we compared HSCs in CB to those in mPB and HSCs of naive samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with culture time (CT-SRGs) and cell source (CS-SRGs), respectively. Interestingly, CT-SRGs and CS-SRGs share genes enriched in the signaling pathways such as mRNA catabolic process, Translational initiation, Ribonucleoprotein complex biogenesis and Cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related purine and ATP metabolic process which is important to initiate hematopoiesis. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs and found a small molecule cucurbitacin I, targeting STAT3/JAK2, can efficiently expand HSCs ex vivo while maintaining its stemness. These results indicate SRGs revealed by scRNA-seq can provide helpful insights to understand the stemness differences under diverse circumstances, and CT-SRGs can be a valuable database to identify candidates enhancing functional HSCs expansion during ex vivo culture.


title: Resolving tricky nodes in the tree of life through amino acid recoding

abstract: Genomic data allowed for a detailed resolution of the tree of life. Yet, tricky nodes such as the root of the animal, plants, eukaryotes, bacterial and archaeal trees remain unresolved. Genomic datasets are heterogeneous as genes and species evolve under different selective pressures, impending the efficacy of evolutionary analyses. Amino acid recodings were developed to reduce heterogeneity, but clear evidence to justify their use is missing. We use simulated genomic-scale datasets and show that recodings can substantially improve phylogenetic accuracy when tackling tricky nodes. We apply our findings to address the root of the animal tree where the debate centers on whether sponges (Porifera) or comb jellies (Ctenophora) branched out first. We show that results from real data follow predictions from simulated data and indicate that a placement of the ctenophores as the first branching animal lineage is most likely artifactual.