title: Interaction between BID and VDAC1 is required for mitochondrial demise and cell death in neurons

abstract:
Mitochondrial damage is a key feature of regulated cell death in neurons. In particular, mitochondrial outer membrane permeabilization (MOMP) has been proposed as a starting point for mitochondrial demise upon cellular stress. Potential mechanisms for MOMP presented in the literature include membrane pore formation by Bcl2-family proteins such as BID and BAX, oligomerization of voltage-dependent anion channels (VDACs) and hetero-oligomer formation of these proteins. In our study, we demonstrate a direct interaction between the voltage-dependent anion channel VDAC1 and the pro-apoptotic protein BID in dying neurons both in vitro and in vivo. Binding of BID to VDAC1 affects anion conductance through VDAC1 and is associated with glutamate-induced cell death in cultured neurons and ischemic brain injury. In cultured neurons, reducing VDAC1 expression significantly attenuates BID-induced hallmarks of mitochondrial damage such as mitochondrial fission, declined mitochondrial respiration, increased ROS production, and mitochondrial membrane potential breakdown. Our data highlight a critical role for VDAC1 as a mitochondrial receptor for activated BID, thereby serving as a key decision point between life and death in neurons.

One Sentence SummaryVDAC1 interacts with BID to mediate mitochondrial membrane permeabilization and neuronal cell death.


title: Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival

abstract: Zygotic Genome Activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance in shifting developmental control from primarily maternal stores in the oocyte to the embryo proper, we know little of the molecular events that initiate ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells (ESCs) have revealed Developmental Pluripotency Associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, while fertile, Dppa2/4 maternal knockout mice had reduced litter sizes, indicating decreased offspring survival. Immunofluorescence and transcriptome analyses of 2-cell embryos revealed while ZGA took place there were subtle defects in embryos lacking maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together our results show that while Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

category: developmental biology


title: A natural genetic variation screen identifies insulin signaling, neuronal communication, and innate immunity as modifiers of hyperglycemia in the absence of Sirt1

abstract: Variation in the onset, progression, and severity of symptoms associated with metabolic disorders such as diabetes impairs the diagnosis and treatment of at-risk patients. Diabetes symptoms, and patient variation in these symptoms, is attributed to a combination of genetic and environmental factors, but identifying the genes and pathways that modify diabetes in humans has proven difficult. A greater understanding of genetic modifiers and the ways in which they interact with metabolic pathways could improve the ability to predict a patients risk for severe symptoms, as well as enhance the development of individualized therapeutic approaches. In this study we use the Drosophila Genetic Reference Panel (DGRP) to identify genetic variation influencing hyperglycemia associated with loss of Sirt1 function. Through analysis of individual candidate functions, physical interaction networks, and Gene Set Enrichment Analysis (GSEA) we identify not only modifiers involved in canonical glucose metabolism and insulin signaling, but also genes important for neuronal signaling and the innate immune response. Furthermore, reducing the expression of several of these candidates suppressed hyperglycemia, making them ideal candidate therapeutic targets. These analyses showcase the diverse processes contributing to glucose homeostasis and open up several avenues of future investigation.

category: genetics


title: Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

abstract: In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize "off-target" sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.


title: Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

abstract: New technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization. However, the sparsity of these measurements and the challenge of integrating multiple binding maps represent significant challenges. Here we introduce scCUT&Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce scChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of individual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states, and identify extensive and cell type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT&Tag datasets.

category: genomics


title: Monomeric IgA antagonizes IgG-mediated enhancement of DENV infection

abstract: Dengue virus (DENV) is a prevalent human pathogen, infecting approximately 400 million individuals per year and causing symptomatic disease in approximately 100 million. A distinct feature of dengue is the increased risk for severe disease in some individuals with preexisting DENV-specific immunity. One proposed mechanism for this phenomenon is antibody-dependent enhancement (ADE), in which poorly-neutralizing IgG antibodies from a prior infection opsonize DENV to increase infection of Fc gamma receptor-bearing cells. While IgM and IgG are the most commonly studied DENV-reactive antibody isotypes, our group and others have described the induction of DENV-specific serum IgA responses during dengue. We hypothesized that monomeric IgA would be able to neutralize DENV without the possibility of ADE. To test this, we synthesized IgG and IgA versions of two different DENV-reactive monoclonal antibodies. We demonstrate that isotype-switching does not affect the antigen binding and neutralization properties of the two mAbs. We show that DENV-reactive IgG, but not IgA, mediates ADE in an Fc gamma receptor-positive K562 cells. Furthermore, we show that IgA potently antagonizes the ADE activity of IgG. These results suggest that levels of serum DENV-reactive IgA induced by DENV infection might regulate the overall ADE activity of DENV-immune plasma in vivo and warrants further study as a predictor of disease risk and/or therapeutic.

category: immunology


title: Inositol hexakisphosphate is a critical regulator of Integrator assembly and function

abstract: Integrator has critical roles in noncoding RNA 3'-end processing as well as transcription attenuation of selected mRNAs. IntS11 is the endonuclease for RNA cleavage, as a part of the IntS4-IntS9-IntS11 complex (Integrator cleavage module, ICM). Our structure of the Drosophila ICM, determined by cryo-electron microscopy at 2.74 [A] resolution, unexpectedly revealed the stable association of an inositol hexakisphosphate (IP6) molecule. The binding site is located in a highly electropositive pocket at an interface among all three subunits of ICM, 55 [A] away from the IntS11 active site and generally conserved in other ICMs. IP6 binding is also confirmed in human ICM. Mutations of residues in this binding site or disruption of IP6 biosynthesis significantly reduced Integrator assembly and activity in snRNA 3'-end processing. Our structural and functional studies reveal that Integrator is subject to intricate cellular control and IP6 is a critical regulator of Integrator assembly and function in Drosophila, humans, and likely other organisms.

category: molecular biology


title: Structural basis for mismatch surveillance by CRISPR/Cas9

abstract: The widespread use of CRISPR/Cas9 as a programmable genome editing tool has been hindered by off-target DNA cleavage (Cong et al., 2013; Doudna, 2020; Fu et al., 2013; Jinek et al., 2013). While analysis of such off-target editing events have enabled the development of Cas9 variants with greater discrimination against mismatches (Chen et al., 2017; Kleinstiver et al., 2016; Slaymaker et al., 2016), the underlying molecular mechanisms by which Cas9 rejects or accepts mismatches are poorly understood (Kim et al., 2019; Liu et al., 2020; Slaymaker and Gaudelli, 2021). Here, we used kinetic analysis to guide cryo-EM structure determination of Cas9 at different stages of mismatch surveillance. We observe a distinct, previously undescribed linear conformation of the duplex formed between the guide RNA (gRNA) and DNA target strand (TS), that occurs in the presence of PAM-distal mismatches, preventing Cas9 activation. The canonical kinked gRNA:TS duplex is a prerequisite for Cas9 activation, acting as a structural scaffold to facilitate Cas9 conformational rearrangements necessary for DNA cleavage. We observe that highly tolerated PAM-distal mismatches achieve this kinked conformation through stabilization of a distorted duplex conformation via a flexible loop in the RuvC domain. Our results provide molecular insights into the underlying structural mechanisms that may facilitate off-target cleavage by Cas9 and provides a molecular blueprint for the design of next-generation high fidelity Cas9 variants that selectively reduce off-target DNA cleavage while retaining efficient cleavage of on-target DNA.

category: biochemistry


title: Structure of a bacterial Rhs effector exported by the type VI secretion system

abstract: The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 [A] resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise {beta}-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs  cocoon where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal  plug domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.


title: Random Forest Factorization Reveals Latent Structure in Single Cell RNA Sequencing Data

abstract: Single-cell RNA sequencing data contain patterns of correlation that are poorly captured by techniques that rely on linear estimation or assumptions of Gaussian behavior. We apply random forest regression to scRNAseq data from mouse brains, which identifies the co-regulation of genes within specific cellular contexts. By analyzing the estimators of the random forest, we identify several novel candidate gene regulatory networks and compare these networks in aged and young mice. We demonstrate that cell populations have cell-type specific phenotypes of aging that are not detected by other methods, including the collapse of differentiating oligodendrocytes but not precursors or mature oligodendrocytes.

category: bioinformatics


title: Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state.

abstract:
MotivationPolyploidy and heterokaryosis are common and consequential genetic phenomena that increase the number of haplotypes in an organism and complicate whole-genome sequence analysis. Allele balance has been used to infer polyploidy and heterokaryosis in diverse organisms using read sets sequenced to greater than 50x whole-genome coverage. However, Sequencing to adequate depth is costly if applied to multiple individuals or large genomes.

ResultsWe developed VCFvariance.pl to utilize the variance of allele balance to infer polyploidy and/or heterokaryosis at low sequence coverage. This analysis requires as little as 10x whole-genome coverage and reduces the allele balance profile down to a single value, which can be used to determine if an individual has two or more haplotypes. This approach was validated on simulated, synthetic, and authentic read sets from an oomycete, fungus, and plant. The approach was deployed to ascertain the genome status of multiple isolates of Bremia lactucae and Phytophthora infestans.

Availability and implementationVCFvariance.pl is a Perl script available at https://github.com/kfletcher88/VCFvariance.


title: Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

abstract: Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the "blind sphere" around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (~14 [A]) than for nitroxide (~11 [A]) in 13C-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron-electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly.

category: biophysics


title: Allosteric modulation of the adenosine A2A receptor by cholesterol

abstract: Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptors active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.


title: Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

abstract: CRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, solution NMR is combined with multi-microsecond molecular dynamics and graph theory-derived models to probe the allosteric role of key enhancement specificity mutations. We show that the mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the HNH domain allosteric structure, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric intercommunication. This differential perturbation of the allosteric signaling reflects the different capabilities of the single mutants to increase Cas9 specificity, with the mutation achieving the highest specificity also strongly perturbing the signaling transfer. These outcomes reveal that the allosteric regulation is critical for the specificity enhancement of the Cas9 enzyme, and are valuable to harness the signaling network to improve the systems specificity.


title: Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer

abstract: CDC20 is a co-activator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with cancer in two families. Characterization of these mutants showed they retain APC/C activation activity but show reduced binding to BUBR1, a component of the SAC. Expression of L151R and N331K promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. CRISPR/Cas9 was used to generate mice carrying N331K. Homozygous mice carrying N331K were non-viable, however, heterozygotes displayed accelerated oncogenicity in Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor promoting genes in humans.

category: cancer biology


title: Elevated phosphorylation of EGFR in NSCLC due to mutations in PTPRH

abstract: The role of EGFR in lung cancer is well described with numerous activating mutations that result in phosphorylation and tyrosine kinase inhibitors that target EGFR. While the role of the EGFR kinase in non-small cell lung cancer (NSCLC) is appreciated, control of EGFR signaling pathways through dephosphorylation by phosphatases is not as clear. In recent work we identified mutations in Protein Tyrosine Phosphatase Receptor Type H (Ptprh, also known as SAP-1) as being associated with elevated phosphorylation of EGFR in a mouse model of breast cancer. We have examined a series of tumors from this mouse model, revealing conserved V483M Ptprh mutations within the FVB background, but a series of varied mutations in other backgrounds. Despite the varied Ptprh mutations in other background strains, matched primary and metastatic tumors largely shared mutational profiles. Profiling the downstream events of Ptprh mutant tumors revealed AKT activation, suggesting a key target of PTPRH was EGFR tyrosine 1197. Given the role of EGFR in lung cancer, we explored TCGA data which revealed that a subset of PTPRH mutant tumors shared gene expression profiles with EGFR mutant tumors, but that EGFR mutations and PTPRH mutations were mutually exclusive. Generation of a PTPRH knockout NSCLC cell line resulted in Y1197 phosphorylation of EGFR, and a rescue with expression of wild type PTPRH returned EGFR phosphorylation to parental line values while a rescue with a D986A catalytically dead mutant PTPRH did not, demonstrating that PTPRH targets EGFR. As expected with active EGFR, the knockout of PTPRH was associated with increased growth rate. Moreover, a dose response curve illustrated that two human NSCLC lines that had naturally occurring PTPRH mutations responded to EGFR tyrosine kinase inhibition. Injection of one of the NSCLC human lines into mice resulted in tumors, and Osimertinib treatment resulted in a reduction of tumor volume relative to vehicle controls. Consistent with prior literature from breast cancer, PTPRH mutation resulted in nuclear pEGFR as seen in immunohistochemistry, suggesting that there may also be a role for EGFR as a transcriptional co-factor. Other roles for PTPRH were explored through a receptor tyrosine kinase array, noting elevated phosphorylation of FGFR1. Knockout of PTPRH in NSCLC cell lines resulted in elevated phosphorylated FGFR1 relative to controls, indicating that PTPRH has a number of targets that may be aberrantly activated in NSCLC with mutations in PTPRH. Together these data suggest that mutations in PTPRH in NSCLC may result in clinically actionable alterations using existing therapies.


title: Predicting Individual Cell Division Events from Single-Cell ERK and Akt Dynamics

abstract: Predictive determinants of stochastic single-cell fates have been elusive, even for the well-studied mammalian cell cycle. What drives proliferation decisions of single cells at any given time? We monitored single-cell dynamics of the ERK and Akt pathways, critical cell cycle progression hubs and anti-cancer drug targets, and paired them to division events in the same single cells using the non-transformed MCF10A epithelial line. Following growth factor treatment, in cells that divide both ERK and Akt activities are significantly higher within the S-G2 time window ([~]8.5-40 hours). Such differences were much smaller in the pre-S-phase, restriction point window which is traditionally associated with ERK and Akt activity dependence, suggesting unappreciated roles for ERK and Akt in S through G2. Machine learning algorithms show that simple metrics of central tendency in this time window are most predictive for subsequent cell division; median ERK and Akt activities classify individual division events with an AUC=0.76. Surprisingly, ERK dynamics alone predict division in individual cells with an AUC=0.74, suggesting Akt activity dynamics contribute little to the decision driving cell division in this context. We also find that ERK and Akt activities are less correlated with each other in cells that divide. Network reconstruction experiments demonstrated that this correlation behavior was likely not due to crosstalk, as ERK and Akt do not interact in this context, in contrast to other transformed cell types. Overall, our findings support roles for ERK and Akt activity throughout the cell cycle as opposed to just before the restriction point, and suggest ERK activity dynamics are substantially more important than Akt activity dynamics for driving cell division in this non-transformed context. Single cell imaging along with machine learning algorithms provide a better basis to understand cell cycle progression on the single cell level.

category: systems biology


title: Core genes can have higher recombination rates than accessory genes within global microbial populations

abstract: Recombination is essential to microbial evolution1-4, and is involved in the spread of antibiotic resistance, antigenic variation, and adaptation to the host niche5-8. Yet quantifying the impact of homologous recombination on different gene classes, which is critical to understanding how selection acts on variation to shape species diversity and genome structure, remains challenging. This is largely due to the dynamic nature of bacterial genomes, whose high intraspecies genome content diversity9 and complex phylogenetic relationships10-12 present difficulties for inferring rates of recombination, particularly for rare genes. In this work, we apply a computationally efficient, non-phylogenetic approach13,14 to measure homologous recombination rates in the core and accessory genome (genes present in all strains and only a subset of strains, respectively) using >100,000 whole genome sequences from 12 microbial species. Our analysis suggests that even well-resolved sequence clusters sampled from global populations interact with overlapping gene pools, which has implications for the role of population structure in genome evolution. We show that in a majority of species, core genes have shorter coalescence times and higher recombination rates than accessory genes, and that gene frequency is often positively correlated with increased recombination. Our results provide a new line of population genomic evidence supporting the hypothesis that core genes are under strong, purifying selection15-17, and indicate that homologous recombination may play a key role in increasing the efficiency of selection in those parts of the genome most conserved within each species.

category: microbiology


title: Experimental confirmation that an uncommon, yet clinically relevant mutation (G878A) in the rrs gene of Mycobacterium tuberculosis confers resistance to streptomycin.

abstract: The effective treatment of patients diagnosed with drug resistant tuberculosis (TB) is highly dependent upon the ability to rapidly and accurately determine the antibiotic resistance/ susceptibility profile of the Mycobacterium tuberculosis isolate(s) involved. Thus, as more and more clinical microbiology laboratories advance towards the routine use of DNA sequence-based diagnostics, it is imperative that their predictive functions extend beyond the well-known resistance-conferring mutations, in order to also encompass as many of the lower-frequency mutations as possible. However, in most cases, the fundamental experimental proof that links these uncommon mutations with phenotypic resistance is still lacking. One such example is the G878A polymorphism within the rrs gene encoding the 16s rRNA. We, and others, have identified this mutation within a small number of drug-resistant M. tuberculosis isolates, although prior to this study a consensus regarding exactly which aminoglycoside antibiotic(s) it conferred resistance toward seems not to have been reached. Here we have employed oligo-mediated recombineering to specifically introduce the G878A polymorphism into the rrs gene of M. bovis BCG - a species very closely related to M. tuberculosis - and demonstrate that it confers low-level resistance to streptomycin alone. In our hands, it does not confer cross-resistance towards amikacin, capreomycin, nor kanamycin. We also demonstrate that the rrsG878A mutation exerts a substantial fitness defect in vitro, that may at least in part explain why clinical M. tuberculosis isolates bearing this mutation appear to be quite rare. Overall, this study provides clarity to the resistance phenotype attributable to the rrsG878A mutation and is relevant to the future implementation of genomics-based diagnostics, as well as the clinical management of patients in situations where this particular polymorphism is encountered.


title: Community diversity determines the evolution of synthetic bacterial communities under artificial selection

abstract: Artificial selection can be conducted at the community level in the laboratory through a differential propagation of the communities according to their level of expression of a targeted function (i.e. community phenotype). Working with communities instead of individuals as selection units brings in additional sources of variation in the considered phenotype that can arise through changes in community structure and influence the outcome of the artificial selection. These sources of variation could even be increased by manipulating species diversity. In this study, we wanted to assess the effect of manipulating initial community richness on artificial selection efficiency, defined as the change in the targeted function over time as compared to a control treatment without artificial selection. We applied artificial selection for a high productivity on synthetic bacterial communities varying for their initial richness level (from one to 16 strains). Our results showed that, overall, the communities that were artificially selected were 16% more productive than the control communities. Community richness positively influenced community productivity and metabolic capacities and was a strong determinant of the dynamics of community evolution. Our results suggested that community richness could influence artificial selection efficiency but a convergence of the community composition might have limited the effect of diversity on artificial selection efficiency. We propose that applying artificial selection on communities varying for their diversity could allow to find communities differing for their level of expression of a function but also for their responsiveness to artificial selection, provided that their initial composition is different enough.

category: evolutionary biology


title: Genome-wide identification and gene expression analysis of clade A protein phosphatase 2C family genes in Brassica juncea var. tumida

abstract: Abscisic acid (ABA) plays crucial roles in plant response to environmental stresses and development. The clade A phosphatases (PP2Cs) play a crucial role in ABA signaling. However, little is known about the details regarding PP2Cs family genes in Brassica juncea var. tumida. Here, 20 clade A PP2Cs family genes were identified in tuber mustard genome, including BjuABI1s, BjuABI2s, BjuAHG1s, BjuAHG3s, BjuHAB1, BjuHAB2s, BjuHAI1s, BjuHAI2s and BjuHAI3. The promoters of BjuPP2Cs family genes contained various of cis-acting elements, such as ABRE, GT1GMSCAM4, ARFAT and MYB1AT. We also analyzed the expression pattern of clade A BjuPP2Cs under abiotic stresses (low temperature, NaCl and ABA) treatment, pathogen Plasmodiophora brassicae treatment and different stages of stem swollen. The results suggested that clade A BjuPP2Cs regulated tuber mustard response to P. brassicae to mediate the formation of clubroot and might play roles in stem swelling and response to abiotic stresses. This study provides valuable information for further functional investigations of clade A PP2Cs family genes in B. juncea var. tumida.


title: Global mapping of the energetic and allosteric landscapes of protein binding domains

abstract: Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of  multidimensional mutagenesis: mutational effects are quantified for multiple molecular phenotypes--here binding and protein abundance--and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering  edgetic variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.


title: The morning burst: Shifting daily patterns of ATP production in Drosophila and temporal windows for their improvement in ageing

abstract: Mitochondria produce energy for cell function via adenosine triphosphate (ATP) and are regulated by a molecular 24h clock. Here we use Drosophila melanogaster to reveal shifts in whole animal ATP production over 24h, showing a marked peak in the morning that declines around midday and remains low from then through to the following morning. Mitochondrial membrane potential and ATP production has been shown previously to improve after long wavelength exposure, but apparently not at all times. Hence, to explore this further we exposed flies to 670nm at different times. Exposures between 08.00 and 11.00 resulted in a significant increase in ATP, while exposures at other times had no effect. Within the morning window, not all times were equally effective, however, 670nm exposure mid-morning when ATP production was maximal did not increase ATP, possibly because mitochondria lacked spare capacity at this time. Hence, in the morning there is a complex dynamic relationship between long wavelength light and mitochondria. Mitochondrial function and the influence of long wavelengths are conserved across species from fly to human, and determining the time points for light administration to improve function in ageing and disease is of key importance. Our data progress this search and reveal the outline of these times.

category: physiology


title: "Understanding Cortical Arousals during Sleep from Leg Movements: A Pilot Study."

abstract: Leg movements during sleep occur in patients with sleep pathology and healthy individuals. Some (but not all) leg movements during sleep are related to cortical arousals which occur without conscious awareness of the patient but have a significant effect of sleep fragmentation. Detecting leg movements during sleep that are associated with cortical arousals can provide unique insight into the nature and quality of sleep in both health and disease. In this study, a novel leg movement monitor is used in conjunction with polysomnography to better understand the relationship between leg movement and electroencephalogram (EEG) defined cortical arousals. In an approach that we call neuro-extremity analysis, graph theoretic, directed connectivity metrics are used to interrogate the causal links between neural activity measured by EEG and leg movements measured by the sensors within the leg movement monitor. The leg movement monitor in this study utilizes novel capacitive displacement sensors, and a 9-axis inertial measurement unit to characterize leg and foot movements. First, the capacitive displacement measures more closely related to EEG-defined cortical arousals than inertial measurements. Second, the neuro-extremity analysis reveals a temporally evolving connectivity pattern that is consistent with a model of cortical arousals in which brainstem dysfunction leads to near-instantaneous leg movements and a delayed, filtered signal to the cortex. Neuro-extremity analysis reveals causal relationships between EEG and leg movement sensor time-series data that may aid researchers to better understand the pathophysiology of cortical arousals associated with leg movements during sleep.

category: neuroscience


title: Human rare variant and zebrafish CRISPR/Cas9-mediated mutant analyses reveal novel functions for API5, HSPB7, and LMO2 in heart failure

abstract: The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.


title: A combined human in silico and CRISPR/Cas9-mediated in vivo zebrafish based approach for supporting gene target validation in early drug discovery


title: Towards precision ecology: Relationships of multiple sampling methods quantifying abundance for comparisons among studies

abstract: Because different sampling techniques will provide different abundance values, it is currently difficult to compare results among many studies to form holistic understandings of how abundance influences ant ecology. Using three sampling methods in the same location we found pitfall traps best confirmed A. gracilipes presence recording the fewest zero values (9.1%), card counts were the least reliable (67.1%), and tuna lures were intermediate (30.1%). The abundance of A. gracilipes from card counts ranged from 0 to 20, in pitfall traps from 0 to 325, and the full range of tuna lure abundance scores (0-7) were sampled. We then determined the relationships between these three standard ant sampling techniques for the abundance of yellow crazy ant Anoplolepis gracilipes. Irrespective of the data transformation method, the strongest relationship was between pitfall traps and tuna lures, and the least strong was between pitfall traps and card counts. We then demonstrate the utility of this knowledge by analysing A. gracilipes abundance reported within published literature to show where the populations in those studies sit on an abundance spectrum. We also comment on insights into the relative utility of the three methods we used to determine A. gracilipes abundance among populations of varying abundance. Pitfall traps was the most reliable method to determine if the species was present at the sample level. Tuna lures were predominantly reliable for quantifying the presence of workers, but were limited by the number of workers that can gather around a spoonful of tuna. Card counts were the quickest method, but were seemingly only useful when A. gracilipes abundance is not low. Finally we discuss how environmental and biological variation needs to be accounted for in future studies to better standardise sampling protocols to help progress ecology as a precision science.

category: ecology


title: Automated satellite remote sensing of giant kelp at the Falkland Islands (Islas Malvinas)

abstract: Giant kelp populations support productive and diverse coastal ecosystems in both hemispheres at temperate and subpolar latitudes but are vulnerable to changing climate conditions as well as direct human impacts. Observations of giant kelp forests are spatially and temporally patchy, with disproportionate coverage in the northern hemisphere, despite the size and comparable density of southern hemisphere kelp forests. Satellite imagery enables the mapping of existing and historical giant kelp populations in understudied regions, but automating the detection of giant kelp in large satellite datasets requires approaches that are robust to the optical complexity of the shallow, nearshore environment. We present and compare two approaches for automating the detection of giant kelp in satellite datasets: one based on crowd sourcing of satellite imagery classifications and another based on a decision tree paired with a spectral unmixing algorithm (automated using Google Earth Engine). Both approaches are applied to satellite imagery (Landsat) of the Falkland Islands or Islas Malvinas (FLK), an archipelago in the southern Atlantic Ocean that supports expansive giant kelp ecosystems. The performance of each method is evaluated by comparing the automated classifications with a subset of expert-annotated imagery cumulatively spanning over 2,700km of coastline. Using the remote sensing approaches evaluated herein, we present the first continuous timeseries of giant kelp observations in the FLK region using Landsat imagery spanning over three decades. We do not detect evidence of long-term change in the FLK region, although we observe a recent decline in total canopy area from 2017-2021. Using a nitrate model based on nearby ocean state measurements obtained from ships and incorporating satellite sea surface temperature products, we find that the area of giant kelp forests in the FLK region is positively correlated with the nitrate content observed during the prior year. Our results indicate that giant kelp classifications using citizen science are approximately consistent with classifications based on a state-of-the-art automated spectral approach. Despite differences in accuracy and sensitivity, both approaches find high interannual variability that impedes the detection of potential long-term changes in giant kelp canopy area, although recent canopy area declines are notable and should continue to be monitored carefully.


title: Ovicidal Activity of 2-Hydroxy-4-Methoxybenzaldehyde, Derivatives and Structural Analogues on Anopheles gambiae eggs

abstract:
BackgroundEffective remedies for disrupting Anopheles gambiae metamorphosis at the egg stage are crucial in suppression of the malaria vector populations that result in the reduction of disease burden. 2-Hydroxy-4-methoxybenzaldehyde (the major component of Mondia whytei roots), its derivatives, structural analogues and their blends were evaluated against the eggs of An. gambiae in the search for ovicidal compounds with potential use in mosquito control programs.

MethodsMature roots were harvested from Mondia whytei plants grown in the Center for African Medicinal & Nutritional Flora and Fauna (CAMNFF) herbal medicinal garden and cleaned with distilled water. 2-Hydroxy-4-methoxybenzaldehyde (1) was isolated by steam distillation of the chopped roots. The selected derivatives and/or analogues were prepared using established chemical procedures and their structures confirmed by NMR spectroscopy and ESI-MS. Ovicidal activity of the pure compounds, derivatives, structural analogues and/or formulated blends was tested at 1, 10, 25 and 50 ppm on An. gambiae eggs..

ResultsEleven mono-substituted (3-7), di-substituted (8-10), tri-substituted (1-2) aromatic compounds were assayed for ovicidal activity against Anopheles gambiae eggs singly or as blends. Benzaldehyde (4) and 4-methoxybenzaldehyde (9) were further converted into 2-hydroxy-1, 2-diphenylethanone (11), 1, 5-diphenylpenta-1, 4-diene-3-one (12) and 1, 5-bis (4-methoxyphenyl) penta-1, 4-diene-3-one (13) and evaluated for ovicidal activity individually or as blends. Of the thirteen compounds evaluated individually, 2-hydroxy-4-methoxybenzaldehyde (1) exhibited the highest ovicidal activity at LC50 0.7075 ppm while anisole had the lowest activity at LC50 40.342 ppm. The derivatives exhibited moderate activity: 2-hydroxy-1, 2-diphenylethanone (LC50 10.599 ppm), 1, 5-diphenylpenta-1, 4-diene-3-one (LC50 9.019 ppm) and 1, 5-bis (4-methoxyphenyl) penta-1, 4-diene-3-one (LC50 15.642 ppm). The blends exhibited intriguingly high ovicidal efficacy with the mixture of benzaldehyde and phenol showing the highest (LC50 0.332 ppm) while phenol and anisole exhibited the lowest activity (LC50 9.9909 ppm).

ConclusionFrom the activity of the blends, it is evident that anisole is antagonistic to the efficacy of phenol and benzaldehyde. It is also apparent that aldehyde and hydroxyl groups, when directly attached to the phenyl ring, provide the critical structural characteristics that contribute to the ovicidal activity of the aromatic compounds.


title: The tomato yellow leaf curl virus C4 protein alters the expression of plant developmental genes correlating to leaf upward cupping phenotype in tomato

abstract: Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus in the family Geminiviridae, is efficiently transmitted by the whitefly, Bemisia tabaci, and causes serious economic losses to tomato crops around the world. TYLCV-infected tomato plants develop distinctive symptoms of yellowing and leaf upper cupping. In recent years, excellent progress has been made in the characterization of TYLCV C4 protein function as a pathogenetic determinant in experimental plants, including Nicotiana benthamiana and Arabidopsis thaliana. However, molecular mechanism leading to disease symptom development in natural host plant tomato has yet to be characterized. The aim of the current study was to generate transgenic tomato plants expressing the TYLCV C4 gene and evaluate differential gene expression through comparative transcriptome analysis between the transgenic C4 plants and the transgenic green fluorescent protein (Gfp) gene control plants. Transgenic tomato plants expressing the TYLCV C4 developed phenotypes, including leaf upward cupping and yellowing that are similar the disease symptom expressed on tomato plants infected with TYLCV. In a total of 241 differentially expressed genes identified in the transcriptome analysis, a series of plant development-related genes, including transcription factors, glutaredoxins, protein kinases, R-genes and microRNA target genes, were significantly altered. These results provide further evidence to support the important function of the C4 protein in begomovirus pathogenicity. These transgenic tomato plants could serve as basic genetic materials for further characterization of plant receptors that are interacting with the TYLCV C4.


title: Title-Effects of Catha Edulis Forsk on Spatial Cognition and correlation with serum electrolytes in Wild- type Male White Albino Rats

abstract:
BackgroundThe burdens of psychostimulant use disorders are becoming a worldwide problem. One of the psychostimulants widely consumed in Ethiopia and East African countries is Catha edulis Forsk (khat). However, no studies have been conducted on the cognitive effects of khat and its correlation with serum electrolytes. The present study was aimed to evaluate the effects of khat on cognitive functions and its correlation with serum electrolytes.

Materials and MethodsA total of 36 adult (7-8 weeks) wild-type male Swiss albino rats weighing between 213 and 229g were used in this study. The rats were received crude khat extract subchronically (kesc) (100 mg/kg, 200 mg/kg and 300 mg/kg b.w), khat juice (khJ 2.5 mL/kg) and 2% tween 80 in distilled water (T80W- v/v, vehicle) and khat extract subacutely (kesa) (300 mg/kg). Spatial learning and memory were measured using Morris water maze model and serum electrolytes were measured using Cobas 6000. The data were analyzed using SPSS version 21.0 and Microsoft Excel.

ResultsSpatial learning was improved with trials across the groups, while average escape latency (s) and swim path-length (cm) of rats that received kesc 200 mg/kg (p<0.001 and p<0.001) and kesc 300 mg/kg (p<0.01 and p<0.001) was significantly greater than rats that received the vehicle. However, there was no significant difference in the latency between rats that received kesa 300mg/kg and vehicle (p>0.05). Thigmotaxis was significantly higher in rats that received all doses of khat extract (p<0.001). The time spent in the target quadrant in rats that received kesc 300 mg/kg was significantly reduced (p<0.05). Serum calcium level was inversely correlated with the escape latency (R=-0.417, p<0.05) in rats that received khat.

Conclusionskhat extract and juice administered subchronically, but not subacute administration, impaired learning and memory in rats and was associated with serum calcium reduction. The neuronal basis for such alteration should be investigated.

category: animal behavior and cognition


title: Pacemaking function of two simplified cell models

abstract: Simplified nonlinear models of biological cells are widely used in computational electrophysiology. The models reproduce qualitatively many of the characteristics of various organs, such as the heart, brain, and intestine. In contrast to complex cellular ion-channel models, the simplified models usually contain a small number of variables and parameters, which facilitates nonlinear analysis and reduces computational load. In this paper, we consider pacemaking variants of the Aliev-Panfilov and Corrado two-variable excitable cell models. We conducted a numerical simulation study of these models and investigated the main nonlinear dynamic features of both isolated cells and 1D coupled pacemaker-excitable systems. Simulations of the 2D sinoatrial node and 3D intestine tissue as application examples of combined pacemaker-excitable systems demonstrated results similar to obtained previously. The uniform formulation for the conventional excitable cell models and proposed pacemaker models allows a convenient and easy implementation for the construction of personalized physiological models, inverse tissue modeling, and development of real-time simulation systems for various organs that contain both pacemaker and excitable cells.


title: Sequential accumulation of dynein and its regulatory proteins at the spindle region in the Caenorhabditis elegans embryo

abstract: Cytoplasmic dynein is responsible for various cellular processes during the cell cycle. The mechanism by which its activity is regulated spatially and temporarily inside the cell remains elusive. There are various regulatory proteins of dynein, including dynactin, NDEL1/NUD-2, and LIS1. Characterizing the spatiotemporal localization of regulatory proteins in vivo will aid understanding of the cellular regulation of dynein. Here, we focused on spindle formation in the Caenorhabditis elegans early embryo, wherein dynein and its regulatory proteins translocated from the cytoplasm to the spindle region upon nuclear envelope breakdown (NEBD). We found that (i) a limited set of dynein regulatory proteins accumulated in the spindle region, (ii) the spatial localization patterns were distinct among the regulators, and (iii) the regulatory proteins did not accumulate in the spindle region simultaneously but sequentially. Furthermore, the accumulation of NUD-2 was unique among the regulators. NUD-2 started to accumulate before NEBD (pre-NEBD accumulation), and exhibited the highest enrichment compared to the cytoplasmic concentration. Using a protein injection approach, we revealed that the C-terminal helix of NUD-2 was responsible for pre-NEBD accumulation. These findings suggest a fine temporal control of the subcellular localization of regulatory proteins.

category: cell biology


title: Regional Cell Atlas of Human Intestine Shapes Distinct Immune Surveillance

abstract: Regional intestinal immune surveillance remains obscure. In this study, we integrated single-cell RNA sequencing and spatial transcriptomics to create a regional atlas of fetal and adult intestines, consisting of 59 cell subsets, of which eight new subsets and ILCs transition states were identified. Results revealed that microenvironment determines in-situ cell differentiation and shapes the regional molecular characteristics, allowing different intestinal segments with diverse functions. We characterized the regional expression of mucins, immunoglobulins, and antimicrobial peptides (AMPs) and their shift during development and in inflammatory bowel disease. Notably, -defensins expressed most abundantly in small intestinal LGR5+ stem cells, rather than in Paneth cells, and down-regulated as cell maturing. Common upstream transcription factors controlled the AMPs expression, illuminating the concurrent change of AMPs during epithelial differentiation, and the spatial co-expression patterns. We demonstrated the correspondence of cell focus of risk genes to diseases location susceptibility and identified distinct cell-cell crosstalk and spatial heterogeneity of immune cell homing in different gut segments. Overall, a cross-spatiotemporal approach to transcriptomes at single-cell resolution revealed that the regional milieu of the human intestine determined cellular and molecular cues of immune surveillance, dictating gut homeostasis and disease.


title: Identification of two pathways mediating protein targeting from ER to lipid droplets

abstract:
Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER.

HIGHLIGHTSO_LIProteins localize to LDs either during formation or later through ER-LD bridges
C_LIO_LISpecific membrane fusion machinery localizes to LDs and mediates protein targeting
C_LIO_LISpecific ER exit site proteins associate with LDs and participate in ERTOLD targeting
C_LIO_LIProteomic studies reveal cargoes for early and late ERTOLD targeting
C_LI


title: Constitutive loss of DNMT3A causes morbid obesity through misregulation of adipogenesis

abstract: DNA Methyltransferase 3A (DNMT3A) is an important facilitator of differentiation of both embryonic and hematopoietic stem cells. Heterozygous germline mutations in DNMT3A lead to Tatton-Brown-Rahman Syndrome (TBRS), characterized by obesity and excessive height. While DNMT3A is known to impact feeding behavior via the hypothalamus, here we investigated a role in adipocyte progenitors utilizing heterozygous knockout mice that recapitulate cardinal TBRS phenotypes. These mice become morbidly obese due to adipocyte enlargement and tissue expansion. Adipose tissue in these mice exhibited defects in preadipocyte maturation and precocious activation of inflammatory gene networks, including interleukin-6 signaling. Adipocyte progenitor cell lines lacking DNMT3A exhibited aberrant differentiation. Furthermore, mice in which Dnmt3a was specifically ablated in adipocyte progenitors showed enlarged fat depots and increased progenitor numbers, partly recapitulating the TBRS obesity phenotypes. Loss of DNMT3A led to constitutive DNA hypomethylation, such that the DNA methylation landscape of young adipocyte progenitors resemble that of older wild-type mice. Together, our results demonstrate that DNMT3A coordinates both the central and local control of energy storage required to maintain normal weight and prevent inflammatory obesity.


title: A cell-based platform for oxidative stress monitoring in motor neurons using genetically encoded biosensors of H2O2

abstract:
BackgroundOxidative stress plays an important role in the development of neurodegenerative diseases: it either can be the initiator or part of a pathological cascade leading to the neurons death. Although a lot of methods are known for oxidative stress study, most of them operate on non-native cellular substrates or interfere with the cell functioning. Genetically encoded (GE) biosensors of oxidative stress demonstrated their general functionality and overall safety in various live systems. However, there is still insufficient data regarding their use for research of disease-related phenotypes in relevant model systems, such as human cells.

MethodsWe applied CRISPR/Cas9 genome editing to introduce mutations (c.272A>C and c.382G>C) in the associated with amyotrophic lateral sclerosis SOD1 gene of induced pluripotent stem cells (iPSC) obtained from a healthy individual. Using CRISPR/Cas9, we modified these mutant iPSC lines, as well as the parental iPSC line, and a patient-specific SOD1D91A/D91A iPSC line with ratiometric GE biosensors of cytoplasmic (Cyto-roGFP2-Orp1) and mitochondrial (Mito-roGFP2-Orp1) H2O2. The biosensors sequences along with a specific transactivator for doxycycline-controllable expression were inserted in the "safe harbor" AAVS1 (adeno-associated virus site 1) locus. We differentiated these transgenic iPSCs into motor neurons and investigated the functionality of the biosensors in such a system. We measured relative oxidation in the cultured motor neurons and its dependence on culture conditions, age, and genotype, as well as kinetics of H2O2 elimination in real-time.

ResultsWe developed a cell-based platform consisting of isogenic iPSC lines with different genotypes associated with amyotrophic lateral sclerosis. The iPSC lines were modified with GE biosensors of cytoplasmic and mitochondrial H2O2. We provide proof-of-principle data showing that this approach may be suitable for monitoring oxidative stress in cell models of various neurodegenerative diseases as the biosensors reflect the redox state of neurons.

ConclusionWe found that the GE biosensors inserted in the AAVS1 locus remain functional in motor neurons and reflect pathological features of mutant motor neurons, although the readout largely depends on the severity of the mutation.


title: High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast

abstract: Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential notably during eukaryotic cell division. Here, we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIPs components Kar9 (functional homologue of APC, MACF, and SLAIN), Bim1 (orthologue of EB1), and Bik1 (orthologue of CLIP-170). Our data indicate that a redundant, multivalent web of interactions links the three +TIPs together to form a "Kar9 body" at the tip of a single cytoplasmic microtubule. They further suggest that this body is a liquid-like condensate, allowing it to persist on both growing and shrinking microtubule tips, and functions as a mechanical coupling device between microtubules and actin cables during mitosis. Our study underlines the power of dissecting the web of low-affinity interactions driving liquid-liquid phase separation of proteins in order to demonstrate the importance and establish the functional roles of condensation processes in vivo.


title: Proteome signatures of reductive stress cardiomyopathy

abstract:
Nuclear factor erythroid 2-related factor 2 (NRF2), a redox sensor, is vital for cellular redox homeostasis. We reported that transgenic mice expressing constitutively active Nrf2 (CaNrf2-TG) exhibit reductive stress (RS). In this study, we identified novel protein biomarkers for RS-induced cardiomyopathy using Tandem Mass Tag (TMT) proteomic analysis in heart tissues of TG (CaNrf2-TG) and non-transgenic (NTg) mice at 6-7 months of age (N= 4/group). A total of 1105 proteins were extracted from 22544 spectra. Of note, about 560 proteins were differentially expressed in TG vs. NTg hearts, indicating a global impact of RS on myocardial proteome. From a closer analysis of the proteome datasets, we identified over 32 proteins that were significantly altered in response to RS. Among these, 20 were upregulated and 12 were downregulated in the hearts of TG vs. NTg mice, suggesting that these proteins could be putative signatures of RS. Scaffold analysis revealed a clear distinction between TG vs NTg hearts. Of note, we observed several proteins with redox (#185; cysteine residues), NEM-adducts (#81), methionine-loss (#21) and acetylation (#1) modifications in TG vs. NTg hearts due to chronic RS. The majority of the differentially expressed proteins (DEPs) that are significantly altered in RS mice were found to be involved in stress related pathways such as antioxidants, NADPH, protein quality control (PQC), etc. Interestingly, proteins that were involved in mitochondrial respiration, lipophagy and cardiac rhythm were dramatically decreased in TG hearts. Of note, we identified the glutathione family of proteins as the significantly changed subset of the proteome in TG heart. Surprisingly, our comparative analysis of NGS based transcriptome and TMT-proteome indicated [~]50% of the altered proteins in TG myocardium was found to be negatively correlated with their transcript levels. Modifications at cysteine/NEM-adducts (redox), methionine or lysine residues in multiple proteins in response to chronic RS might be associated with impaired PQC mechanisms, thus causing pathological cardiac remodeling.

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title: Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends

abstract: Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.


title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

abstract: During the progression of osteoarthritis (OA), degradation of the extracellular matrix alters the biomechanical properties of cartilage, especially the compressive modulus. The mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) is required for chondrocyte mechanotransduction However, how OA-mediated cartilage degradation influences TRPV4 signaling remains unknown. To determine if ATDC5 cells alter TRPV4-mediated calcium signaling and cell phenotype in response to softer substrates, we created PEGDA-RGDS hydrogels with Youngs moduli that simulated healthy (~350 kPa), OA (~175 kPa) and severe OA (~35 kPa) tissue. We found that softer substrates reduced the influx of calcium through TRPV4 when challenging chondrocytes with hypotonic swelling (HTS). Chondrocyte apoptosis also increased on the OA and severe OA gels due to elevated basal [Ca2+]i, which is attenuated with pharmacological agonism of TRPV4. Pharmacological agonism of TRPV4 rescued the expression of aggrecan and TRPV4 in chondrocytes cultured on OA gels and enhanced the type II collagen (col2) expression in cells on the normal and OA gels. These data suggest that the biomechanical properties of degraded cartilage alter TRPV4-mediated mechanotransduction in chondrocytes. Given that TRPV4 reduced apoptosis and improved the chondrogenic capacity of cells, TRPV4 stimulation could provide a potential therapeutic target in patients with early to moderate OA.


title: Activation of the adhesion GPCR GPR133 (ADGRD1) by antibodies targeting the N-terminus

abstract: We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor (aGPCR) whose canonical signaling raises cytosolic cAMP, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We showed that dissociation of autoproteolytically generated N-terminal and C-terminal fragments (NTF and CTF) of GPR133 at the plasma membrane promotes receptor activation and increases signaling. Toward developing biologics modulating GPR133 function, we tested antibodies against the N-terminus of GPR133 for effects on receptor signaling. Treatment of HEK293T cells overexpressing GPR133 with such antibodies increased cAMP levels in a concentration-dependent manner. Analysis of supernatants following antibody treatment revealed complexes of the antibodies with the autoproteolytically cleaved NTF of GPR133. Cells expressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. The antibody-mediated stimulation of wild-type GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that NTF-CTF dissociation promotes receptor activation and signaling.


title: Activation of endoplasmic reticulum stress via clustering of inner nuclear membrane proteins

abstract: One of the major cellular mechanisms to ensure protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is typically triggered by accumulation of misfolded proteins in the ER lumen. Here we describe activation of ER stress via protein aggregation in the cell nucleus. We find in the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) activation of ER stress due to the aggregation of the diseases-causing progerin protein at the nuclear envelope. The presence of nucleoplasmic protein aggregates is sensed and signaled to the ER lumen via immobilization and clustering of the inner nuclear membrane protein SUN2, leading to activation of the Unfolded Protein Response (UPR). These results identify a nuclear trigger of ER stress and they provide insight into the molecular disease mechanisms of HGPS.


title: Exocytosis of the silicified cell wall of diatoms involves extensive membrane disintegration

abstract:
Diatoms are unicellular algae, characterized by silica cell walls. The silica elements are formed intracellularly in a membrane-bound silica deposition vesicle (SDV), and are exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements is a long-standing enigma. We studied membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during the formation of the mineral phase it is in tight association with the SDV membranes, which are forming a precise mold of the delicate geometrical patterns. During exocytosis, the distal SDV membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. Within the cell, there is no evidence for the formation of a new plasma membrane, thus the proximal SDV membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.

Significance StatementExocytosis is a fundamental process for cell metabolism, communication, and growth. During exocytosis, an intracellular vesicle fuses with the plasma membrane to release its contents. In classical exocytosis, where the exocytosed vesicles are much smaller than the cell, membrane homeostasis is maintained by recycling excess membranes back into the cell. However, an extreme case of exocytosis is the extrusion of large and rigid cell wall elements by unicellular marine algae. During this process, the cell needs to deal with a potential doubling of its plasma membrane. This study reports on a unique exocytosis mechanism used by these organisms, in which the cells cope with the geometrical and physical challenges of exocytosis by releasing a significant amount of membranes to the extracellular space.


title: Evolutionary dynamics of pseudoautosomal region 1 in humans and great apes

abstract: The pseudoautosomal region 1 (PAR1) is a 2.7 Mb telomeric region of human sex chromosomes. As the largest point of contact between the X and Y, PAR1 has a crucial role in ensuring proper segregation of sex chromosomes during male meiosis, exposing it to extreme recombination and associated mutational processes. We investigate PAR1 evolution using population genomic datasets of extant humans, eight populations of great apes and two archaic human genome sequences. We find that the PAR1 sequence is closer to nucleotide equilibrium than autosomal telomeric sequences. We detect a difference between long-term substitution patterns and extant diversity in PAR1 that is mainly driven by the conflict between strong mutation and recombination-associated fixation bias at CpG sites. Additionally, we detect excess C[->]G mutations in PAR1 of all great ape species, specific to the mutagenic effect of male recombination. Analysis of differences between frequencies of alleles segregating in females and males provided no evidence for sexually antagonistic selection in this region. Furthermore, despite recent evidence for Y chromosome introgression from humans into Neanderthals, we find that the Neanderthal PAR1 retained similarity to the Denisovan sequence, as is the case for the X chromosome and the autosomes. Lastly, we study repeat content and double-strand break hotspot regions in PAR1 and find that they may play roles in ensuring the obligate X-Y recombination event during male meiosis. Our study provides an unprecedented quantification of population genetic forces and insight into evolutionary processes governing PAR1 biology.


title: Human mitochondrial protein complexes revealed by large-scale coevolution analysis and deep learning-based structure modeling

abstract: Recent development of deep-learning methods has led to a breakthrough in the prediction accuracy of 3-dimensional protein structures. Extending these methods to protein pairs is expected to allow large-scale detection of protein-protein interactions and modeling protein complexes at the proteome level. We applied RoseTTAFold and AlphaFold2, two of the latest deep-learning methods for structure predictions, to analyze coevolution of human proteins residing in mitochondria, an organelle of vital importance in many cellular processes including energy production, metabolism, cell death, and antiviral response. Variations in mitochondrial proteins have been linked to a plethora of human diseases and genetic conditions. RoseTTAFold, with high computational speed, was used to predict the coevolution of about 95% of mitochondrial protein pairs. Top-ranked pairs were further subject to the modeling of the complex structures by AlphaFold2, which also produced contact probability with high precision and in many cases consistent with RoseTTAFold. Most of the top ranked pairs with high contact probability were supported by known protein-protein interactions and/or similarities to experimental structural complexes. For high-scoring pairs without experimental complex structures, our coevolution analyses and structural models shed light on the details of their interfaces, including CHCHD4-AIFM1, MTERF3-TRUB2, FMC1-ATPAF2, ECSIT-NDUFAF1 and COQ7-COQ9, among others. We also identified novel PPIs (PYURF-NDUFAF5, LYRM1-MTRF1L and COA8-COX10) for several proteins without experimentally characterized interaction partners, leading to predictions of their molecular functions and the biological processes they are involved in.


title: Molecular and cellular adaptations in hippocampal parvalbumin neurons mediate behavioral responses to chronic social stress

abstract: Parvalbumin-expressing interneurons (PV neurons) maintain inhibitory control of local circuits implicated in behavioral responses to environmental stressors. However, the roles of molecular and cellular adaptations in PV neurons in stress susceptibility or resilience have not been clearly established. Here, we show behavioral outcomes of chronic social defeat stress (CSDS) are mediated by differential neuronal activity and gene expression in hippocampal PV neurons in mice. Using in vivo electrophysiology and chemogenetics, we find increased PV neuronal activity in the ventral dentate gyrus is required and sufficient for behavioral susceptibility to CSDS. PV neuron-selective translational profiling indicates mitochondrial oxidative phosphorylation is the most significantly altered pathway in stress-susceptible versus resilient mice. Among differentially expressed genes associated with stress-susceptibility and resilience, we find Ahnak, an endogenous regulator of L-type calcium channels which are implicated in the regulation of mitochondrial function and gene expression. Notably, Ahnak deletion in PV neurons impedes behavioral susceptibility to CSDS. Altogether, these findings indicate behavioral effects of chronic stress can be controlled by selective modulation of PV neuronal activity or a regulator of L-type calcium signaling in PV neurons.


title: Incidental reinstatement of faces and scenes in medial temporal lobe subregions during word recognition

abstract: During associative retrieval, the brain reinstates neural representations that were present during encoding. The human medial temporal lobe (MTL) with its subregions hippocampus (HC), perirhinal cortex (PRC), and parahippocampal cortex (PHC) plays a central role in neural reinstatement. Previous studies have given compelling evidence for reinstatement in the MTL during explicitly instructed associative retrieval. High-confident recognition may be similarly accompanied by recollection of associated information from the encoding context. It is unclear, however, whether high-confident recognition memory elicits reinstatement in the MTL even in the absence of an explicit instruction to retrieve associated information. Here, we addressed this open question using high-resolution fMRI. Twenty-eight male and female human volunteers engaged in a recognition memory task for words that they had previously encoded together with faces and scenes. Using complementary uni- and multivariate approaches, we show that MTL subregions including the PRC, PHC, and HC differentially reinstate category-specific representations during high-confident word recognition, even though no explicit instruction to retrieve the associated category was given. This constitutes novel evidence that high-confident recognition memory is accompanied by incidental reinstatement of associated category information in MTL subregions, and supports a functional model of the MTL that emphasises content-sensitive representations during both encoding and retrieval.


title: Early stage of Spinocerebellar Ataxia Type 1 (SCA1) progression exhibits region- and cell-specific pathology and is partially ameliorated by Brain Derived Neurotrophic Factor (BDNF)

abstract:
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by an abnormal expansion of CAG repeats in the gene Ataxin1 (ATXN1) and characterized by motor deficits, cognitive decline, and premature lethality. Due to the severe cerebellar degeneration seen in SCA1, the pathogenesis of Purkinje cells has been the main focus of previous studies. However, mutant ATXN1 is expressed throughout the brain, and pathology in brain regions beyond the cerebellar cortex likely contributes to the symptoms of SCA1. Here, using immunohistochemistry and gene expression analysis we investigated early-stage SCA1 alterations in neurons, astrocytes, and microglia in clinically relevant SCA1 brain regions including the cerebellum, medulla oblongata, hippocampus, and motor cortex of Atxn1154Q/2Q mice, a knock-in mouse model of SCA1 expressing mutant ATXN1 broadly.

Our results indicate a spectrum of astrocyte and microglial phenotypes across brain regions early in SCA1. We found reduced expression of homeostatic astrocytic genes Kcnj10, Aqp4, Slc1a2 and Gja1 in 12 week old Atxn1154Q/2Q mice, all of which are key for neuronal function, in the hippocampus and medulla. At the same age, the expression of these genes was increased in the cortex, indicating region-specific astrocyte reactive phenotypes. We have found altered microglial density throughout the brain, with decreases in cerebellar white matter but increases in medulla, motor cortex and corpus callosum. On the other hand, neuronal pathology was evident only in cerebellar Purkinje cells at this early stage of SCA1.

We have previously shown that expression of brain-derived neurotrophic factor (BDNF), an astrocyte-released growth factor important for the survival and function of neurons, is reduced in the cerebellum, and exogenous BDNF treatment has therapeutic potential for cerebellar aspects of disease in Purkinje cells specific transgenic mouse model of SCA1 (Pcp2-ATXN1[82Q] line). We report here that BDNF expression is decreased in the cerebellar Bergmann glia of patients with SCA1.

Moreover, we found brain region specific changes in BDNF expression in Atxn1154Q/2Q mice with decreased expression in the cerebellum and hippocampus, and increased expression in the medulla and cortex. Using ALZET pumps to deliver BDNF, we have found that BDNF treatment ameliorated several aspects of SCA1 pathogenesis, including motor deficits, learning and problem-solving strategy development, hippocampal neurogenesis, and expression of astrocyte homeostatic genes in the hippocampus.


title: Spectrum of BDNF expression changes and reactive glial phenotypes across brain regions precede neuronal loss and are associated with therapeutic effects of BDNF in SCA1


title: Disruption of the autism-associated gene SCN2A alters synaptic development and neuronal signaling in patient iPSC-glutamatergic neurons

abstract:
SCN2A is an autism spectrum disorder (ASD) risk gene and encodes a voltage-gated sodium channel. However, the impact of autism-associated SCN2A de novo variants on human neuron development is unknown. We studied SCN2A using isogenic SCN2A-/- induced pluripotent stem cells (iPSCs), and patient-derived iPSCs harboring a p.R607* or a C-terminal p.G1744* de novo truncating variant. We used Neurogenin2 to generate excitatory glutamatergic neurons and found that SCN2A+/p.R607* and SCN2A-/- neurons displayed a reduction in synapse formation and excitatory synaptic activity using multielectrode arrays and electrophysiology. However, the p.G1744* variant, which leads to early-onset seizures in addition to ASD, altered action-potential dynamics but not synaptic activity. Proteomic and functional analysis of SCN2A+/p.R607* neurons revealed defects in neuronal morphology and bioenergetic pathways, which were not present in SCN2A+/p.G1744* neurons. Our study reveals that SCN2A de novo variants can have differential impact on human neuron function and signaling.

HIGHTLIGHTS- Isogenic SCN2A-/- neurons display intrinsic hyperexcitability and impaired excitatory synapse function
- SCN2A+/p.R607* variant reduces excitatory synapse function in patient neurons
- C-terminal SCN2A+/p.G1744* variant enhances action potential properties but not synaptic transmission in patient neurons
- SCN2A+/p.R607* variant display impacts on morphological and bioenergetic signaling networks through proteomic and functional analysis

eTOC- Brown et al. examined Autism-associated SCN2A variants using patient-derived iPSC NGN2-neurons. They discover that genetic variants differentially impact neuronal development and synaptic function, and highlight neuronal and bioenergetic signaling networks underlying SCN2A loss-of-function.


title: Theta oscillations in frontal cortex differentially modulate accuracy and speed in flexible stimulus- and action-based learning

abstract: Flexible reward learning relies on frontal cortex, with substantial evidence indicating the anterior cingulate cortex (ACC) and orbitofrontal cortex (OFC) play important roles. Recent studies in both rat and macaque suggest theta oscillations (5-10 Hz) may be a spectral signature that coordinates this learning. However, causal network-level interactions between ACC and OFC in flexible learning remain unclear. We investigated the learning of stimulus-outcome (S-O) and action-outcome (A-O) associations using a combination of simultaneous in-vivo electrophysiology in dorsal ACC and ventral OFC, partnered with bilateral inhibitory DREADDs in ACC. In freely-behaving male and female rats and using a within-subject design, we examined accuracy and speed of response across distinct and precisely-defined trial epochs including correct choice, incorrect choice, and reward collection. We report significant modulation of accuracy by theta power in both ACC and OFC. Both ACC and OFC theta oscillations consistently signaled accuracy in the initial discrimination phase, whereas it was OFC theta alone in the reversal phases of both S-O and A-O learning. Theta power in either region was not correlated with deliberation speed. Indeed, theta modulation of accuracy was dissociable from its involvement in speed of response which was affected by ACC inhibition, most prominently in A-O learning. Results are discussed with reference to the nonhuman primate literature, where there is by contrast more reported specialization of OFC and ACC in flexible learning of stimuli and actions. The present results also point to a specific role of OFC theta in signaling a reversal of either kind.


title: Nucleolin loss-of-function leads to aberrant FGF signaling and craniofacial anomalies

abstract: rRNA transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies, however the cellular and molecular reasons for this are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. We discovered that Nucleolin is dynamically expressed during embryonic development with high enrichment in the craniofacial tissues. Consistent with this pattern of expression, ncl homozygous mutant (ncl-/-) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observe that ncl-/- mutants exhibit decreased rRNA synthesis and p53-dependent neuroepithelial cell death. In addition, the half-life of fgf8a mRNA is reduced in ncl-/- mutants, which perturbs Fgf signaling, resulting in misregulation of Sox9a mediated chondrogenesis and Runx2 mediated osteogenesis. Exogenous addition of human recombinant FGF8 to the mutant zebrafish significantly rescues the cranioskeletal phenotype, suggesting that Nucleolin regulates osteochondroprogenitor differentiation during craniofacial development by post-transcriptionally regulating Fgf signaling. Our work has therefore uncovered a novel tissue-specific function for Nucleolin in rRNA transcription and growth factor signaling during embryonic craniofacial development.

category: developmental biology


title: Microsaccades as a marker not a cause for attention-related modulation

abstract: Recent evidence suggests that microsaccades are causally linked to the attention-related modulation of neurons - specifically, that microsaccades towards the attended location are required for the subsequent changes in firing rate. These findings have raised questions about whether attention-related modulation is due to different states of attention as traditionally assumed or might instead be a secondary effect of microsaccades. Here, in two rhesus macaques, we tested the relationship between microsaccades and attention-related modulation in the superior colliculus, a brain structure crucial for allocating attention. We found that attention-related modulation emerged even in the absence of microsaccades, was already present prior to microsaccades towards the cued stimulus, and persisted through the suppression of activity that accompanied all microsaccades. Nonetheless, consistent with previous findings, we also found significant attention-related modulation when microsaccades were directed towards, rather than away from, the cued location. Thus, in contrast to the prevailing hypothesis, microsaccades are not necessary for attention-related modulation, at least not in the superior colliculus. They do, however, provide an additional marker for the state of attention, especially at times when attention is shifting from one location to another.


title: Common micro- and macroscale principles of connectivity in the human brain

abstract: The brain requires efficient information transfer between neurons and between large-scale brain regions. Brain connectivity follows predictable organizational principles: at the cellular level, larger supragranular pyramidal neurons have larger dendritic trees, more synapses, more complex branching and perform more complex neuronal computations; at the macro-scale, region-to-region connections are suggested to display a diverse architecture with highly connected hub-areas facilitating complex information integration and computation. Here, we explore the hypothesis that the branching structure of large-scale region-to-region connectivity follows similar organizational principles as known for the neuronal scale. We examine microscale connectivity of basal dendritic trees of supragranular pyramidal neurons (300+) across ten cortical areas in five human donor brains (1M/4F). Dendritic complexity was quantified as the number of branch points, tree length, spine count, spine density and overall branching complexity. High-resolution diffusion-weighted MRI was used to construct  white matter trees of cortico-cortical wiring. Examining the complexity of the resulting white matter trees using the same measures as for dendritic trees shows multimodal association areas to have larger, more complexly branched white matter trees than primary areas (all p<0.0001) and regional macroscale complexity to run in parallel with microscale measures, in terms of number of inputs (r=0.677, p=0.032), branch points (r=0.790, p=0.006), total tree length (r=0.664, p=0.036) and branching complexity (r=0.724, p=0.018). Our findings support the integrative theory that brain connectivity is structured following similar  principles of connectivity at the neuronal and macroscale level, and provide a framework to study connectivity changes in brain conditions at multiple levels of brain organization.


title: Assessment of CAR-T mediated and targeted cytotoxicity in 3D microfluidic TBNC co-culture models for combination therapy

abstract: Chimeric antigen receptor (CAR)-T cell therapy is efficacious against many hematological malignancies; however, their therapeutic application to treat solid tumors presents further challenges. A better understanding of how the solid tumor microenvironment (TME) impacts CAR-T anti-tumor effects would enable the selection of effective combination therapies to decipher the optimal course of treatment for patients and to better engineer CAR-Ts. Classical 2D in vitro models do not provide sufficient recapitulation of the native human TME, and in vivo models, such as patient-derived xenografts, are costly, complex and labor intensive. Here, we present a novel 3D, miniaturized assay for the evaluation of EGFR-targeted CAR-T cell cytotoxicity and specificity on tumor-stroma triple-negative breast cancer models in microfluidic devices. CAR-T cells were shown to home towards EGFR-expressing cancer cells to elicit a cytotoxic effect, whilst leaving low EGFR-expressing fibroblasts viable, an effect which was enhanced through combination anti-PD-L1 therapy and carboplatin chemotherapy. Hence, we propose this proof-of-concept immunoassay as a future preclinical screening tool for the development of novel immunotherapeutics and for use in personalized medicine.

category: bioengineering


title: Axial multi-layer perceptron architecture for automatic segmentation of choroid plexus in multiple sclerosis

abstract: Choroid plexuses (CP) are structures of the brain ventricles which produce most of the cerebrospinal fluid (CSF). Several postmortem and in vivo studies have pointed towards their role in the inflammatory processes in multiple sclerosis (MS). Automatic segmentation of CP from MRI thus has high value for studying their characteristics in large cohorts of patients. To the best of our knowledge, the only freely available tool for CP segmentation is FreeSurfer but its accuracy for this specific structure is poor. In this paper, we propose to automatically segment CP from non-contrast enhanced T1-weighted MRI. To that end, we introduce a new model called "Axial-MLP" based on an assembly of Axial multi-layer perceptrons (MLPs). This is inspired by recent works which showed that the self-attention layers of Transformers can be replaced with MLPs. This approach is systematically compared with a standard 3D U-Net, nnU-Net, Freesurfer and FastSurfer. For our experiments, we make use of a dataset of 141 subjects (44 controls and 97 patients with MS). We show that all the tested deep learning (DL) methods outperform FreeSurfer (Dice around 0.7 for DL vs 0.33 for FreeSurfer). Axial-MLP is competitive with U-Nets even though it is slightly less accurate. The conclusions of our paper are two-fold: 1) the studied deep learning methods could be useful tools to study CP in large cohorts of MS patients; 2) Axial-MLP is a potentially viable alternative to convolutional neural networks for such tasks, although it could benefit from further improvements.


title: Disentangling dimensions of animacy in human brain and behaviour

abstract: The perception of animate things is of great behavioural importance to humans. Despite the prominence of the distinct brain and behavioural responses to animate and inanimate things, however, it remains unclear which of several commonly confounded properties underlie these observations. Here, we investigate the importance of five dimensions of animacy: "being alive", "looking like an animal", "having agency", "having mobility", and "being unpredictable" in brain (fMRI, EEG) and behaviour (property and similarity judgments) of 19 subjects using a stimulus set of 128 images that disentangles the five dimensions (optimized by a genetic algorithm). The living/non-living distinction ("being alive") was prominent in subjects judgments, but despite its prominence in the neuroscience literature, did not explain variance in brain representations. The other dimensions of animacy explained variance in both brain and behaviour. The "having agency" dimension explained more variance in higher-level visual areas, consistent with higher cognitive contributions. The "being unpredictable" dimension was reflected in representations in both lower and higher-level visual cortex, possibly because unpredictable things require attention. We conclude that the multiple dimensions commonly conflated in the notion of animacy are distinctly reflected in different brain and behavioural responses.


title: Disentangling five dimensions of animacy in human brain and behaviour


title: Brain stimulation boosts perceptual learning by altering sensory GABAergic plasticity and functional connectivity

abstract: Interpreting cluttered scenes --a key skill for successfully interacting with our environment-- relies on our ability to select relevant sensory signals while filtering out noise. Training is known to improve our ability to make these perceptual judgements by altering local processing in sensory brain areas. Yet, the brain-wide network mechanisms that mediate our ability for perceptual learning remain largely unknown. Here, we combine transcranial direct current stimulation (tDCS) with multi-modal brain measures to modulate cortical excitability during training on a signal-in-noise task (i.e. detection of visual patterns in noise) and test directly the link between processing in visual cortex and its interactions with decision-related areas (i.e. posterior parietal cortex). We test whether brain stimulation alters inhibitory processing in visual cortex, as measured by magnetic resonance spectroscopy (MRS) of GABA and functional connectivity between visual and posterior parietal cortex, as measured by resting state functional magnetic resonance imaging (rs-fMRI). We show that anodal tDCS during training results in faster learning and decreased GABA+ during training, before these changes occur for training without stimulation (i.e. sham). Further, anodal tDCS decreases occipito-parietal interactions and time-varying connectivity across the visual cortex. Our findings demonstrate that tDCS boosts learning by accelerating visual GABAergic plasticity and altering interactions between visual and decision-related areas, suggesting that training optimises gain control mechanisms (i.e. GABAergic inhibition) and functional inter-areal interactions to support perceptual learning.