title: Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche

abstract: Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Discrete AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.


title: Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex

abstract: Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC.

category: biophysics


title: A Notch-dependent transcriptional mechanism controls expression of temporal patterning factors in Drosophila medulla

abstract: Temporal patterning is an important mechanism for generating a great diversity of neuron subtypes from a seemingly homogenous progenitor pool in both vertebrates and invertebrates. Drosophila neuroblasts have been shown to be temporally patterned by sequentially expressed Temporal Transcription Factors (TTFs). These TTFs are proposed to form a transcriptional cascade based on mutant phenotypes, although direct transcriptional regulation between TTFs has not been verified in most cases. Furthermore, it is not known how the temporal transitions are coupled with generation of the appropriate number of neurons at each stage. We use neuroblasts of the Drosophila optic lobe medulla to address these questions, and show that the expression of TTFs Sloppy-paired 1/ 2 (Slp1/2) is regulated at transcriptional level directly by two other TTFs and the cell-cycle dependent Notch signaling through two cis-regulatory elements. We also show that supplying transcriptional active Notch can rescue the delayed transition into the Slp stage in cell cycle arrested neuroblasts. Our findings reveal a novel Notch-pathway dependent mechanism through which the cell cycle progression regulates the timing of a temporal transition within a TTF transcriptional cascade.

category: developmental biology


title: Mispatterned motile cilia beating causes flow blockage in the epileptic brain

abstract:
Beating of motile cilia at the brain ventricular surface generates rapid flow in an evolutionary conserved pattern mediating the transport of cerebrospinal fluid, but its functional importance has yet to be demonstrated. Here we show disturbance of this transport may contribute to seizure susceptibility. Mice haploinsufficient for FoxJ1, transcription factor regulating motile cilia exhibited cilia-driven flow blockage and increased seizure susceptibility. Mutations in two epilepsy-associated kinases, Cdkl5 and Yes1, in mice resulted in similar cilia-driven flow blockage and increased seizure susceptibility. We showed this arises from disorganized cilia polarity associated with disruption in the highly organized basal body anchoring meshwork. Together these findings suggest mispatterning of cilia-generated flow may contribute to epilepsy and thus might account for seizures unresponsive to current seizure medications.

One sentence summaryEpilepsy is associated with disturbance of cilia motion and mispatterning of fluid transport in the brain ventricles.

category: neuroscience


title: Polarization and cell-fate decision facilitated by the adaptor Ste50 in Saccharomyces cerevisiae

abstract: In response to pheromone, many proteins localize on the plasma membrane of yeast cell to reform it into a polarized shmoo structure. The adaptor protein Ste50, long known as a pheromone signal enhancer critical for shmoo polarization, has never been explored systematically for its localization and function in the polarization process. Time-lapse single-cell imaging and quantitation shown here characterizes Ste50p involvement in the establishment of cell polarity. We found early localization of Ste50p patches on the cell cortex that mark the point of shmoo initiation, these polarity sites move, and patches remain associated with the growing shmoo tip in a pheromone concentration timedependent manner until shmoo maturation. By quantitative analysis we show that polarization corelates with the rising levels of Ste50p, enabling rapid individual cell responses to pheromone that corresponds to a critical level of Ste50p at the initial G1 phase. We exploited the quantitative differences in the pattern of Ste50p expression to corelate with the cell-cell phenotypic heterogeneity showing Ste50p involvement in the cellular differentiation choice. Taken together, these findings present spatiotemporal localization of Ste50p during yeast polarization, suggesting that Ste50p is a component of the polarisome, and plays a critical role in regulating the polarized growth of shmoo during pheromone response.

category: cell biology


title: Vocal tract allometry in a mammalian vocal learner

abstract: Acoustic allometry occurs when features of animal vocalisations can be predicted from body size measurements. Despite this being considered the norm, allometry sometimes breaks, resulting in species sounding smaller or larger than expected for their size. A recent hypothesis suggests that allometry-breaking mammals cluster into two groups: those with anatomical adaptations to their vocal tracts and those capable of learning new sounds (vocal learners). Here we test which mechanism is used to escape from acoustic allometry by probing vocal tract allometry in a proven mammalian vocal learner, the harbour seal (Phoca vitulina). We test whether vocal tract structures and body size scale allometrically in 68 young individuals. We find that both body length and body mass accurately predict vocal tract length and one tracheal dimension. Independently, body length predicts vocal fold length while body mass predicts a second tracheal dimension. All vocal tract measures are larger in weaners than in pups and some structures are sexually dimorphic within age classes. We conclude that harbour seals do comply with anatomical allometric constraints. However, allometry between body size and vocal fold length seems to emerge after puppyhood, suggesting that ontogeny may modulate the anatomy-learning distinction previously hypothesised as clear-cut. We suggest that seals, like other species producing signals that deviate from those expected from their vocal tract dimensions, may break allometry without morphological adaptations. In seals, and potentially other vocal learning mammals, advanced neural control over vocal organs may be the main mechanism for breaking acoustic allometry.

category: animal behavior and cognition


title: Correcting the Hebbian Mistake: Toward a Fully Error-Driven Hippocampus

abstract: The hippocampus plays a critical role in the rapid learning of new episodic memories. Many computational models propose that the hippocampus is an autoassociator that relies on Hebbian learning (i.e., "cells that fire together, wire together"). However, Hebbian learning is computationally suboptimal as it modifies weights unnecessarily beyond what is actually needed to achieve effective retrieval, causing more interference and resulting in a lower learning capacity. Our previous computational models have utilized a powerful, biologically plausible form of error-driven learning in hippocampal CA1 and entorhinal cortex (EC) (functioning as a sparse autoencoder) by contrasting local activity states at different phases in the theta cycle. Based on specific neural data and a recent abstract computational model, we propose a new model called Theremin (Total Hippocampal ERror MINimization) that extends error-driven learning to area CA3 -- the mnemonic heart of the hippocampal system. In the model, CA3 responds to the EC monosynaptic input prior to the EC disynaptic input through dentate gyrus (DG), giving rise to a temporal difference between these two activation states, which drives error-driven learning in the EC[->]CA3 and CA3{leftrightarrow}CA3 projections. In effect, DG serves as a teacher to CA3, correcting its patterns into more pattern-separated ones, thereby reducing interference. Results showed that Theremin, compared with our original model, has significantly increased capacity and learning speed. The model makes several novel predictions that can be tested in future studies.


title: Mapping neurotransmitter systems to the structural and functional organization of the human neocortex

abstract: Neurotransmitter receptors support the propagation of signals in the human brain. How receptor systems are situated within macroscale neuroanatomy and how they shape emergent function remains poorly understood, and there exists no comprehensive atlas of receptors. Here we collate positron emission tomography data from >1 200 healthy individuals to construct a whole-brain 3-D normative atlas of 19 receptors and transporters across 9 different neurotransmitter systems. We find that receptor profiles align with structural connectivity and mediate function, including neurophysiological oscillatory dynamics and resting state hemodynamic functional connectivity. Using the Neurosynth cognitive atlas, we uncover a topographic gradient of overlapping receptor distributions that separates extrinsic and intrinsic psychological processes. Finally, we find both expected and novel associations between receptor distributions and cortical thinning patterns across 13 disorders. We replicate all findings in an independently collected autoradiography dataset. This work demonstrates how chemoarchitecture shapes brain structure and function, providing a new direction for studying multi-scale brain organization.


title: Stratification of the Gut Microbiota Composition Landscape Across the Alzheimer's Disease Continuum in a Turkish Cohort

abstract:
Alzheimers disease (AD) is a heterogeneous neurodegenerative disorder that spans over a continuum with multiple phases including preclinical, mild cognitive impairment, and dementia. Unlike most other chronic diseases there are limited number of human studies reporting on AD gut microbiota in the literature. These published studies suggest that the gut microbiota of AD continuum patients varies considerably throughout the disease stages, raising expectations for existence of multiple microbiota community types. However, the community types of AD gut microbiota were not systematically investigated before, leaving important research gap for diet-based intervention studies and recently initiated precision nutrition approaches aiming at stratifying patients into distinct dietary subgroups. Here, we comprehensively assessed the community types of gut microbiota across the AD continuum. We analyze 16S rRNA amplicon sequencing of stool samples from 27 mild cognitive patients, 47 AD, and 51 non-demented control subjects using tools compatible with compositional nature of microbiota. To characterize gut microbiota community types, we applied multiple machine learning techniques including partitioning around the medoid clustering, fitting probabilistic Dirichlet mixture model, Latent Dirichlet Allocation model, and performed topological data analysis for population scale microbiome stratification based on Mapper algorithm. These four distinct techniques all converge on Prevotella and Bacteroides partitioning of the gut microbiota across AD continuum while some methods provided fine scale resolution in partitioning the community landscape. The Signature taxa and neuropsychometric parameters together robustly classify the heterogenous groups within the cohort. Our results provide a framework for precision nutrition approaches and diet-based intervention studies targeting AD cohorts.

IMPORTANCEThe prevalence of AD worldwide is estimated to reach 131 million by 2050. Most disease modifying treatments and drug trials have failed due partly to the heterogeneous and complex nature of the disease. Unlike other neurodegenerative diseases gut microbiota of AD patients is poorly studied. Recently initiated ambitious precision nutrition initiative or other diet-based interventions can potentially be more effective if the heterogeneous disease such as AD is deconstructed into multiple strata allowing for better identification of biomarkers across narrower patient population for improved results. Because gut microbiota is inherently integral part of the nutritional interventions there is unmet need for microbiota-informed stratification of AD clinical cohorts in nutritional studies. Our study fills in this gap and draws attention to the need for microbiota stratification as one of the essential steps for precision nutrition interventions. We demonstrate that while Prevotella and Bacteroides clusters are the consensus partitions the newly developed probabilistic methods can provide fine scale resolution in partitioning the AD gut microbiome landscape.

category: microbiology


title: The indoor mycobiome of daycare centers is affected by occupancy and climate

abstract:
Many children spend considerable time in daycare centers and may here be influenced by indoor microorganisms, including fungi. In this study, we investigate the indoor mycobiome of 125 daycare centers distributed along strong environmental gradients throughout Norway. Dust samples were collected from doorframes outside and inside buildings using a citizen science sampling approach. Fungal communities in the dust samples were analyzed using DNA metabarcoding of the ITS2 region. We observed a marked difference between the outdoor and indoor mycobiomes. The indoor mycobiome included considerably more yeasts and molds compared to the outdoor samples, with Saccharomyces, Mucor, Malassezia and Penicillium among the most dominant fungal genera. Changes in the indoor fungal richness and composition correlated to numerous variables related to both outdoor and indoor conditions; there was a clear geographic structure in the indoor mycobiome composition that mirrored the outdoor climate, ranging from humid areas in western Norway to drier and colder areas in eastern Norway. Moreover, the number of children in the daycare centers, as well as various building features, influenced the indoor mycobiome composition. We conclude that the indoor mycobiome in Norwegian daycare centers is structured by multiple factors and is dominated by yeasts and molds. This study exemplifies how citizen science sampling enables DNA-based analyses of a high number of samples covering wide geographic areas.

ImportanceWith an alarming increase in chronic diseases like childhood asthma and allergies, there is an increased focus on the exposure of young children to indoor biological and chemical air pollutants. Our study of 125 daycares throughout Norway demonstrates that the indoor mycobiome not only reflects co-occurring outdoor fungi but includes a high abundance of yeast and mold fungi with an affinity for indoor environments. A multitude of factors influence the indoor mycobiome in daycares, including building type, inhabitants, as well as the outdoor environment. Many of the detected yeasts and molds are likely associated with the human body, where some have been coupled to allergies and respiratory problems. Our results call for further studies investigating the potential impact of the identified daycare-associated mycobiomes on children health.


title: Saturated Proteostasis and Nuclear Injuries Defeat Homeostatic Potentials of α-Synuclein Filaments

abstract:
Biogenesis of inclusion bodies (IBs) contributes to protein quality control (PQC). Perinuclear IBs like aggresomes/JUNQs serve as sites for ubiquitin-proteasome mediated protein degradation. The other canonical IB, IPOD, does not degrade but sequesters non-ubiquitinated terminally aggregated proteins to prevent their promiscuous interactions and interferences with other cellular functions. Here, we show that as a deviation from this convention, misfolding-prone -Synuclein is simultaneously deposited into two distinct IBs - Syn-aggresomes and seeding based filamentous inclusions (Syn-filaments), both acting as sites for ubiquitin-proteasome mediated protein degradation. Syn-aggresomes buffer the spontaneous turnover of -Synuclein. Syn-filaments serve the dual purpose of self-sequestration and opportunistic degradation. Counterintuitively, overgrowth of perinuclear Syn-filaments titrates out cellular PQC-pool and challenges the turnover and solubility of other misfolding-prone proteins. Moreover, large Syn-filaments associate with LaminB1, mount mechanical stress on nuclear envelope via dynein, disrupt nuclear integrity, and deregulate stress-triggered transcription of chaperones failing their homeostatic potential.

HighlightsO_LI-Synuclein accumulates into two distinct inclusion bodies for subsequent quality control - cytoskeleton-driven aggresomes and seeding-based filamentous inclusions
C_LIO_LIRepurposing of proteostasis-factors into overgrown filamentous inclusions saturates protein homeostasis
C_LIO_LIGrowing perinuclear filamentous inclusions compromises nuclear integrity by exerting dynein-mediated mechanical stress on nuclear lamina
C_LIO_LIOpportunistic degradation of filamentous inclusions restores cellular equilibrium
C_LI


title: Blocking chondrocyte hypertrophy in conditional Evc knockout mice does not modify osteoarthritis progression

abstract:
BackgroundChondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2 genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO) model of OA.

MethodsOA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n=10 knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1{beta} as an inflammatory insult.

ResultsTamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. Hypertrophic - IHH - and inflammatory - COX-2 - markers co-localized in OA cartilage samples.

ConclusionsTamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

category: pathology


title: Evaluation of small non-coding RNAs as a possible epigenetic mechanism mediating the transition from biotrophy to necrotrophy in the life cycle of Phytophthora infestans

abstract: Phytophthora infestans, causal agent of late blight disease of potatoes, causes billion-dollar losses worldwide each year. This plant pathogen is a hemibiotroph, first feeding on the host and later killing it. Even though the transcription dynamics of this transition are characterized, the role that small non-coding RNAs (sRNAs) might have is still unknown. Furthermore, a bioinformatic pipeline to search and analyze sRNAs in P. infestans, is needed. Using our proposed pipeline, 146 sRNAs were found to be significantly differentially expressed between the evaluated stages of the pathogens life cycle. One hundred of these sRNAs were successfully annotated and classified into nine functional categories. The expression of the genes associated to ten of these sRNAs was validated via qRT-PCR. Among these, the expression levels of genes encoding for effectors were inversely correlated to that of the sRNAs aligning to them, which is expected if sRNAs are indeed regulating their expression. This correlation was not clear for sRNAs in other functional categories and should not be confused with strict causality. This study works as a starting point for considering sRNAs as role players in the transition from biotrophy to necrotrophy in P. infestans when infecting Solanum tuberosum.


title: Silencing of an efflux pump coding gene decreases the efflux rate of pyrazinoic acid in Mycobacterium smegmatis

abstract:
BackgroundTuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). The recommended treatment for TB is based on the use of first-line drugs, including pyrazinamide (PZA). PZA is also a drug used in the treatment of multidrug-resistant TB (MDR-TB) because of its main effect against the latent stage. The main cause of resistance to PZA is mutations in the pncA gene, which compromise the activity of the encoded enzyme pyrazinamidase (PZAse), which hydrolyzes PZA into POA, the active antituberculosis molecule. The mechanism of action of PZA requires that POA is expelled from the bacterium by an efflux mechanism. After that, if the extracellular medium is sufficiently acidic, POA is protonated and returns to the cytosol, releasing the proton and repeating the cycle, resulting lethal to the bacteria. The efflux pump responsible for extruding the POA to the extracellular environment is not yet known. Mycobacterium smegmatis is naturally resistant to PZA and has a 900-fold faster POA efflux rate than MTB, and has the advantage to be a faster growing mycobacterium.

MethodsIn the present study we have silenced the transcription of several genes encoding efflux pumps in M. smegmatis by CRISPRi (CRISPR interference). These genes (MSMEG_0250, MSMEG_3815, MSMEG_0241, MSMEG_5046 and MSMEG_0410) were homologous to efflux pump genes in MTB. POA efflux rate was measured, and a quantitative Waynes test was performed after silencing each gene.

ResultsSilencing of MSMEG_0250, resulted in approximately 5-fold decrease in the POA efflux rate in M. smegmatis (P<0.0001). None of the other silenced genes showed a notable decrease in POA efflux rate.

category: molecular biology


title: Liberibacter solanacearum interacts with host psyllid vitellogenin with its membrane proteins

abstract: Candidatus Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica is a major constraint in carrot production in Israel. Understanding the molecular interactions between the psyllid vector and CLso can facilitate non-chemical approaches for controlling CLso caused-diseases. In this study, we used CLso outer membrane protein (OmpA) and flagellin as baits to screen for psyllid interacting proteins in a yeast-two hybrid assay. We identified psyllid vitellogenin (Vg) protein to interact with both OmpA and flagellin of CLso. As Vg is often involved in innate immunity with its expression tightly linked to autophagy, a major component of the immune response in the cell, we also analyzed the expression of autophagy-related genes to further elucidate this interaction. We used the juvenile hormone (JH-III) to induce the expression of Vg, thapsigargin for suppressing autophagy, and rapamycin for inducing autophagy. The results revealed that Vg negatively regulates autophagy and vice versa. JH-III induced Vg expression significantly suppressed autophagy and, the levels of CLso significantly increased resulting in a significant mortality of the insect. Although the specific role of Vg remains obscure, the findings presented here identify Vg as an important component in the insect immune responses against CLso and may help in understanding the initial molecular response in the vector against Liberibacter.


title: mTOR inhibition via Rapamycin treatment partially reverts the deficit in energy metabolism caused by FH loss in RPE cells

abstract: Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including ageing, genetics and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), that leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. While the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by Mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found, that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of them are associated with UPS. As inhibition of mTORC1 has been previoulsy shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.


title: Impaired lamin localization to the nuclear envelope is responsible for nuclear damage in LMNA mutant iPSC-derived cardiomyocytes

abstract: The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy (LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of LMNA-DCM remains incompletely understood. Using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared to healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and illustrate that defects in nuclear lamina organization can contribute to the nuclear and cellular dysfunction in LMNA-DCM.


title: Plasmodium DDI1 is an essential chromatin-associated protein with a role in DNA-protein crosslink repair

abstract: DDI1 proteins are involved in a variety of cellular processes, including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral aspartyl protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other Apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is essential, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating potential of DDI1 knock-down parasites as a whole organism vaccine. P. falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. Hence, Plasmodium DDI1 could be a dual-therapeutic target for malaria control.


title: Plasmodium DDI1 is a potential dual-therapeutic target and essential chromatin-associated protein


title: Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

abstract: High-throughput short-read sequencing has taken on a central role in research and diagnostics. Literally hundreds of different assays exist today to take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities, and the inertia associated with the research enterprise as a whole have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the long-read ONT MinION by circularizing and rolling circle amplifying the short library molecules using the R2C2 method. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into millions of high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, as well as regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.

category: genomics


title: A simple secretion assay for assessing new and existing myocilin variants

abstract: With the increasing use of molecular genetics approaches for determination of potential disease-causing mutations, it is becoming more important to be able to interpret and act upon the provided results. As an example of such an instance, nearly 300 mutations have been identified in the myocilin (MYOC) gene, which is the most commonly mutated gene causing primary open angle glaucoma. Yet a lack of sufficient information exists for many of these variants, hindering their definitive classification. While the function of MYOC is unclear, biochemically, the vast majority of glaucoma-causing MYOC mutations result in protein non-secretion and intracellular insoluble aggregate formation in cultured cells. Previously we generated a Gaussia luciferase-based MYOC fusion protein to sensitively track secretion of the protein. Herein we applied this same assay to fourteen clinically-derived MYOC variants with varying degrees of predicted pathogenicity and compared the luciferase secretion results with the better established MYOC assay of western blotting. Eight of the variants (G12R, V53A, T204T, P254L, T325T, D380H, D395_E396insDP, and P481S) had not been biochemically assessed previously. Of these, P254L and D395_E396insDP demonstrated significant secretion defects from human embryonic kidney (HEK-293A) cells reminiscent of glaucoma-causing mutations. Overall, we found that the luciferase assay results agreed with western blotting for thirteen of the fourteen variants (93%), suggesting a strong concordance. These results suggest that the Gaussia luciferase assay may be used as a complementary or standalone assay for quickly assessing MYOC variant behavior, and anticipate that these results will be useful in MYOC variant curation and reclassification.

category: biochemistry


title: Lipid phase separation in vesicles enhances TRAIL-mediated cytotoxicity

abstract: Ligand spatial presentation and density play important roles in many signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Currently, Janus particles are most often used to spatially control ligand conjugation, but the technological challenge of manufacturing Janus particles limits adoption for translational applications. Here, we demonstrate that lipid phase separation can be used to spatially control protein presentation onto lipid vesicles. We used this system to study the density dependence of TNF-related apoptosis inducing ligand (TRAIL), a model therapeutic protein that exhibits greater cytotoxicity to cancer cells when conjugated onto a vesicle surface than when administered as a soluble protein. Using assays for apoptosis and caspase activity, we show that phase separated TRAIL vesicles induced higher cytotoxicity to Jurkat cancer cells than uniformly-conjugated TRAIL vesicles, and enhanced cytotoxicity was dependent on the TRAIL domain density. We then assessed this relationship in other cancer cell lines and demonstrated that phase separated TRAIL vesicles only enhanced cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, was unaffected by the TRAIL density. These results indicate unique signaling requirements for each TRAIL receptor and how TRAIL therapy could be tailored depending on the relative levels of expression for cancer receptors of interest. Overall, this work demonstrates a readily adoptable method to control protein conjugation and density on bilayer vesicles that can be easily adopted to other therapeutic nanoparticle systems to improve receptor signaling of nanoparticles targeted to cancer and diseased cells.

category: bioengineering


title: Effect of G4C2-Repeat Expansions on the Motion of Lysosomes Inside Neurites.

abstract: The G4C2 hexanucleotide repeat expansion in the c9orf72 locus is one among a plethora of mutations associated with amyotrophic lateral sclerosis. It accounts for the majority of disease cases. The exact processes underlying the pathology of this mutation remain elusive, yet recent evidence suggests a mechanism that disrupts axonal trafficking. Here, we used a neuronal cell line with and without the G4C2 repeats, and implemented time-resolved local mean squared displacement analysis to characterize the motion of lysosomes inside neurites. Neurites were either aligned along chemically patterned lines, or oriented randomly on the substrate. We confirmed that in the presence of the G4C2 repeats, lysosome motion was affected. Lysosomes had a smaller reach exhibited lower velocity, especially inside aligned neurites. At the same time they became more active with increasing length of the G4C2 repeats when the neurites were randomly oriented. The duration of diffusive and super-diffusive lysosome transport remained unaffected for both neurite geometries and for all lengths of the repeats, but the displacement and velocity was decreased on varying the repeat number and neurite geometry. Lastly, the ratio of anterograde/retrograde/neutral trajectories was affected disparately for the two neurite geometries. Our observations support the hypothesis that impaired axonal trafficking emerges in the presence of the G4C2 hexanucleotide repeat expansion.


title: Characterization of heterologously expressed fibril filaments, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma

abstract: Fibril is a constitutive filament forming cytoskeletal protein of unidentified fold, exclusive to members of genus Spiroplasma. It is hypothesized to undergo conformational changes necessary to bring about Spiroplasma motility through changes in body helicity. However, in the absence of a cofactor such as nucleotide that binds to the protein and drives polymerization, the mechanism driving conformational changes in fibril remains unknown. Sodium dodecyl sulphate (SDS) solubilized the fibril filaments and facilitated fibril purification by affinity chromatography. An alternate protocol for obtaining enriched insoluble fibril filaments has been standardized using density gradient centrifugation method. Visualization of purified protein using electron microscopy demonstrated that it forms filament bundles. Probable domain boundaries of fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of both -helical and {beta}-sheet signatures in FT-IR measurements suggests that fibril filaments consist of assembly of folded globular domains, and not a {beta}-strand based aggregation similar to amyloid fibrils.


title: Proteins mediating different DNA topologies block RNAP elongation with different efficiency

abstract: DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA carrying specific binding sites, while the phage {lambda} CI repressor binds in units of dimers to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by E. coli RNA polymerase through these protein complexes. 186 CI or {lambda} CI bound along unlooped DNA negligibly interfered with transcription by RNAP. More complex topologies induced by scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with rather high affinity, these repressors are not effective roadblocks to transcription.


title: Transcription disrupts DNA-scaffolded bacteriophage repressor complexes


title: Natural transformation protein ComFA exhibits single-stranded DNA translocase activity

abstract:
Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a handful of proteins required for natural transformation in gram-positive bacteria. Its structural resemblance to the DEADbox helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5 to 3. However, this translocase activity does not lead to DNA unwinding in the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake.

ImportanceCompetence, or the ability of bacteria to take up and incorporate foreign DNA in a process called natural transformation is common in the bacterial kingdom, but understanding of the mechanism is still limited. Increasing evidence in several bacteria confirms that long, contiguous stretches of DNA are imported into cells, and yet how bacteria power processive transformation remains unclear. Our finding that ComFA, a DExD-box helicase required for competence in gram-positive bacteria, translocates on single-stranded DNA from 5 to 3, supports the long held hypothesis that ComFA may be the motor powering DNA transport during natural transformation. Moreover, ComFA may be a previously unidentified type of DExD-box helicase--one with the capability of extended translocation on single-stranded DNA.


title: Proteome-wide cross-linking mass spectrometry to identify specific virus capsid-host interactions between tick-borne encephalitis virus and neuroblastoma cells

abstract:
Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. In this work, we demonstrate a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for identification by mass spectrometry. Using the intraviral protein cross-links as an internal control to assess cross-link confidence levels, we identified 22 high confidence unique intraviral cross-links and 59 high confidence unique virus-host protein-protein interactions. The identified host proteins were shown to interact with eight distinct sites on the outer surface of the virus. Notably, we identified an interaction between the substrate-binding domain of heat shock protein family A member 5, an entry receptor for four related flaviviruses, and the hinge region of the viral envelope protein. We also identified host proteins involved in endocytosis, cytoskeletal rearrangement, or located in the cytoskeleton, suggesting that entry mechanisms for tick-borne encephalitis virus could include both clathrin-mediated endocytosis and macropinocytosis. Additionally, cross-linking of the viral proteins showed that the capsid protein forms dimers within tick-borne encephalitis virus, as previously observed with purified C proteins for other flaviviruses. This method enables the identification and mapping of transient virus-host interactions, under near-physiological conditions, without the need for genetic manipulation.

Author summaryTick-borne encephalitis virus is an important human pathogen that can cause severe infection often resulting in life-long neurological complications or even death. As with other viruses, it fully relies on the host cells, and any successful infection starts with interactions between the viral structural proteins and cellular surface proteins. Mapping these interactions is essential both for the fundamental understanding of viral entry mechanisms, and for guiding the design of new antiviral drugs and vaccines. Here, we stabilise the interactions between tick-borne encephalitis virus and human proteins by chemical cross-linking. We then detect the interactions using mass spectrometry and analyse the data to identify protein-protein complexes. We demonstrate that we can visualise the protein interaction interfaces by mapping the cross-linked sites onto the host and viral protein structures. We reveal that there are eight distinct sites on the outer surface of the viral envelope protein that interact with host. Using this approach, we mapped interactions between the tick-borne encephalitis virus envelope protein, and 59 host proteins, identifying a possible new virus receptor. These results highlight the potential of chemical cross-linking coupled with mass spectrometry to identify and map interactions between viral and host proteins.


title: Identification, Characterization and Synthesis of Natural Parasitic Cysteine Protease Inhibitors -- More Potent Falcitidin Analogs

abstract: Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogs of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogs by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfeys analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogs followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and additionally low-micromolar inhibition of -chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.


title: Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile

abstract: The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or  pdcB switch, determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes. These results indicate that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.


title: CTCF blocks anti-sense transcription initiation at divergent gene promoters

abstract: Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is largely unknown. Here by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters, the great majority of which bear proximal CTCF binding sites. Genome editing, chromatin conformation studies, and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its chromatin architectural function. Primary transcript RNA FISH revealed co-bursting of sense and anti-sense transcripts is disfavored, suggesting CTCF-regulated competition for transcription initiation. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.


title: Maintenance of copy number variation at the human salivary agglutinin gene (DMBT1) by balancing selection driven by host-microbe interactions

abstract: Most genetic variation in humans occurs in a pattern consistent with neutral evolution, but a small subset is maintained by balancing selection. Identifying loci under balancing selection is important not only for understanding the processes explaining variation in the genome, but also to identify loci with alleles that affect response to the environment and disease. Several genome scans using genetic variation data have identified the 5 end of the DMBT1 gene as a region undergoing balancing selection. DMBT1 encodes the pattern-recognition glycoprotein DMBT1, also known as SALSA, gp340 or salivary agglutinin. It binds to a wide variety of pathogens through a tandemly-arranged scavenger receptor cysteine-rich (SRCR) domain, with the number of SRCR domains varying in humans. Here we use expression analysis, linkage in pedigrees, and long range single transcript sequencing, to show that the signal of balancing selection is driven by one haplotype usually carrying shorter SRCR repeats, and another usually carrying a longer SRCR repeat, within the coding region of DMBT1. The DMBT1 protein size isoform encoded by a shorter SRCR domain repeat allele showed complete loss of binding of a cariogenic and invasive Streptococcus mutans strain in contrast to the long SRCR allele. Taken together, our results suggest that balancing selection at DMBT1 is due to host-microbe interactions of encoded SRCR tandem repeat alleles.

category: genetics


title: Promoter sequence and architecture determine expression variability and confer robustness to genetic variants

abstract: Genetic and environmental exposures cause variability in gene expression. Although most genes are affected in a population, their effect sizes vary greatly, indicating the existence of regulatory mechanisms that could amplify or attenuate expression variability. Here, we investigate the relationship between the sequence and transcription start site architectures of promoters and their expression variability across human individuals. We find that expression variability is largely determined by a promoters DNA sequence and its binding sites for specific transcription factors. We further demonstrate that flexible usage of transcription start sites within a promoter attenuates variability, providing transcriptional and mutational robustness.


title: Chimeric chromosome landscapes of human somatic cell cultures show dependence on stress and regulation of genomic repeats by CGGBP1.

abstract: Genomes of somatic cells in culture are prone to spontaneous mutations due to errors in replication and DNA repair. Some of these errors, such as chromosomal fusions, are not rectifiable and subject to selection or elimination in growing cultures. Somatic cell cultures are thus expected to generate background levels of potentially stable chromosomal chimeras. A description of the landscape of such spontaneously generated chromosomal chimeras in cultured cells will help us understand the factors affecting somatic mosaicism. Here we show that short homology-associated non-homologous chromosomal chimeras occur in normal human fibroblasts and HEK293T cells at genomic repeats. The occurrence of chromosomal chimeras is enhanced by heat stress and depletion of a repeat regulatory protein CGGBP1. We also present evidence of homologous chromosomal chimeras between allelic copies in repeat-rich DNA obtained by methylcytosine immunoprecipitation. The formation of homologous chromosomal chimeras at Alu and L1 repeats increases upon depletion of CGGBP1. Our data are derived from de novo sequencing from three different cell lines under different experimental conditions and our chromosomal chimera detection pipeline is applicable to long read as well as short read sequencing platforms. These findings present significant information about the generation, sensitivity and regulation of somatic mosaicism in human cell cultures.


title: Sulfotyrosine, an interaction specificity determinant for extracellular protein-protein interactions

abstract: Tyrosine sulfation, a post-translational modification, can enhance and often determine protein-protein interaction specificity. Sulfotyrosyl residues (sTyr) are formed by tyrosyl- protein sulfotransferases (TPSTs) during maturation of certain secreted proteins. Here we consider three contexts for sTyr function. First, a single sTyr residue is critical for high-affinity peptide-receptor interactions in plant peptide hormones and animal receptors for glycopeptide hormones. Second, structurally flexible anionic segments often contain a cluster of two or three sTyr residues within a six-residue span. These sTyr residues are essential for coreceptor binding of the HIV-1 envelope spike protein during virus entry and for chemokine interactions with many chemokine receptors. Third, several proteins that interact with thrombin, central to normal blood-clotting, require the presence of sTyr residues in the context of acidic sequences termed hirudin- like motifs. Consequently, many proven and potential therapeutic proteins derived from blood-consuming invertebrates depend on sTyr residues for their activity. Technical advances in generating and documenting site-specific sTyr substitutions facilitate discovery and analysis, and promise to enable engineering of defined interaction determinants.


title: Modulatory potentials of zerumbone isolated from ginger (Zingiber zerumbet) on eicosanoids: evidence from LPS induced peripheral blood leukocytes.

abstract: Several bioactive molecules from plant origin have been studied for their anti-inflammatory properties. In this study, we deciphered the anti-eicosanoid properties of zerumbone (sesquiterpene) isolated from ginger (Zingiber zerumbet) in LPS induced peripheral blood leukocytes from rats. Molecular interaction between zerumbone (Z) and eicosanoid metabolizing enzymes (COX-2, 5-LOX, FLAP, and LTA4-hydrolase) and receptors (EP-4, BLT-1, and ICAM-1) along with NOS-2 were assessed using Auto-Dock 4.2 docking software. Further, the rat peripheral blood leukocytes were isolated and treated with zerumbone (5M) and activated using bacterial lipopolysaccharide (10nM). Oxidative stress (OS) markers, reactive oxygen species, antioxidant enzymes, COX-2, 5-LOX, BLT-1, EP-4 were assessed along with the activity of COX-2. Zerumbone showed a higher binding affinity with mPGES-1, NOS-2, FLAP, COX-2, LTA-4-hydrolase, and BLT-1 mediators of the eicosanoid pathway. Further, zerumbone significantly (p<0.05) inhibited COX-2, 5-LOX, NOS-2, EP-4, BLT-1, and ICAM-1 expression in LPS induced peripheral blood leukocytes from rats. Zerumbone positively modulates critical enzymes and receptors of eicosanoids in leukocytes activated with lipopolysaccharides. Thus, zerumbone offers a promising therapeutic strategy in the management of inflammation.


title: Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells Are Controlled by pH Balance

abstract: Many biological processes are controlled by cell death and proliferation. The present study found that intracellular pH positively correlated with proliferation but negatively with apoptosis. Acid treatments lowered intracellular pH and preferentially depleted highly proliferating lymphocytes, whereas alkaline treatments increased intracellular pH and enhanced lymphocyte proliferation. Likewise, acid treatments induced apoptosis and alkaline treatments maintained proliferation of tumor cells. These findings support a unified theory of regulation of cell death and proliferation where an intracellular pH balance controls both events, and the mitochondria act as the pH-stats of the cell. Accordingly, the Warburg effect is explained as necessary for proliferating cells to have a high intracellular pH environment to accelerate proliferation and avoid low pH-induced death.


title: Cell Intrinsic Control of Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells by pH


title: A comprehensive evaluation of CRISPR lineage recorders using TraceQC

abstract: The CRISPR-Cas9 genome editing-based lineage tracing system is emerging as a powerful tool to track cell lineages at unprecedented scale and resolution. However, the complexity of CRISPR-Cas9 induced mutations has raised challenges in lineage reconstruction, which requires a unique computational analysis framework. Meanwhile, multiple distinctive CRISPR-based high-throughput lineage recorders have been developed over the years in which the data analysis is incompatible across platforms. To address these challenges, first, we present the TraceQC, a cross-platform open-source package for data processing and quality evaluation of CRISPR lineage tracing data. Second, by using the TraceQC package, we performed a comprehensive analysis across multiple CRISPR lineage recorders to uncover the speed and distribution of CRISPR-induced mutations. Together, this work provides a computational framework for the CRISPR lineage tracing system that should broadly benefit the design and application of this promising technology.

category: bioinformatics


title: The amino acid transporter CG1139 is required for retrograde transport and fast recovery of gut enterocytes after Serratia marcescens intestinal infection

abstract: The intestinal tract is constantly exposed to microbes. Severe infections can arise following the ingestion of pathogenic microbes from contaminating food or water sources. The host directly fights off ingested pathogens with resistance mechanisms, the immune response, or withstands and repairs the damages inflicted either by virulence factors or the host immune effectors, through tolerance/resilience mechanisms. In a previous study, we reported the existence in Drosophila melanogaster of a novel evolutionarily conserved resilience mechanism to intestinal infections with a hemolysin-positive Serratia marcescens strain (SmDb11), the purge of the apical cytoplasm of enterocytes. The epithelium becomes very thin and recovers rapidly, regaining its normal thickness within several hours. Here, we found that this recovery of gut enterocyte morphology is based on the host internal reserves and not on ingested food. Indeed, we observed a retrograde transport of amino acids from the host hemolymph to the enterocytes. We have identified several amino acid transporters required for recovery and we focused on the SLC36 family transporter CG1139. CG1139 is required for the retrograde transport of amino acids. RNA sequencing revealed that genes involved in the positive regulation of growth were observed in wild-type but not CG1139 mutant guts, in which the expression of Myc and genes involved in Insulin signaling is down-regulated. Functional analysis revealed that Myc is also required for the recovery of the thick gut epithelium after infection. Altogether, our results show the importance of an amino acid transporter in the fast regrowth of the enterocytes upon infection. Unexpectedly, we found that this transporter acts non cell-autonomously and can regulate the transcription of other genes, suggesting a signaling function of CG1139 that therefore appears to act as a transceptor.

category: physiology


title: Resistance-guided mining of bacterial genotoxins defines a family of DNA glycosylases

abstract: Unique DNA repair enzymes that provide self-resistance against genotoxic natural products have been discovered recently in bacterial biosynthetic gene clusters (BGCs). The DNA glycosylase AlkZ belongs to a superfamily of uncharacterized proteins found in antibiotic producers and pathogens, but despite its importance to azinomycin B resistance, the roles of AlkZ orthologs in production of other natural products are unknown. Here, we analyze the genomic distribution and use a resistance-based genome mining approach to identify Streptomyces AlkZ homologs associated with known and uncharacterized BGCs. We show that the ortholog associated with synthesis of the alkylating agent hedamycin excises hedamycin-DNA adducts and provides resistance to the genotoxin in cells. Our results define AlkZ in self-resistance to specific antimicrobials and implicate a related but distinct homolog, which we name AlkX, in protection against an array of genotoxins. This work provides a framework for targeted discovery of new genotoxic compounds with therapeutic potential.


title: Endogenous retroviruses mediate transcriptional rewiring in response to oncogenic signaling in colorectal cancer

abstract: Cancer cells exhibit rewired transcriptional regulatory networks that promote tumor growth and survival. However, the processes that configure these pathological networks remain poorly understood. Through a pan-cancer epigenomic analysis, we found that primate-specific endogenous retroviruses (ERVs) are an abundant source of enhancers that show cancer-specific activity. In colorectal cancer and other epithelial tumors, oncogenic MAPK/AP1 signaling drives activation of enhancers derived from the primate-specific ERV family LTR10. Functional studies in colorectal cancer cells revealed that LTR10 elements are involved in regulating multiple genes associated with tumorigenesis, including ATG12. Within the human population, individual LTR10 elements show frequent structural variation resulting from a highly mutable internal tandem repeat region, which affects AP1 binding activity. Our findings reveal that ERV-derived enhancers mediate transcriptional dysregulation in response to oncogenic signaling, and shape the evolution of cancer-specific regulatory networks.


title: Double-edged role of resource competition in gene expression noise and control

abstract: Despite extensive investigation demonstrating that resource competition can significantly alter the circuits deterministic behaviors, a fundamental issue is how resource competition contributes to the gene expression noise and how the noise can be controlled. Utilizing a two-gene circuit as a prototypical system, we uncover a surprising double-edged role of resource competition in gene expression noise: the competition decreases noise through a resource constraint but generates its own type of noise which we name as "resource competitive noise." Utilization of orthogonal resources enables retaining the noise reduction conferred by resource constraint while removing the added resource competitive noise. The noise reduction effects are studied using three negative feedback controller types: negatively competitive regulation (NCR), local, and global controllers, each having four placement architectures in the protein biosynthesis pathway (mRNA or protein inhibition on transcription or translation). Our results show that both local and NCR controllers with mRNA-mediated inhibition are efficacious at reducing noise, with NCR controllers demonstrating a superior noise-reduction capability. We also find that combining negative feedback controllers with orthogonal resources can improve the local controllers. This work provides deep insights into the origin of stochasticity in gene circuits with resource competition and guidance for developing effective noise control strategies.

category: synthetic biology


title: Onset and stepwise extensions of recombination suppression are common in mating-type chromosomes of Microbotryum anther-smut fungi

abstract: Sex chromosomes and mating-type chromosomes can display large genomic regions without recombination. Recombination suppression often extended stepwise with time away from the sex- or mating-type-determining genes, generating evolutionary strata of differentiation between alternative sex or mating-type chromosomes. In anther-smut fungi of the Microbotryum genus, recombination suppression evolved repeatedly, linking the two mating-type loci and extended multiple times in regions distal to the mating-type genes. Here, we obtained high-quality genome assemblies of alternative mating types for four Microbotryum fungi. We found an additional event of independent chromosomal rearrangements bringing the two mating-type loci on the same chromosome followed by recombination suppression linking them. We also found, in a new clade analysed here, that recombination suppression between the two mating-type loci occurred in several steps, with first an ancestral recombination suppression between one of the mating-type locus and its centromere; later, completion of recombination suppression up to the second mating-type locus occurred independently in three species. The estimated dates of recombination suppression between the mating-type loci ranged from 0.15 to 3.58 million years ago. In total, this makes at least nine independent events of linkage between the mating-type loci across the Microbotryum genus. Several mating-type locus linkage events occurred through the same types of chromosomal rearrangements, where similar chromosome fissions at centromeres represent convergence in the genomic changes leading to the phenotypic convergence. These findings further highlight Microbotryum fungi as excellent models to study the evolution of recombination suppression.

category: evolutionary biology


title: A neural substrate for Bayesian integration in human parietal cortex

abstract: Making adaptive decisions often requires inferring unobservable states based on unreliable information. Bayesian logic prescribes that individuals form probabilistic beliefs about a state by integrating the likelihood of new evidence with their prior beliefs, but human neuroimaging studies on probability representations have not typically examined this integration process. We developed an inference fMRI task in which participants estimated the posterior probability of a hidden state while we parametrically modulated the prior probability of the state, the likelihood of the supporting evidence, and a monetary penalty for estimation inaccuracy. Consistent with a neural substrate for Bayesian integration, activation in left posterior parietal cortex tracked the estimated posterior probability of the solicited state and its components of prior probability and likelihood, all independently of expected value. This activation further reflected deviations in individual reports from objective probabilities. Thus, this region may provide a neural substrate for humans ability to approximate Bayesian inference.


title: Mllt11 regulates the migration and neurite outgrowth of cortical projection neurons during development

abstract: The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are generally two types of cortical projection neurons: those that project locally and interhemispherically, and those that project to sub-cerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate, particularly during the formation of the upper or superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is a required for neuritogenesis and proper migration of upper layer cortical projection neurons. Loss of Mllt11 in the superficial cortex leads to a severe reduction in fibres crossing the corpus callosum, a progressive loss in the maintenance of upper layer projection neuron gene expression, and dysplasia of dendritic arborisation patterns. Proteomic analysis revealed that Mllt11 associates with cytoskeletal components including stabilized microtubules consistent with a role in neuronal migration and neuritogenesis. Taken together, our findings support a role for Mllt11 in promoting the formation of mature projection neuron morphologies and connectivity in the cerebral cortex.


title: Molecular mechanism of Arp2/3 complex inhibition by Arpin

abstract: Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpins mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-[A] resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within this helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.


title: Near-infrared carbon nanotube tracking reveals the nanoscale extracellular space around synapses

abstract: We provide evidence of a local synaptic nano-environment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nano-environment might play a role in the regulation of brain activity.


title: Neuronally Produced Betaine Acts via a Novel Ligand Gated Ion Channel to Control Behavioural States

abstract: Trimethyl glycine, or betaine, is an amino acid derivative found in diverse organisms, from bacteria to plants and animals. It can function as an osmolyte to protect cells against osmotic stress, and building evidence suggests betaine may also play important functional roles in the nervous system. However, despite growing interest in betaines roles in the nervous system, few molecular mechanisms have been elucidated. Here we identify the expression of betaine synthesis pathway genes in the nervous system of the nematode worm, C. elegans. We show that betaine, produced in a single pair of interneurons, the RIMs, can control complex behavioural states. Moreover, we also identify and characterise a new betaine-gated inhibitory ligand gated ion channel, LGC-41, which is required for betaine related behavioural changes. Intriguingly we observed expression of LGC-41 in punctate structures across several sensory and interneurons, including those synaptically connected to the RIMs. Our data presents a neuronal molecular mechanism for the action of betaine, via a specific receptor, in the control of complex behaviour within the nervous system of C. elegans. This may suggest a much broader role for betaine in the regulation of animal nervous systems than previously recognised.


title: Chlamydomonas ARMC2/PF27 is an obligate cargo adapter for IFT of radial spokes

abstract: Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. Chlamydomonas ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 comigrated on anterograde trains. In contrast, a cargo and an adapter of inner and outer dynein arms moved independently of ARMC2, indicating that unrelated cargoes distribute stochastically onto the IFT trains. After concomitant unloading at the flagellar tip, RSP3 attached to the axoneme whereas ARMC2 diffused back to the cell body. In armc2/pf27 mutants, IFT of radial spokes was abolished and the presence of radial spokes was limited to the proximal region of flagella. We conclude that ARMC2 is a cargo adapter required for IFT of radial spokes to ensure their assembly along flagella. ARMC2 belongs to a growing class of cargo-specific adapters that enable flagellar transport of preassembled axonemal substructures by IFT.


title: SETD2 negatively regulates cell size through its catalytic activity and SRI domain

abstract: Cell size varies between cell types but is tightly regulated by cell-intrinsic and extrinsic mechanisms. Cell-size control is important for cell function and changes in cell size are frequently observed in cancer cells. Here we uncover a non-canonical role of SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription regulation, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Although most of SETD2s cellular functions have been linked to this activity, several non-histone substrates of SETD2 have recently been identified - some of which have been linked to novel functions of SETD2 beyond chromatin regulation. Using multiple, independent perturbation strategies we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that this function is dependent on the catalytic activity of SETD2 but independent of H3K36 methylation. Paradoxically, ectopic overexpression of a decoy SRI domain also increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.


title: Netrins and receptors control Drosophila optic lobe organization and transmedullary neuron axon targeting

abstract: During development, integration of temporal patterning and spatial patterning of neural progenitors as well as Notch-dependent binary fate choice between sister neurons contribute to generation of neural diversity. How these upstream neural fate specification programs regulate downstream effector genes to control axon targeting and neuropil assembly remains less well-understood. Here we show that Notch-dependent binary fate choice in Drosophila medulla neurons regulates the expression of Netrin, and that Netrin pathway controls axon guidance of transmedullary (Tm) neurons and contributes to the organization of optic lobe neuropils. Netrins are enriched in the lobula where Tm axons target, and the attractive receptor Frazzled is expressed broadly in medulla neurons, while the repulsive receptor Unc-5 is excluded from Tm neurons and this is necessary for their correct targeting to the lobula. Frazzled is required collectively in a group of early-born Tm neurons to establish the inner optic chiasm (IOC) through which Tm axons target lobula. In addition, Frazzled acts in the layer-specific targeting step of Tm3 and Tm4 cell-autonomously, and is also required for the formation of the lobula branch of TmY3. Moreover, we show that the diffusibility of Netrins is necessary for Netrin enrichment in the lobula, the IOC formation and layer-specific targeting of Tm3 and Tm4. Netrin enrichment in the lobula is promoted by Frazzled expressed in Tm neurons, while Unc-5 appears to have an opposite role in Netrin distribution. Furthermore, we show that Netrin B is expressed in the Notch-on hemilineage of medulla neurons including most Tm and TmY neurons that target lobula, and loss of Su(H) abolished NetB expression in the medulla. Without medulla-originated NetB, Tm axons from late-born medulla columns cannot join the IOC. Therefore, the Notch-dependent binary fate choice regulates the assembly of the optic lobe neuropils by controlling the expression of Netrin.


title: Mem3DG: Modeling Membrane Mechanochemical Dynamics in 3D using Discrete Differential Geometry

abstract:
Biomembranes adopt varying morphologies that are vital to cellular functions. Many studies use computational modeling to understand how various mechanochemical factors contribute to membrane shape transformations. Compared to approximation-based methods (e.g., finite element method), the class of discrete mesh models offers greater flexibility to simulate complex physics and shapes in three dimensions; its formulation produces an efficient algorithm while maintaining coordinate-free geometric descriptions. However, ambiguities in geometric definitions in the discrete context have led to a lack of consensus on which discrete mesh model is theoretically and numerically optimal; a bijective relationship between the terms contributing to both the energy and forces from the discrete and smooth geometric theories remains to be established. We address this and present an extensible framework, Mem3DG, for modeling 3D mechanochemical dynamics of membranes based on Discrete Differential Geometry (DDG) on triangulated meshes. The formalism of DDG resolves the inconsistency and provides a unifying perspective on how to relate the smooth and discrete energy and forces. To demonstrate, Mem3DG is used to model a sequence of examples with increasing mechanochemical complexity: recovering classical shape transformations such as 1) biconcave disk, dumb-bell, and unduloid and 2) spherical bud on spherical, flat-patch membrane; investigating how the coupling of membrane mechanics with protein mobility jointly affects phase and shape transformation. As high-resolution 3D imaging of membrane ultrastructure becomes more readily available, we envision Mem3DG to be applied as an end-to-end tool to simulate realistic cell geometry under user-specified mechanochemical conditions.

Why it mattersCellular membranes have shapes and shape changes which characterize cells/organelles, and support nutrient trafficking among other critical processes. Modeling membrane shape changes using mechanical principles can provide insight into how cells robustly bend membranes to support life. Mathematical and computational strategies to solve the equations describing membrane shape evolution can be complex and challenging without simplifying assumptions. Here, we present a new, general, numerical approach to model arbitrary 3D membrane shapes in response to interaction with curvature sensing and generating membrane proteins. The accompanying implementation, Mem3DG, is a software tool to make computational membrane mechanics accessible to the general researcher.

HighlightsO_LIIntroduces a discrete theory for membrane mechanics which connects with smooth theory
C_LIO_LIDiscrete energy/force are functions of basic geometric quantities
C_LIO_LIRecipes for extending the discrete framework with additional physics are provided
C_LIO_LIDescribes a user-friendly software implementation called Mem3DG
C_LIO_LIMem3DG is designed to facilitate modeling in tandem with experimental studies
C_LI

Significance and noveltyHelfrich Hamiltonian is widely used to model biomembranes. Many numerical methods have been developed to solve the geometric PDE. Compared to approximation-based methods, discrete-mesh-based models have many advantages when incorporating multiphysics in 3D due to their flexibility, efficiency, and straightforward implementation. However, there is no consensus on which mesh-based model is optimal, and a connection between the discrete and smooth geometric theory remains obscure. In this work, we provide a unifying perspective by identifying fundamental geometric invariants of the discrete force derived from a discrete energy. As a demonstration of generality of the framework to various physics, we follow a formulaic procedure to derive additional physics such as interfacial line tension, surface-bulk adsorption, protein lateral diffusion, and curvature-dependent protein aggregation.


title: Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering

abstract:
The robust modularity of biological components that are assembled into complex functional systems is central to synthetic biology. Here we apply modular "plug and play" design principles to a microscale solid phase protein display system that enables protein purification and functional assays for biotherapeutics. Specifically, we capture protein molecules from cell lysates on polyacrylamide hydrogel display beads ( PHD beads), made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA). Anchors form covalent bonds with fusion proteins bearing cognate tag recognition (SNAP and Halo-tags) in specific, orthogonal and stable fashion. Given that these anchors are copolymerised throughout the 3D structure of the beads, proteins are also distributed across the entire bead sphere, allowing attachment of [~]109 protein molecules per bead (O 20 m). This mode of attachment reaches a higher density than possible on widely used surface-modified beads, and additionally mitigates surface effects that often complicate studies with proteins on beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher and SnpTag). Proteins can be displayed in their monomeric forms, but also reformatted as a multivalent display (using secondary capture modules that create branches) to test the contributions of avidity and multivalency towards protein function. Finally, controlled release of modules by irradiation of light is achieved by incorporating the photocleavable protein PhoCl: irradiation severs the displayed protein from the solid support, so that functional assays can be carried out in solution. As a demonstration of the utility of valency engineering, an antibody drug screen is performed, in which an anti-TRAIL-R1 scFv protein is released into solution as monomers-hexamers, showing a [~]50-fold enhanced potency in the pentavalent format. The ease of protein purification on solid support, quantitative control over presentation and release of proteins and choice of valency make this experimental format a versatile, modular platform for large scale functional analysis of proteins, in bioassays of protein-protein interactions, enzymatic catalysis and bacteriolysis.

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title: Pan-cancer mapping of single T cell profiles reveals a TCF1:CXCR6-CXCL16 regulatory axis essential for effective anti-tumor immunity

abstract: Unleashing cytotoxic CD8+ T cells for effective cancer treatment requires understanding T cell states across different tumor microenvironments. Here, we developed an algorithm to recover both shared and tumor type specific programs and used it to analyze a scRNA-seq compendium of 38,852 CD8+ T cells from 141 patients spanning nine different human cancers. We uncovered a pan-cancer T cell dysfunction program that was predictive of clinical responses to immunotherapy and highlighted CXCR6 as a pan-cancer marker of dysfunctional T cells. In mouse models, CXCR6 increased following checkpoint blockade and was repressed by TCF1. Its ligand, CXCL16, was expressed primarily by myeloid cells, and was co-regulated with antigen presentation genes. CXCR6 deletion decreased Tox, CX3CR1, and Bcl2 expression, predisposing dysfunctional PD-1+Tim3+CD8+ T cells to apoptosis, and compromising tumor growth control. Our approach discovered a TCF1:CXCR6-CXCL16 regulatory axis essential for effective anti-tumor immunity, revealing a new perspective on T cell dysfunction and new opportunities for modulating this cell state.

category: immunology


title: Identification and tracking of alloreactive T cell clones in Rhesus Macaques through the RM-scTCR-Seq platform.

abstract: T cell receptor clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cells transcriptional program with its T cell receptor (TCR) identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques, a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline,  RM-scTCR-Seq, which, for the first time, enables RM specific single cell TCR amplification, reconstitution and pairing of RM TCRs with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.


title: Polyamines mediate enterovirus attachment directly and indirectly through cellular heparan sulfate synthesis.

abstract: Productive viral infection begins with attachment to a susceptible cell, and viruses have evolved complex mechanisms to attach to and subsequently enter cells. Prior to engagement with a cellular receptor, viruses frequently interact with nonspecific attachment factors that can facilitate virus-receptor interactions and viral entry. Polyamines, small positively-charged molecules abundant in mammalian cells, mediate viral attachment, though the mechanism was not fully understood. Using the Coxsackievirus B3 (CVB3) enterovirus model system, we show that polyamines mediate viral attachment both directly and indirectly. The polyamine putrescine specifically enhances viral attachment to cells depleted of polyamines. Putrescines positive charge mediates its ability to enhance viral attachment, and polyamine analogs are less efficient at mediating viral attachment. In addition to this direct role of polyamines in attachment, polyamines facilitate the cellular expression of heparan sulfates, negatively-charged molecules found on the cell surface. In polyamine-depleted cells, heparan sulfates are depleted from the surface of cells, resulting in reduced viral attachment. We find that this is due to polyamines role in the process of hypusination of eukaryotic initiation factor 5A, which facilitates cellular translation. These data highlight the important role of polyamines in mediating cellular attachment, as well as their function in facilitating cellular heparan sulfate synthesis.


title: Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

abstract: Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding--analysis of extra-organismal DNA released into the environment--allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set "Amph16S" had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

category: ecology


title: Worldwide reef surveys highlight different adaptive responses to heat between coral taxa

abstract: Coral reefs around the world are under threat due to widespread decline of hard corals caused by anomalous heat waves. Different coral taxa are known to have different sensitivities to heat. However, little is known about how long-term effects of heat exposure differ between coral taxa, nor in particular how such effects might drive adaptive processes. Here, we combined worldwide reef survey data with remotely sensed thermal anomalies to evaluate how local rates of taxa-specific coral cover were associated with heat stress. We found a negative association between coral cover and heat stress: weaker for massive and encrusting Poritidae, for meandroid Favidae and Mussidae; and stronger for branching, tabular and corymbose Acroporidae. Additionally, we found that coral cover decline driven by recent heat stress (measured as the year before a survey) was mitigated by long-term past heat (measured since 1985) in branching, tabular and corymbose Acroporidae and Pocilloporidae, but not in Poritidae, Favidae and Mussidae. This mitigation of coral decline was not observed under the effect of short-term past heat (measured during the two previous years), suggesting that these presumptive adaptive processes are the result of evolutionary adaptation, rather than of physiological acclimatization. We finally queried the Coral Trait Database to characterize morphological, physiological and reproductive traits of different taxa showing similar heat-responses. Further genomics studies should confirm the evolutionary origin of such divergent adaptive responses to heat stress.


title: Worldwide analysis of reef surveys sorts coral taxa by (adaptive) response to heat stress


title: The colonial legacy of herbaria

abstract: Herbarium collections shape our understanding of the worlds flora and are crucial for addressing global change and biodiversity conservation. The formation of such natural history collections, however, are not free from sociopolitical issues of immediate relevance. Despite increasing efforts addressing issues of representation and colonialism in natural history collections, herbaria have received comparatively less attention. While it has been noted that the majority of plant specimens are housed in the global North, the extent of this disparity has not been rigorously quantified to date. Here, by analyzing over 85 million specimen records and surveying herbaria across the globe, we assess the colonial legacy of botanical collections and how we may move towards a more inclusive future. We demonstrate that colonial exploitation has contributed to an inverse relationship between where plant biodiversity exists in nature and where it is housed in herbaria. Such disparities persist in herbaria across physical and digital realms despite overt colonialism having ended over half a century ago, suggesting ongoing digitization and decolonization efforts have yet to alleviate colonial-era discrepancies. We emphasize the need for acknowledging the inconvenient history of herbarium collections and the implementation of a more equitable, global paradigm for their collection, curation, and use.


title: Dimensionality and modularity of adaptive variation: Divergence in threespine stickleback from diverse environments

abstract: In nature, populations are subjected to a wide variety of environmental conditions that affect fitness and induce adaptive or plastic responses in traits, resulting in phenotypic divergence between populations. The dimensionality of that divergence, however, remains contentious. At the extremes, some contend that populations diverge along a single axis of trait covariance with greatest availability of heritable variation, even if this does not lead a population directly to its fitness optimum. Those at the other extreme argue that selection can push populations towards their fitness optima along multiple phenotype axes simultaneously, resulting in divergence in numerous dimensions. Here, we address this debate using populations of threespine stickleback (Gasterosteus aculeatus) in the Cook Inlet region of southern Alaska from lakes with contrasting ecological conditions. We calculated effective dimensionality of divergence in several trait suites (defensive, swimming, and trophic) thought to be under correlated selection pressures, as well as across all traits. We also tested for integration among the trait suites and between each trait suite and the environment. We found that populations in the Cook Inlet radiation exhibit dimensionality of phenotype high enough to preclude a single axis of divergence.


title: A macromutation eliminates colour patterning in captive butterflies

abstract: Captive populations often harbor variation that is not present in the wild due to artificial selection. Recent efforts to map this variation have provided insights into the genetic and molecular basis of variation. Heliconius butterflies display a large array of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here we sought to identify the genetic basis of an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78kb deletion at the cortex wing patterning locus as the ivory mutation. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5 region of the cortex gene that includes a facultative 5UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.