title: The genetics of assisted gene flow: immediate costs and long-term benefits

abstract: Plant and animal populations are facing several novel risks such as human-mediated habitat fragmentation and climate change that threaten their long-term productivity and persistence. With the genetic health of many populations deteriorating due to climate change outpacing physiological adaptation, human interventions in the form of assisted gene flow (AGF) may provide genetic variation to adapt populations to predicted climate change scenarios and result in more robust and productive populations. We ran genetic simulations to mimic a variety of AGF scenarios and measured their outcomes on population-level fitness to answer the question: in which circumstances is it worthwhile to perform AGF? Based on the parameters we explored, AGF may be harmful in certain situations over the short term (e.g., the first [~]10-20 generations), due to outbreeding depression and introducing deleterious genetic variation. Moreover, under many parameter sets, the benefits of AGF were relatively weak or took many generations to accrue. In general, when the adaptive trait is controlled by many loci of small effect, the benefits of assisted gene flow take much longer to realize-potentially too long for most climate-related management decisions. We also show that when translocation effort is divided across several generations and outbreeding depression is strong, the recipient population experiences a smaller decrease in fitness as compared to moving all individuals in a single effort. Importantly, in most cases, we show that the genomic integrity of the recipient population remains relatively intact following AGF; the amount of genetic material from the donor population typically ends up constituting no more of the recipient populations genome than the fraction introduced. Our results will be useful for conservation practitioners and silviculturists, for instance, aiming to intervene and adaptively manage so that populations maintain a robust genetic health and maintain productivity into the future given anthropogenic climate change.


title: Comparative anatomy and phylogenetic contribution of intracranial osseous canals and cavities in armadillos and glyptodonts (Xenarthra, Cingulata).

abstract: The evolutionary history of the Cingulata, as for many groups, remains a highly debated topic to this day, particularly for one of their most emblematic representatives: the glyptodonts. There is no consensus among morphological and molecular phylogenies relative to their position within Cingulata. As demonstrated by recent works, the study of the internal anatomy constitutes a promising path for enriching morphological matrices for the phylogenetic study of armadillos. However, internal cranial anatomy remains under-studied in the Cingulata. Here we explored and compared the anatomy of intracranial osseous canals and cavities in a diverse sample of extant and extinct cingulates, including the earliest well-preserved glyptodont crania. The virtual 3D reconstruction (using X-ray microtomography) of selected canals, i.e., the nasolacrimal canal, the palatine canal, the sphenopalatine canal, the canal for the frontal diploic vein, the transverse canal, the orbitotemporal canal, the canal for the capsuloparietal emissary vein and the posttemporal canal, and alveolar cavities related to cranial vascularization, innervation or tooth insertion allowed us to compare the locations, trajectories and shape of these structures and to discuss their potential interest for cingulate systematics. We tentatively reconstructed evolutionary scenarios for eight selected traits on these structures, in which glyptodonts often showed a greater resemblance to pampatheres, to the genus Proeutatus and/or to chlamyphorines. This latter pattern was partly congruent with recent molecular hypotheses, but more research is needed on these resemblances and on the potential effects of development and allometry on the observed variations. Overall, these comparisons have enabled us to highlight new anatomical variation that may be of great interest to further explore the evolutionary history of cingulates and the origins of glyptodonts on a morphological basis.

category: evolutionary biology


title: A systematic benchmark of Nanopore long read RNA sequencing for transcript level analysis in human cell lines

abstract: The human genome contains more than 200,000 gene isoforms. However, different isoforms can be highly similar, and with an average length of 1.5kb remain difficult to study with short read sequencing. To systematically evaluate the ability to study the transcriptome at a resolution of individual isoforms we profiled 5 human cell lines with short read cDNA sequencing and Nanopore long read direct RNA, amplification-free direct cDNA, PCR-cDNA sequencing. The long read protocols showed a high level of consistency, with amplification-free RNA and cDNA sequencing being most similar. While short and long reads generated comparable gene expression estimates, they differed substantially for individual isoforms. We find that increased read length improves read-to-transcript assignment, identifies interactions between alternative promoters and splicing, enables the discovery of novel transcripts from repetitive regions, facilitates the quantification of full-length fusion isoforms and enables the simultaneous profiling of m6A RNA modifications when RNA is sequenced directly. Our study demonstrates the advantage of long read RNA sequencing and provides a comprehensive resource that will enable the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution.

category: genomics


title: Structures of active melanocortin-4 receptor--Gs-protein complexes with NDP-α-MSH and setmelanotide

abstract: The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R- Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP--MSH and setmelanotide, with 2.9 [A] and 2.6 [A] resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP--MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor- Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.

category: biochemistry


title: Relationship between Deceleration Morphology and Phase Rectified Signal Averaging-based Parameters during Labor

abstract: During labor, uterine contractions trigger the response of the autonomic nervous system (ANS) of the fetus, producing sawtooth-like decelerations in the fetal heart rate (FHR) series. Under chronic hypoxia, ANS is known to regulate FHR differently with respect to healthy fetuses. In this study, we hypothesized that such different ANS regulation might also lead to a change in the FHR deceleration morphology. The hypothesis was tested in an animal model comprising 7 normoxic and 5 chronically hypoxic fetuses that underwent a protocol of umbilical cord occlusions (UCOs). Deceleration morphologies in the fetal inter-beat time interval (FRR) series were modeled using a trapezoid with four parameters, i.e., baseline b, deceleration depth a, UCO response time{tau} u and recovery time{tau} r. Comparing normoxic and hypoxic sheep, we found a clear difference for{tau} u (24.8 {+/-} 9.4 vs 39.8 {+/-} 9.7 s; p < 0.05), a (268.1 {+/-} 109.5 vs 373.0 {+/-} 46.0 ms; p < 0.1) and {Delta}{tau} ={tau} u -{tau} r (13.2 {+/-} 6.9 vs 23.9 {+/-} 7.5 s; p < 0.05). Therefore, the animal model supported the hypothesis that hypoxic fetuses have a longer response time{tau} u and larger asymmetry {Delta}{tau} as a response to UCOs. Assessing these morphological parameters during labor is challenging due to non-stationarity, phase desynchronization and noise. For this reason, in the second part of the study, we quantified whether acceleration capacity (AC), deceleration capacity (DC), and deceleration reserve (DR), computed through Phase-Rectified Signal Averaging (PRSA, known to be robust to noise), were correlated with the morphological parameters. DR and DC correlated with {Delta}{tau} and{tau} u for a wide range of the PRSA parameter T (max Pearsons correlation{rho} = 0.9, p < 0.05, and{rho} = 0.6, p < 0.1, respectively). In conclusion, deceleration morphologies have been found to differ between normoxic and hypoxic sheep fetuses during UCOs. The same difference can be assessed through PRSA based parameters, further motivating future investigations on the translational potential of this methodology on human data.

category: bioengineering


title: 3D Flexible Refinement: Structure and Motion of Flexible Proteins from Cryo-EM

abstract: Single particle cryo-EM excels in determining static structures of biological macromolecules such as proteins. However, many proteins are dynamic, with their motion inherently linked to their function. Recovering the continuous motion and detailed 3D structure of flexible proteins from cryo-EM data has remained an open challenge. We introduce 3D Flexible Refinement (3DFlex), a motion-based deep neural network model of continuous heterogeneity. 3DFlex directly exploits the knowledge that conformational variability of a protein is often the result of physical processes that transport density over space and tend to conserve mass and preserve local geometry. From 2D image data, the 3DFlex model jointly learns a single canonical 3D map, latent coordinate vectors that specify positions on the proteins conformational landscape, and a flow generator that, given a latent position as input, outputs a 3D deformation field. This deformation field convects the canonical map into appropriate conformations to explain experimental images. Applied to experimental data, 3DFlex learns non-rigid motion spanning several orders of magnitude while preserving high-resolution details of secondary structure elements. Further, 3DFlex resolves canonical maps that are improved relative to conventional refinement methods because particle images contribute to the maps coherently regardless of the conformation of the protein in the image. Together, the ability to obtain insight into motion in macromolecules, as well as the ability to resolve features that are usually lost in cryo-EM of flexible specimens, will provide new insight and allow new avenues of investigation into biomolecular structure and function.

category: biophysics


title: The beta-catenin-target Fascin-1, altering hepatocyte differentiation, is a new marker of immature cells in hepatoblastomas

abstract:
BACKGROUND & AIMS{beta}-catenin is a well-known effector of the Wnt pathway and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of {beta}-catenin are highly frequent in pediatric liver primary tumors. Those mutations are mostly heterozygous allowing the co-expression of wild-type (WT) and mutated {beta}-catenins in tumor cells. We investigated the interplay between WT and mutated {beta}-catenins in liver tumor cells, and searched for new actors of the {beta}-catenin pathway.

METHODSUsing an RNAi strategy in {beta}-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of {beta}-catenin, carried mainly by, respectively, WT and mutated proteins. Their impact was characterized using transcriptomic and functional analyses. We studied mice that develop liver tumors upon activation of {beta}-catenin in hepatocytes (APCKO and {beta}-catenin{Delta}exon3 mice). We made use of transcriptomic data from mouse and human HB specimens and analyzed samples by immunohistochemistry.

RESULTSWe highlighted an antagonist role of WT and mutated {beta}-catenins on hepatocyte differentiation as attested by alteration of hepatocyte markers expression and bile canaliculi formation. We characterized Fascin-1 as a target of {beta}-catenin involved in hepatocyte differentiation. Using mouse models that allow the formation of two phenotypically distinct tumors (differentiated or undifferentiated), we found that Fascin-1 expression is higher in undifferentiated tumors. Finally, we found that Fascin-1 is a specific marker of the embryonal component in human HBs.

CONCLUSIONSIn mice and human, Fascin-1 expression is linked to loss of differentiation and polarity of hepatocytes. Thus, we highlighted Fascin-1 as a new player in the modulation of hepatocyte differentiation associated to {beta}-catenin pathway alteration in the liver.

Data Transparency Statementstudy materials will be made available to other researchers upon request.

category: cancer biology


title: Defining function of wild-type and patient specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

abstract: In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss or gain of function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding TP53 effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet TP53 mutations when present in tumors are associated with poor prognosis. Employing a kRASG12D-driven ERMS tumor model and newly generated tp53 null (tp53-/-) zebrafish, we define both wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumor initiation, where tp53 loss expands tumors initiation from <35% to >97% of animals. Next, characterizing three patient-specific mutants finds that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, while the TP53P153{Delta} and TP53Y220C mutants define two structural mutations that predispose to head musculature ERMS.


title: The memory of airway epithelium damage in smokers and COPD patients

abstract:
BackgroundChronic obstructive pulmonary disease (COPD) is a devastating lung disease, representing the third cause of mortality worldwide. In COPD, the bronchial epithelium displays several structural and functional abnormalities affecting barrier integrity, cell polarity, and differentiation, as well as epithelial-to-mesenchymal transition (EMT) and inflammation. Although COPD is currently considered an irreversible disease, the (ir)reversible nature of epithelium changes ex vivo remains poorly known.

MethodsThe persistence of COPD epithelial abnormalities was addressed in very long-term (10 weeks) primary cultures of air/liquid interface (ALI)-reconstituted airway epithelium from non-smoker controls, smoker controls, and COPD patients. The role of inflammation was also explored by stimulating ALI cultures with a cytokine mix consisting of TNF-, IL-6 and IL-1{beta}. Finally, the cellular niche holding epithelial memory was studied by exploiting a single cell RNA-sequencing database.

ResultsAlmost all epithelial defects (barrier dysfunction, impaired polarity, lineage abnormalities) observed in smokers and COPD patients persisted in vitro up to week 10, except IL-8/CXCL-8 release and EMT which declined over time. Cytokine treatment induced COPD-like changes and reactivated EMT in COPD cells. Progenitor cells of large and small airways, namely basal and club cells, exhibited EMT-related signatures reminiscent of features observed in situ.

ConclusionsThe airway epithelium from smokers and COPD patients displays a memory of its native state and previous injuries by cigarette smoking, which is multidimensional and sustained for years. This memory probably resides in progenitor cells of the airway epithelium.

category: pathology


title: Integrative analysis of the hydroxypyruvate reductases revealing their distinct roles in photorespiration of Chlamydomonas

abstract:
Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a different photorespiration system from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductase in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together we identify five proteins to be good candidates as CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a NADH-dependent enzyme in mitochondria, may function as the major component of photorespiration, and deletion of CrHPR1 causes severe photorespiratory defects. CrHPR2 takes parts in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, our results reveal that the CrHPRs are far more complex than previously recognized, and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate the genetic engineering for crop improvement by synthetic biology.

Brief summaryIdentification and characterization of genes encoding hydroxypyruvate reductases in Chlamydomonas, demonstrating difference in the enzymatic activity, subcellular location, as well as function in photorespiration.

category: plant biology


title: General object-based features account for letter perception better than specialized letter features

abstract:
After years of experience, humans become experts at perceiving letters. Is this visual capacity attained by learning specialized letter features, or by reusing general visual features previously learned in service of object categorization? To explore this question, we first measured the perceptual similarity of letters in two behavioral tasks, visual search and letter categorization. Then, we trained deep convolutional neural networks on either 26-way letter categorization or 1000-way object categorization, as a way to operationalize possible specialized letter features and general object-based features, respectively. We found that the general object-based features more robustly correlated with the perceptual similarity of letters. We then operationalized additional forms of experience-dependent letter specialization by altering object-trained networks with varied forms of letter training; however, none of these forms of letter specialization improved the match to human behavior. Thus, our findings reveal that it is not necessary to appeal to specialized letter representations to account for perceptual similarity of letters. Instead, we argue that it is more likely that the perception of letters depends on domain-general visual features.

AUTHOR SUMMARYIn literate societies, the ability to visually recognize letters is a highly important skill. For over a century, scientists have conducted behavioral experiments to investigate how the visual system recognizes letters, but it has proven difficult to propose a model of the feature space underlying this capacity. Here we leveraged recent advances in machine learning to model a wide variety of features ranging from specialized letter features to general object-based features. Across two large-scale behavioral experiments we find that general object-based features account well for letter perception, and that adding letter specialization did not improve the correspondence to human behavior. It is plausible that the ability to recognize letters largely relies on general visual features unaltered by letter learning.

category: neuroscience


title: General object-based features account for letter perception


title: SKAP2 as a new regulator of oligodendroglial migration and myelin sheath formation

abstract:
Oligodendroglial progenitor cells (OPC) are highly proliferative and migratory bipolar cells, which differentiate into complex myelin forming and axon ensheathing mature oligodendrocytes during myelination. Recent studies indicate that the oligodendroglial cell population is heterogeneous on transcriptional and functional level depending on the location in the CNS. Here, we compared intrinsic properties of OPC from spinal cord and brain on functional and transcriptional level. Spinal cord OPC demonstrated increased migration as well as differentiation capacity. Moreover, transcriptome analysis revealed differential expression of several genes between both OPC populations. In spinal cord OPC we confirmed upregulation of SKAP2, a cytoplasmatic adaptor protein known for its implication in cytoskeletal remodelling and migration in other cell types. Recent findings suggest that actin dynamics determine not only oligodendroglial migration, but also differentiation: Whereas actin polymerization is important for process extension, actin destabilization and depolymerization is required for myelin sheath formation. Downregulation or complete lack of SKAP2 in OPC resulted in reduced migration and impaired morphological maturation in oligodendrocytes. In contrast, overexpression of SKAP2 as well as constitutively active SKAP2 increased OPC migration suggesting that SKAP2 function is dependent on activation by phosphorylation. Furthermore, lack of SKAP2 enhanced the positive effect on OPC migration after integrin activation suggesting that SKAP2 acts as modulator of integrin dependent migration. In summary, we demonstrate the presence of intrinsic differences between spinal cord and brain OPC and identified SKAP2 as a new regulator of oligodendroglial migration and sheath formation.

Significance statementOPC play an important role in many still incurable diseases, such as multiple sclerosis, leukodystrophies or neurodegenerative diseases. Their heterogeneity in different CNS regions has recently been identified. Here, we observed increased migration and differentiation capabilities of OPC isolated from the spinal cord compared to brain OPC and confirmed differences in the transcriptome between these two cell populations. Furthermore, we identified SKAP2 as potential modulator of actin dynamics in oligodendrocytes. Whereas knockdown or lack of SKAP2 impairs migration and myelin sheath formation in mouse and human oligodendrocytes, overexpression of wildtype or constitutively active SKAP2 enhances the migratory capacity of murine oligodendrocytes. In summary, we present SKAP2 as modulator of cytoskeletal dynamics regulating OPC migration, differentiation and myelin sheath formation.


title: Place Cells in the Claustrum Remap Under NMDA Receptor Control

abstract: Place cells are cells exhibiting location-dependent responses; they have mostly been studied in the hippocampus. Place cells have also been reported in the rat claustrum, an underexplored paracortical region with extensive corto-cortical connectivity. It has been hypothesised that claustral neuronal responses are anchored to cortical visual inputs. We show rat claustral place cells remap when visual inputs are eliminated from the environment and that this remapping is NMDA-receptor-dependent. Eliminating visual input enhances delta-band oscillatory activity in the claustrum, without affecting simultaneously-recorded visual cortical activity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs. (106 words)


title: Characterization of a fiber-coupled EvenField illumination system for fluorescence microscopy

abstract: Fluorescence microscopy benefits from spatially and temporally homogeneous illumination with illumination area matched to the shape and size of the camera sensor. Fiber-coupled illumination schemes have the added benefit of straightforward and robust alignment and ease of installation compared to free-space coupled illumination. Commercial and open-source fiber-coupled, homogenized illumination schemes have recently become available to the public; however, there have been no published comparisons of speckle reduction schemes to date. We characterize three different multimode fibers in combination with two laser speckle reduction devices and compare spatial and temporal profiles to a commercial unit. This work yields a new design, the EvenField Illuminator, which is freely available along for researchers to integrate into their own imaging systems.


title: Marburg and Ebola virus mRNA 3 untranslated regions contain negative regulators of translation that are modulated by ADAR1 editing

abstract:
The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5 and 3 untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3UTRs are prone to editing by ADAR1. A reporter mRNA approach, in which different 5 or 3UTRs were inserted into luciferase encoding mRNAs, demonstrates that MARV 3UTRs yield different levels of reporter gene expression suggesting modulation of translation. The modulation occurs in cells unable to produce miRNAs and can be recapitulated in a minigenome assay. Deletion mutants identified negative regulatory regions at end of the MARV NP and L 3UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3UTR, but not the edited or deletion mutant NP 3UTRs, induce a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the VP40 3UTR, VP40 3UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3UTR edits also enhanced translation but neither the wildtype nor the edited 3UTRs induced IFN. These findings implicate filoviral mRNA 3UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1.

ImportanceUTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and of ADAR1 editing have been uncertain. This study demonstrates that MARV and EBOV 3UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3UTRs, relieves the negative effects of the UTRs. These data indicate that filovirus 3UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.

category: microbiology


title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of SNAR-A noncoding RNA

abstract: Dysregulation of the RNA polymerase III (Pol III) transcription program, which synthesizes tRNA and other classes of small noncoding RNA critical for cell growth and proliferation, is associated with cancer and human disease. Previous studies have identified two distinct Pol III isoforms defined by the incorporation of either subunit POLR3G (RPC7) during early development, or POLR3GL (RPC7{beta}) in specialized tissues. Though POLR3G is re-established in cancer and immortalized cell lines, the contributions of these isoforms to transcription potential and transcription dysregulation in cancer remain poorly defined. Using an integrated Pol III genomic profiling approach in combination with in vitro differentiation and subunit disruption experiments, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. In particular, we observe that a specific class of small noncoding RNA, SNAR-A, is exquisitely sensitive to the availability of subunit POLR3G in proliferating cells. Taken further, large-scale analysis of Pol III subunit expression and downstream chromatin features identifies concomitant loss of POLR3G and SNAR-A activity across a multitude of differentiated primary immune cell lineages, and conversely, coordinate re-establishment of POLR3G expression and SNAR-A features in a variety of human cancers. These results altogether argue against strict redundancy models for subunits POLR3G and POLR3GL, and instead support a model in which Pol III identity itself functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.

category: molecular biology


title: Redox sensitive E2 Rad6 controls cellular response to oxidative stress via K63 ubiquitination of ribosomes.

abstract: Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Among its non-proteolytic functions, K63 ubiquitin accumulates in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63 ubiquitin conjugates accumulate because of redox inhibition of the deubiquitinating enzyme Ubp2, however, the role and regulation of ubiquitin conjugating enzymes in this pathway remained unclear. Here we found that the E2 Rad6 binds and modifies elongating ribosomes during oxidative stress. We elucidated a mechanism by which Rad6 and its human homolog UBE2A are redox-regulated by forming reversible disulfides with the E1 activating enzyme, Uba1. We further showed that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress. Finally, we showed that Rad6 is required to induce phosphorylation of the translation initiation factor eIF2, providing a novel link for K63 ubiquitin, elongation stalling, and the integrated stress response.

category: cell biology


title: Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow

abstract: Advanced three-dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the workflow in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3{beta} phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.


title: Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells

abstract: Platelet deficiency, known as thrombocytopenia, can cause haemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of 5 surface markers which reproducibly purify MKPs, allowing us an insight into their transcriptional and epigenetic profiles. Finally, we performed culture optimisation, increasing MKP production. Altogether, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products towards the clinic.

category: developmental biology


title: Costs, Benefits, Parasites and Mutualists: The Use and Abuse of the Mutualism-Parasitism Continuum Concept for Epichloë Fungi

abstract: The species comprising the fungal endophyte genus Epichloe are symbionts of cool season grasses. About half the species in this genus are strictly vertically transmitted, and evolutionary theory suggests that these species must be mutualists. Nevertheless, Faeth and Sullivan (e.g. 2003) have argued that such vertically transmitted endophytes are "usually parasitic," and Muller and Krauss (2005) have argued that such vertically transmitted endophytes fall along a mutualism-parasitism continuum. These papers (and others) have caused confusion in the field. We used a systematic review to show that close to half of the published papers in this field incorrectly categorize the interaction. Here, we develop the argument that advantages and disadvantages are not the same things as mutualism and parasitism and that experimental evidence must be interpreted in the context of theory. We argue that, contra Faeth and Sullivan, it is highly unlikely that such strictly vertically transmitted endophytes can be parasites, and that, contra Muller and Krauss, it is inappropriate to apply the continuum concept to strictly vertically transmitted endophytes. We develop a mathematical model to support and illustrate our arguments. We use the model to clarify that parasitism requires some degree of horizontal transmission, and that while it is appropriate to use the continuum concept when referring to the genus as a whole, or to species that possess horizontal transmission, we argue that it is inappropriate to use the concept when referring to species that are strictly vertically transmitted.

category: ecology


title: Assessment of dependency and consumption pattern of different forest products by the forest fringe villages of Shivalik Himalaya, Uttarakhand, India

abstract:
Forest are essential for human beings for the enormous services it gives for livelihood and subsistence in the developing countries. We estimated the consumption and extraction levels of three major forest products viz., timber, fuelwood, and fodder in 20 forest fringe villages of the Timli forest range of Uttarakhand, India. We used a questionnaire-based household-level survey to collect information on the household economy and dependence of 380 households selected through stratified random sampling. We estimated that 69% of the overall yearly timber consumption of 20 villages, which comes around 2750 cubic meters (cum), was extracted from the nearby forest. The average timber consumption was 0.52 {+/-} 0.22 cum household-1 year-1. We estimated the total annual fuelwood and fodder consumption to be 298913.89, and 204475 Quintal (Qt). The average fuelwood and fodder consumption were estimated to be 417.6 Quintal household-1 year-1 and 49 {+/-} 9.1 Qt. household-1 year-1. We did general linear regression analysis to assess major biophysical and socio-economic determinants of villages and households for dependency on timber, fuelwood, and fodder. We found that the population of the village, distance from forest, distance from market, and annual average income are the major determining factors for timber, fodder, and fuelwood demand of the villages. Extraction of timber and non-timber forest products was the primary cause of depletion of forest biomass and forest carbon emission. Conservation effective management strategies in collaboration with all the stakeholder departments are needed to conserve forest resources with minimum extraction pressure from forest fringe villages of the study area.

HighlightsO_LIQuestionnaire based household level survey was done to assess extraction and consumption patter of fuelwood, fodder and timber in Shivalik range of Uttarakhand, India.
C_LIO_LIFuelwood and Timber consumption was found high compared to other studies in the low altitude areas.
C_LIO_LI69% of total timber and 62% of the total fodder requirement met from the adjacent forest areas.
C_LIO_LIPopulation of the village, distance from the forest and nearby market place, and annual average income were the major determinants of dependency of different forest products.
C_LI


title: Disentangling the effects of past logging and ongoing cryptic anthropogenic disturbance on vegetation structure and composition in Himalayan Foothills, India

abstract:
Most tropical forests have undergone commercial logging. Even where logging has ceased, subsistence harvest of forest resources often persists especially in South-East Asia. Understanding of impacts of frequent forest resources extraction in areas recovering from past selective logging would be essential for designing the appropriate management interventions.

We studied the impacts of current chronic anthropogenic disturbances (hereafter CAD) and past selective logging on vegetation structure, diversity, and regeneration, and the invasion of a non-native shrub, Lantana camara, in three major forest types in the Himalayan foothills, India. We analyzed field data on intensity of CAD and vegetation variables, collected from 269 stratified random plots, using ordination and generalized linear (mixed) modeling approaches.

Our results, based on 2758 adult trees of 54 species, showed that forest types differed in disturbance regimes depending on protection level and availability of fodder tree species. Intensity of CAD depended on proximity to settlements (for livestock related disturbances). Whereas selective logging, including firewood collection, was associated with land protection status. Selective logging reduced the extent of mature forest but facilitated regeneration, thereby promoting secondary forest features such as tree density and canopy cover. In contrast, the interaction between lopping and selective logging was negatively associated with regeneration. Past logging facilitated L. camara invasion in Dry and Hill forests but not in Moist forest. Finally, while selective logging marginally enhanced tree diversity, CAD reduced native shrub diversity.

Our study demonstrates that selective logging followed by CAD arrest forest recovery, as evident from the suppression of mature forest elements, loss of shrub biomass, reduced regeneration rate, and facilitation of invasive species. To abate these impacts, alternative livelihood/subsistence options that sustain forests and local communities should be explored. Additionally, CAD management should be site-specific as local ecological contexts modify their impacts on forests.


title: Climate-induced forest dieback drives compositional change in insect communities that is concentrated amongst rare species

abstract: Marked decline in insect species richness, abundance and biomass have recently been quantified in Europe. We metabarcoded 224 Malaise-trap samples to investigate whether drought-induced forest dieback and subsequent salvage logging have an impact on flying insects (ca. 3000 insect species) in silver fir Pyrenean forests. We found no evidence that climate-induced forest dieback impacted species richness of flying insects but revealed compositional turnover patterns consistent with those seen during natural forest succession, given that the key covariates explaining compositional variation were canopy openness versus microhabitat diversity and deadwood amount at local and landscape scales, respectively. Importantly, most change was driven by rare species. In contrast, observed levels of salvage logging did not explain change in species richness or composition. Hence, although forest dieback appears to cause changes in species assemblages mimicking natural forest succession, it also increases the risk of catastrophic loss of rare species through homogenization of environmental conditions.


title: The genetic basis of structural colour variation in mimetic Heliconius butterflies

abstract: Structural colours, produced by the reflection of light from ultrastructures, have evolved multiple times in butterflies. Unlike pigmentary colours and patterns, little is known about the genetic basis of these colours. Reflective structures on wing-scale ridges are responsible for iridescent structural colour in many butterflies, including the Mullerian mimics Heliconius erato and Heliconius melpomene. Here we quantify aspects of scale ultrastructure variation and colour in crosses between iridescent and non-iridescent subspecies of both of these species and perform quantitative trait locus (QTL) mapping. We show that iridescent structural colour has a complex genetic basis in both species, with offspring from crosses having wide variation in blue colour (both hue and brightness) and scale structure measurements. We detect two different genomic regions in each species that explain modest amounts of this variation, with a sex-linked QTL in H. erato but not H. melpomene. We also find differences between species in the relationships between structure and colour, overall suggesting that these species have followed different evolutionary trajectories in their evolution of structural colour. We then identify genes within the QTL intervals that are differentially expressed between subspecies and/or wing regions, revealing likely candidates for genes controlling structural colour formation.


title: The genetic architecture underlying body-size traits plasticity over different temperatures and developmental stages in Caenorhabditis elegans

abstract: Most ectotherms obey the temperature-size rule, meaning they grow larger in a colder environment. This raises the question of how the interplay between genes and temperature affect the body size of ectotherms. Despite the growing body of literature on the physiological life-history and molecular genetic mechanism underlying the temperature-size rule, the overall genetic architecture orchestrating this complex phenotype is not yet fully understood. One approach to identify genetic regulators of complex phenotypes is Quantitative Trait Locus (QTL) mapping. Here, we explore the genetic architecture of body size phenotypes, and plasticity of body-size phenotypes in different temperatures using Caenorhabditis elegans as a model ectotherm. We used 40 recombinant inbred lines (RILs) derived from N2 and CB4856, which were reared at four different temperatures (16{degrees}C, 20{degrees}C, 24{degrees}C, and 26{degrees}C) and measured at two developmental stages (L4 and adult). The animals were measured for body length, width at vulva, body volume, length/width ratio, and seven other body-size traits. The genetically diverse RILs varied in their body-size phenotypes with heritabilities ranging from 0.0 to 0.99. We detected 18 QTL underlying the body-size traits across all treatment combinations, with the majority clustering on Chromosome X. We hypothesize that the Chromosome X QTL could result from a known pleiotropic regulator - npr-1 - known to affect the body size of C. elegans through behavioral changes. We also found five plasticity QTL of body-size which three of them colocalized with some body-size QTL at certain temperature. In conclusion, our findings shed more light on multiple loci affecting body size plasticity and the possibility of co-regulation of traits and traits plasticity by the same loci under different environment.

category: genetics


title: Leveraging probability concepts for genotype by environment recommendation

abstract: Statistical models that capture the phenotypic plasticity of a genotype across environments are crucial in plant breeding programs to potentially identify parents, generate offspring, and obtain highly productive genotypes for distinct environments. In this study, our aim is to leverage concepts of Bayesian models and probability methods of stability analysis to untangle genotype-by-environment interaction (GEI). The proposed method employs the posterior distribution obtained with the No-U-Turn sampler algorithm to get Monte Carlo estimates of adaptation and stability probabilities. We applied the proposed models in two empirical tropical datasets. Our findings provide a basis to enhance our ability to consider the uncertainty of cultivar recommendation for global or specific adaptation. We further demonstrate that probability methods of stability analysis in a Bayesian framework are a powerful tool for unraveling GEI given a defined intensity of selection that results in a more informed decision-making process towards cultivar recommendation in multi-environment trials.


title: The fall and rise of group B Streptococcus in dairy cattle: reintroduction due to human-to-cattle host jumps?

abstract:
Group B Streptococcus (GBS; Streptococcus agalactiae) is a major neonatal and opportunistic bacterial pathogen of humans and an important cause of mastitis in dairy cattle with significant impacts on food security. Following the introduction of mastitis control programs in the 1950s, GBS was nearly eradicated from the dairy industry in northern Europe, followed by re-emergence in the 21st century. Here, we sought to explain this re-emergence based on short and long read sequencing of historical (1953-1978; n = 44) and contemporary (1997-2012; n = 76) bovine GBS isolates. Our data show that a globally distributed bovine-associated lineage of GBS was commonly detected among historical isolates but never among contemporary isolates. By contrast, tetracycline resistance, which is present in all major GBS clones adapted to humans, was commonly and uniquely detected in contemporary bovine isolates. These observations provide evidence for strain replacement and suggest a human origin of newly emerged strains. Three novel GBS plasmids were identified, including two showing >98% homology with plasmids from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis, which co-exist with GBS in the human oropharynx. Our findings support introduction of GBS into the dairy population due to human to-cattle jumps on multiple occasions and demonstrate that reverse zoonotic transmission can erase successes of animal disease control campaigns.

IMPACT STATEMENTPathogens can jump between humans and animals. Animal domestication and intensification of livestock production systems have caused multiple human to animal spill-over events, sometimes with significant impact on animal health and food production. The most common production-limiting disease of dairy cattle is mastitis, inflammation of the mammary gland, which can be caused by group B Streptococcus, a common commensal and pathogen of humans. Using genomic data from historical and recent isolates, we show that re-emergence of this pathogen in the dairy industry in northern Europe is due to strains with genomic signatures of human host-adaptation, including antimicrobial resistance genes and plasmids. This shows how elimination of animal diseases may be hampered by humans serving as a reservoir of multi-host pathogens, and reverse zoonotic transmission.

REPOSITORIESReads for all isolates sequenced in this study have been submitted to the ENA Sequence Read Archive (SRA). SRA accession numbers are included in Table S1, supplementary material, available in the online version of this article.


title: M&Ms: A software for building realistic Microbial Mock communities

abstract:
MotivationAdvances in sequencing technologies have triggered the development of many bioinformatic tools aimed to analyze these data. As these tools need to be tested, it is important to simulate datasets that resemble realistic conditions. Although there is a large amount of software dedicated to produce reads from  in silico microbial communities, often the simulated data diverge widely from real situations.

ResultsHere, we introduce M&Ms, a user-friendly open-source bioinformatic tool to produce realistic amplicon datasets from reference sequences, based on pragmatic ecological parameters. This tool creates sequence libraries for  in silico microbial communities with user-controlled richness, evenness, microdiversity, and source environment. M&Ms allows the user to generate simple to complex read datasets based on real parameters that can be used in developing bioinformatic software or in benchmarking current tools. M&Ms also provides additional figures and files with extensive details on how each synthetic community is composed, so that users can make informed choices when designing their benchmarking pipelines.

AvailabilityThe source code of M&Ms is freely available from https://github.com/ggnatalia/MMs

Contactngarcia@cnb.csic.es

category: bioinformatics


title: Interaction of the TonB dependent transporter HasR with its cognate TonB-like protein HasB in a membrane environment

abstract: The envelope of Gram-negative bacteria is composed of a double membrane separated by the periplasmic space. This organization imposes geometrical and distance constraints that are key for the mechanism of action of multicomponent systems spanning the envelope. However, consideration of all three compartments by experimental approaches is still elusive. Here we used the state-of-the-art molecular dynamics simulation in an Escherichia coli envelope model to obtain a dynamic view of molecular interactions between the outer membrane heme transporter HasR and the inner membrane TonB-like protein HasB. Their interaction allows the transfer of the inner membrane proton motive force derived energy to the transporter for heme internalization. The simulations which incorporate both membranes show the key role of periplasmic domains of both proteins, and their dynamics in the complex formation and stability. They revealed a previously unidentified atomic network of interactions, as well as the sequences of the interactions and their variations with the presence of external substrates. Experimental validation (mutations, phenotypic and in vitro assays) confirms the robustness of our approach and provides verification of the simulation-predicted interactions. Based on structural and sequence conservation, the network of interaction revealed in this study is expected to occur in other nutrient import systems. The integrative approach presented here is highly useful to study the dynamic interplay between components of any other bacterial transmembrane systems.


title: Dynamic interplay between a TonB dependent heme transporter and a TonB protein in a membrane environment


title: Unperturbed dormancy recording reveals stochastic awakening strategies in endocrine treated breast cancer cells

abstract: Hormone dependent breast cancer (HDBC) is the most commonly diagnosed tumor type in women. Adjuvant endocrine therapies (ET) have been the cornerstone in the clinical management of HDBC patients for over forty years. A vast proportion of HDBC patients incur long periods of clinical dormancy following ET, with tumour awakening appearing at a steady pace for up to 25 years (Pan et al., 2017). Extensive genomic studies have demonstrated that 15-30% of clinical relapses develop recurrent genomic changes which contribute to drug resistance (i.e. ESR1 activating mutations) (Bertucci et al., 2019; Magnani et al., 2017; Razavi et al., 2018). However, even in these cases, there is no conclusive evidence around the pre-existence vs. de novo nature of these events. We previously showed that ETs can trigger and select for dormancy in subpopulations of breast cancer(Hong et al., 2019). In this work we took two novel approaches to investigate the dormancy and awakening roadmap of HDBC cells in unprecedented detail. Firstly, we leveraged a rare cohort of n=5 patients which were treated with primary adjuvant ETs in the absence of surgery (TRACING-HT) to dissect the contribution of genomic aberrations to tumor awakening. Next, we developed a first of its kind evolutionary study in vitro to systematically annotate cancer cells adaptive strategies at single cell level in unperturbed systems during a period of several months (TRADITIOM). Collectively our data suggest that ETs steer HDBC cells into an inherently unstable dormant state. Over time, routes to awakening emerge sporadically and spontaneously in single lineages. Each dormant cell retains an intrinsic awakening probability which we propose is a function of epigenetic decay. Awakening occurs without an external trigger and involves multiple apparent endpoint phenotypes that cannot be fully explained by conventional Darwinian genetic selection processes. Finally, our data show that common genetic hits associated with resistance happen downstream of awakening. Overall, our data have uncovered previously unsuspected roles for stochastic nongenetic events during dormancy with profound clinical implications.


title: Effects of TeaCrine(R) (theacrine), Dynamine™ (methylliberine), and caffeine on gamer psychomotor performance in a first-person shooter video game scenario

abstract:
PurposeTo compare the effects of purported cognitive enhancing dietary bioactive ingredients on subjective and objective measures of cognitive and motor performance during a first-person shooter video game.

MethodsUsing a placebo-controlled crossover design, nine healthy men (23.4{+/-}5.7 yr, 178.9{+/-}5.8cm, 86.0{+/-}17.1kg) completed four 20-minute gaming sessions designed to assess cognitive, motor, and perceptual skills via artificial intelligence-driven battery of tasks (Aim Lab). Participants ingested either a placebo (PL), caffeine (CAFF), or caffeine + methylliberine (Dynamine) + theacrine (TeaCrine(R)) (CMT). Before and after each gaming session participants rated various feelings of affect. Data were analyzed using mixed factorial ANOVA, bootstrapping post-hoc tests with 95% confidence intervals, and effect sizes.

ResultsCompared to PL, self-assessed performance was significantly increased in CMT vs. PL (p=0.035) and self-assessed jitteriness was increased by CAFF vs. PL (p=0.043). CMT was associated with a greater improvement of participants visuo-spatial working memory from baseline vs. PL (p=0.04) and CAFF (p=0.033). CAFF had a greater decrease in reaction time for false alarms (indicating diminished cognitive control) from baseline vs. PL (p=0.002) and CMT (p=0.001) and a greater increase for time on target tracking vs. PL (p=0.008) and CMT (p=0.047). Compared to PL, CMT was associated with a greater decrease in median kill time (indicating improved speed) (p=0.017). Compared to PL, systolic blood pressure was significantly increased by CAFF (p=0.025) and CMT (p=0.020) but remained within normal limits.

ConclusionsAcute CMT supplementation improved cognitive and motor abilities in recreational gamers. The addition of theacrine and methylliberine to caffeine may lessen some undesirable effects of isolated caffeine ingestion on cognitive control and jitteriness.

category: physiology


title: NAC transcription factor RD26 is a regulator of root hair morphogenic plasticity

abstract:
Root hairs are outgrowths of epidermal cells central for water and nutrient acquisition. Root hair growth is plastically modified by environmental cues. A frequent response to water limitation is active shortening of root hairs, involving largely unknown molecular mechanisms. A root hair-specific cis-regulatory element (RHE) integrates developmental cues with downstream signalling of root hair morphogenesis. Here, we demonstrate NAC transcription factor RD26 to be a key expressional regulator of this drought stress-triggered developmental response in Arabidopsis thaliana. RD26 directly represses RSL4 and RSL1, two master transcription regulators of root hair morphogenesis, by binding RHE. RD26 further represses core cell wall modification genes including expansins (EXPA7, EXPA18), hydroxyproline-rich glycoproteins (LRX1), xyloglucan endotransglucosylases/hydrolases (XTH12, 13, 14, 26), class III peroxidases (PRX44) and plasma membrane H+-ATPase (AHA7) through RHE. Of note, several RD26-repressed genes are activated by RSL4. Thus, by repressing RSL4 and numerous cell wall-related genes, RD26 governs a robust gene regulatory network for restricting root hair growth under drought. A similar regulatory network exists in tomato, indicating evolutionary conservation across species.

Significance statementIn plants, root hairs play a vital role for water and nutrient acquisition, soil anchorage, and microbial interactions. During drought stress, root hair growth is suppressed as an adaptive strategy to save cellular energy. We identified NAC transcription factor RD26 as a key regulator of this developmental plasticity in the model plant Arabidopsis thaliana. RD26 directly and negatively controls the transcriptional activity of key root hair developmental genes, RSL1 and RSL4. Furthermore, RD26 suppresses the expression of several functional genes underlying root hair development including numerous cell wall-related genes. RD26 thus governs a robust gene regulatory network underlying the developmental response to drought stress. A similar regulatory network exists in tomato indicating evolutionary conservation of this mechanism across species.


title: RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control

abstract: The brain expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntingtons disease where it promotes the clearance of mutant Huntingtin protein. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.


title: Musical experience partially counteracts temporal speech processing deficits in mild cognitive impairment

abstract: Mild cognitive impairment (MCI) commonly impacts older adults resulting in more rapid cognitive and behavioral declines than typical aging. Individuals with MCI can exhibit impaired receptive speech abilities that may reflect neurophysiological changes in auditory-sensory processing prior to usual cognitive deficits. Benefits from current interventions targeting communication difficulties in MCI are limited. Yet, neuroplasticity associated with musical experience has been implicated in improving neural representations of speech and offsetting age-related declines in perception. Here, we asked whether these experience-dependent effects of musicianship might extend to aberrant aging and offer some degree of cognitive protection against MCI. During a vowel categorization task, we recorded single-channel EEGs in older adults with putative MCI to evaluate speech encoding across subcortical and cortical levels of the auditory system. Critically, listeners varied in their duration of formal musical training experience (0-21 years). Older musicians exhibited sharpened temporal precision in auditory cortical responses suggesting musical experience produces more efficient processing of acoustic features by offsetting age-related neural delays. Additionally, we found robustness of brainstem responses predicted severity of cognitive decline suggesting early speech representations are sensitive to pre-clinical stages of cognitive impairment. Our preliminary results extend prior studies by demonstrating positive benefits of musical experience in older adults with emergent cognitive impairments.


title: The Autophagy Receptor TAX1BP1 (T6BP) is a novel player in antigen presentation by MHC-II molecules.

abstract: CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented on the surface of antigen presenting cells (APCs) by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and a rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. We determined the interactome of T6BP, identified calnexin as a T6BP partner and show that CANX cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway and we propose one potential mechanism of action.

category: immunology


title: Absolute quantification and single-cell dose-response of cytosolic siRNA delivery

abstract: Endosomal escape and subsequent cytosolic delivery of small inhibitory RNA (siRNA) therapeutics is believed to be highly inefficient. Since, it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determination of delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to detect cytosolic delivery of fluorescently labelled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.


title: Pinhead antagonizes Admp to promote notochord formation

abstract: Dorsoventral patterning of a vertebrate embryo critically depends on the activity of Smad1 that mediates signaling by several BMP proteins, anti-dorsalizing morphogenetic protein (Admp), and their antagonists. Pinhead (Pnhd), a cystine-knot-containing secreted protein, is expressed in the ventrolateral marginal zone during Xenopus gastrulation, however, its molecular targets and signaling mechanisms have not been fully elucidated. An unbiased mass spectrometry-based screen of the gastrula secretome identified Admp as a primary Pnhd-associated protein. We show that Pnhd binds Admp and inhibits its ventralizing activity by reducing Smad1 phosphorylation and suppressing its transcriptional targets. By contrast, Pnhd did not affect the signaling activity of BMP4. Importantly, the Admp gain-of-function phenotype and phospho-Smad1 levels have been enhanced after Pnhd depletion. Furthermore, Pnhd strongly synergized with Chordin and a truncated BMP4 receptor in the induction of notochord markers in ectoderm cells, and Pnhd-depleted embryos displayed notochord defects. Our findings suggest that Pnhd binds and inactivates Admp to promote notochord development. We propose that the interaction between Admp and Pnhd refines Smad1 activity gradients during vertebrate gastrulation.


title: Human-derived alleles in SOST and RUNX2 3'UTRs cause differential regulation in a bone cell-line model

abstract: The inquiry into the phenotypic features that set apart human species, such as a light, gracile skeleton and a rounded skull characteristic of Homo sapiens, can now benefit from the examination of ancient genomes. These have added a new layer of analysis allowing the identification of genetic differences between species like ours and our closest extinct relatives. Most of these genetic differences are non-coding changes with unknown functional consequences, and dissecting their putative regulatory effect remains challenging. Here we focus on the three prime untranslated regions (3UTR), known to play a critical role in messenger RNA regulation and a plausible locus for divergent regulation between Homo species. We report a set of genes with derived 3UTR changes in either the Homo sapiens or the Neanderthal/Denisovan lineages and experimentally evaluate the impact of 3UTR variants in four genes: E2F6, GLI3, RUNX2 and SOST. We performed a luciferase reporter assay in a bone cell-line model and found a statistically significant difference for the 3UTR variants of SOST (Homo sapiens-derived) and RUNX2 (Neanderthal/Denisovan-derived). The differential expression caused by these variants in our experimental model points to species differences in bone mineral density. Thus, this study adds insights into the functional effects of regulatory variants that emerged in recent human evolution.

category: evolutionary biology


title: AGO104 is an RdDM effector of paramutation at the maize b1 locus

abstract:
Paramutation is an exception among eukaryotes, in which epigenetic information is conserved through mitosis and meiosis. It has been studied for over 70 years in maize, but the mechanisms involved are largely unknown. Previously described actors of paramutation encode components of the RNA-dependent DNA-methylation (RdDM) pathway all involved in the biogenesis of 24-nt small RNAs. However, no actor of paramutation have been identified in the effector complex of RdDM. Here, through a combination of reverse genetics, immunolocalization and immunoprecipitation (siRNA-IP) we found that ARGONAUTE104 (AGO104), AGO105 and AGO119 are members of the RdDM effector complex in maize and bind siRNAs produced from the tandem repeats required for paramutation at the b1 locus. We also showed that AGO104 is an effector of the b1 paramutation in maize.

Author summaryReprogramming of epigenetic information has been described in both plants and mammals. Here, we show that maize ARGONAUTE (AGO) AGO104 and AGO105/AGO119, respectively the close homologs of A. thaliana AGO9 and AGO4, are required to enable paramutation at the b1 locus in maize. Paramutation is an epigenetic phenomenon that is stable over many generations (both mitotically and meiotically). A classic example is the booster1 (b1) gene in maize, where the weakly expressed Booster (B) allele stably decreases the expression of the Booster-Intense (B-I) allele, and changes it into a new B allele. This new B allele will in turn change B-I into new B in subsequent crosses. Previous research demonstrated that paramutation requires several proteins involved in the biosynthesis of small interfering RNAs (siRNAs) all related to the RNA-dependent DNA-methylation (RdDM) pathway. Yet, few members of the RdDM were functionally identified in maize. Here, we identify two new members of the maize RdDM pathway, and provide evidence that they are also involved in paramutation at the b1 locus.


title: CMOS electrochemical imaging arrays for thedetection and classification of microorganisms

abstract: Microorganisms account for most of the biodiversity on earth. Yet while there are increasingly powerful tools for studying microbial genetic diversity, there are fewer tools for studying microorganisms in their natural environments. In this paper, we present recent advances in CMOS electrochemical imaging arrays for detecting and classifying microorganisms. These microscale sensing platforms can provide non-optical measurements of cell geometries, behaviors, and metabolic markers. We review integrated electronic sensors appropriate for monitoring microbial growth, and present measurements of single-celled algae using a CMOS sensor array with thousands of active pixels. Integrated electrochemical imaging can contribute to improved medical diagnostics and environmental monitoring, as well as discoveries of new microbial populations.


title: A 'Variant Viewer' for investigating population-level variation in Arabidopsis thaliana

abstract: High throughput sequencing has opened the doors for investigators to probe genetic variation present in large populations of organisms. In plants, the 1001 Genomes Project (1001genomes.org) is one such effort that sought to characterize the extant worldwide variation in Arabidopsis thaliana for future analyses to compare and draw upon. We developed a web application that accesses the 1001 Genomes database called The Variant Viewer, for investigators to view variants in any A. thaliana gene and within gene families. These variants may be visualized in the context of alignments of queried genes, across splice isoforms of these genes and in relation to conserved domains.


title: Optical Probing of Local Membrane Potential with Fluorescent Polystyrene Beads

abstract: Studying the electrical activity in single cells and in local circuits of excitable cells, like neurons, requires an easy to use and high throughput methodology that enables the measurement of membrane potential. Studying the electrical properties in particular sub-compartments of neurons, or in a specific type of neurons produces additional complexity. An optical voltage-imaging technique that allows high spatial and temporal resolution could be an ideal solution. However, most of the valid voltage imaging techniques are nonspecific; The ones that are more site-directed require much pre-work and specific adaptations in addition to other disadvantages. Here, a new technique for membrane voltage imaging, based on FRET between fluorescent polystyrene (FPS) beads and Dipicrylamine (DPA) is explored. Not only fluorescent intensity is demonstrated to be correlated with membrane potential, but more importantly, single particle voltage detection is demonstrated. Among other advantages, FPS beads can be synthesized with functional surface groups, and be further targeted to specific proteins via conjugation of recognition molecules. Therefore, FPS beads, in the presence of DPA, constitute single-particle detectors for membrane voltage, with a potential to be localized to specific membrane compartments. This new and accessible platform for targeted optical voltage imaging may further elucidate the mechanisms of neuronal electrical activity.


title: No evidence that HLA genotype influences the driver mutations that occur in cancer patients

abstract: The major histocompatibility (MHC) molecules are capable of presenting neoantigens resulting from somatic mutations on cell surfaces, potentially directing immune responses against cancer. This led to the hypothesis that cancer driver mutations may occur in gaps in the capacity to present neoantigens that are dependent on MHC genotype. If this is correct, it has important implications for understanding oncogenesis and may help to predict driver mutations based on genotype data. In support of this hypothesis, it has been reported that driver mutations that occur frequently tend to be poorly presented by common MHC alleles and that the capacity of a patients MHC alleles to present the resulting neoantigens is predictive of the driver mutations that are observed in their tumour. Here we show that these reports of a strong relationship between driver mutation occurrence and patient MHC alleles are a consequence of unjustified statistical assumptions. Our reanalysis of the data provides no evidence of an effect of MHC genotype on the oncogenic mutation landscape.


title: Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase

abstract: The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

category: synthetic biology


title: Scalable total-evidence inference from molecular and continuous characters in a Bayesian framework

abstract: AO_SCPLOWBSTRACTC_SCPLOWTime-scaled phylogenetic trees are both an ultimate goal of evolutionary biology and a necessary ingredient in comparative studies. While accumulating genomic data has moved the field closer to a full description of the tree of life, the relative timing of certain evolutionary events remains challenging even when this data is abundant, and absolute timing is impossible without external information such as fossil ages and morphology. The field of phylogenetics lacks efficient tools integrating probabilistic models for these kinds of data into unified frameworks for estimating phylogenies. Here, we implement, benchmark and validate popular phylogenetic models for the study of paleontological and neontological continuous trait data, incorporating these models into the BEAST2 platform. Our methods scale well with number of taxa and of characters. We tip-date and estimate the topology of a phylogeny of Carnivora, comparing results from different configurations of integrative models capable of leveraging ages, as well as molecular and continuous morphological data from living and extinct species. Our results illustrate and advance the paradigm of Bayesian, probabilistic total evidence, in which explanatory models are fully defined, and inferential uncertainty in all their dimensions is accounted for.

category: systems biology


title: Daily rhythms in the transcriptomes of the human parasite Schistosoma mansoni

abstract:
BackgroundThe consequences of the earths daily rotation have led to 24-hour biological rhythms in most organisms. Even parasites have daily rhythms, which, when in synchrony with host rhythms, optimize their fitness. Understanding these rhythms may enable development of novel control strategies that take advantage of rhythmic vulnerabilities. Recent work on blood-dwelling protozoan parasites has revealed daily rhythms in gene expression, physiology, drug sensitivity and the presence of an intrinsic circadian clock. However, similar studies on metazoan parasites are lacking. The aims of this study were to investigate if a metazoan parasite has daily molecular oscillations, whether they give us insight into how these longer-lived organisms can survive host daily cycles over a life-span of many years and to determine whether canonical metazoan circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni, that lives in the vasculature for decades and causes the serious neglected tropical disease schistosomiasis.

ResultsUsing round-the-clock transcriptomics of male and female adult worms we discovered that [~]2% of its genes followed a daily pattern of expression. Rhythmic processes included a night-time stress response and a day-time metabolic  rush hour. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include drug targets and a vaccine candidate. These 24hr rhythms may be driven by host rhythms and/or generated by a circadian clock. However, core clock genes are missing and orthologues of secondary clock genes show no 24hr rhythmicity in transcript abundance.

ConclusionsThe daily rhythms identified here reveal temporally compartmentalised internal processes and host interactions over the daily cycle, including processes relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other Metazoa. Most importantly, knowing which gene transcripts oscillate at this temporal scale is relevant to functional genomic studies that will lead to the development and delivery of therapeutics against schistosomiasis.


title: Pre-existing chromatin accessibility and gene expression differences among naïve CD4+ T cells influence effector potential

abstract: CD4+ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probed the heterogeneity present among naive CD4+ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and epigenetic differences. Using single-cell RNA sequencing, we showed that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a read-out of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4+ T cells impact follicular helper cell (TFH) versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4 T cell chromatin landscapes that ultimately shape their effector potential.


title: Host regulator PARP1 contributes to sex differences and immune responses in a mouse model of tuberculosis

abstract: Tuberculosis (TB) is a devastating infectious disease responsible for nearly 2 million deaths annually that has a poorly understood male bias. Elucidating the basis of this male bias may enable precision medicine interventions for TB treatment and prevention. Here, we identify the master regulator Poly(ADP-ribose) Polymerase 1 (PARP1) as a driver of TB sex differences. We found that infection with M. tuberculosis (M. tb) triggers robust PARP activation in mouse lungs, suggesting that PARP1 activation is a fundamental host response to TB. Remarkably, PARP1 deletion abolished known sex differences in TB cytokine responses and blunted the early induction of TNF, IL-1{beta}, IFN{gamma}, MCP-1, and IL-6, particularly in male mice. In contrast, PARP1 was required for IL-10 induction in male or female mice. PARP1 deletion was protective against TB in female mice, resulting in significantly prolonged survival and reduced bacterial burden, but impaired TB containment in male mice. Our findings indicate that PARP1 contributes to TB sex differences via sexually divergent immune regulation and uniquely enhances early proinflammatory responses in males that prove beneficial for TB containment.


title: Wzc and WcaJ mutations in hypervirulent Klebsiella pneumoniae lead to phage resistance at the cost of reduced virulence

abstract:
Hypervirulent Klebsiella pneumoniae (hvKp) is one of the major community-acquired pathogens, which can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment of Klebsiella pneumoniae, but the characteristics of the phage resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. In vitro and in vivo experiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations of wzc and wcaJ genes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule, meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. The findings can contribute to a better understanding of that bacteriophage therapy can not only kill bacteria directly, but also reduce the virulence of bacteria by phage screening.

ImportanceBacteriophages are considered potential therapeutic alternative to antibiotics; however host-evolved phage resistance has accounted for the resurgences of pathogens, meaning further measures are need to improve the efficacy of phage therapy. This study showed two phages capable of infecting hypervirulent K. pneumoniae and identified phage-resistant mutants whose virulence was significantly reduced. Gene sequencing analysis revealed that mutations of wzc and wcaJ gene, related to capsule synthesis, recovered phage resistance but reduced the virulence of hypervirulent K. pneumoniae.


title: Viromes in Marine Ecosystems Reveal Remarkable Invertebrate RNA Virus Diversity

abstract: Ocean viromes remain poorly understood and little is known about the ecological factors driving aquatic RNA virus evolution. In this study, we used a meta-transcriptomic approach to characterize the viromes of 58 marine invertebrate species across three seas. This revealed the presence of 315 newly identified RNA viruses in nine viral families or orders (Durnavirales, Totiviridae, Bunyavirales, Hantaviridae, Picornavirales, Flaviviridae, Hepelivirales, Solemoviridae and Tombusviridae), with most of them are sufficiently divergent to the documented viruses. With special notice that we first time revealed an ocean virus rooting to mammalian hantaviruses. We also found evidence for possible host sharing and switch events during virus evolution. In sum, we demonstrated the hidden diversity of marine invertebrate RNA viruses.


title: Tracheal motile cilia require CAMSAP3 for formation of central microtubule pair and coordinated beating

abstract: Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a  transition zone. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.


title: Tracheal motile cilia in mice require CAMSAP3 for formation of central microtubule pair and coordinated beating


title: What basal membranes can tell us about viscous forces in Drosophila ventral furrow formation

abstract: Ventral furrow (VF) formation in Drosophila melanogaster is an important model of epithelial folding. Past studies of VF formation focus on apical forces in driving folding, while other forces are largely unexplored. When basal membrane formation is genetically blocked using anillin knockdown (scra RNAi), the VF can still fold. To interpret our observations, we developed a computational model of VF formation that explicitly simulates the flows of viscous cytoplasm. The viscosity included in our model is required for tissue invagination in the absence of basal membranes. This strongly suggests that epithelial folding requires the presence of viscous shear forces from cytoplasm. Our model characterizes folding during VF formation as a "swimming phenomenon", where tissue deforms by pushing against the ambient viscous surroundings. Since VF formation is successful in scra RNAi embryos, we propose that models of gastrulation should be able to replicate this folding in the absence of basal membranes.


title: Genomic time-series data show that gene flow maintains high genetic diversity despite substantial genetic drift in a butterfly species

abstract: Effective population size affects the efficacy of selection, rate of evolution by drift, and neutral diversity levels. When species are subdivided into multiple populations connected by gene flow, evolutionary processes can depend on global or local effective population sizes. Theory predicts that high levels of diversity might be maintained by gene flow, even very low levels of gene flow, consistent with species long-term effective population size, but tests of this idea are mostly lacking. Here, we show that Lycaeides butterfly populations maintain low contemporary (variance) effective population sizes (e.g., [~]200 individuals) and thus evolve rapidly by genetic drift. Contemporary effective sizes were consistent with local census populations sizes. In contrast, populations harbored high levels of genetic diversity consistent with an effective population size several orders of magnitude larger. We hypothesized that the differences in the magnitude and variability of contemporary versus long-term effective population sizes were caused by gene flow of sufficient magnitude to maintain diversity but only subtly affect evolution on generational time scales. Consistent with this hypothesis, we detected low but non-trivial gene flow among populations. Furthermore, using population-genomic time-series data, we documented patterns consistent with predictions from this hypothesis, including a weak but detectable excess of evolutionary change in the direction of the mean (migrant gene pool) allele frequencies across populations, and consistency in the direction of allele frequency change over time. The documented decoupling of diversity levels and short-term change by drift in Lycaeides has implications for our understanding of contemporary evolution and the maintenance of genetic variation in the wild.


title: Evidence for continent-wide convergent evolution and stasis throughout 150 years of a biological invasion

abstract: The extent to which evolution can rescue a species from extinction, or facilitate range expansion, depends critically on the rate, duration, and geographical extent of the evolutionary response to natural selection. Adaptive evolution can occur quickly, but the duration and geographical extent of contemporary evolution in natural systems remains poorly studied. This is particularly true for species with large geographical ranges and for timescales that lie between  long-term field experiments and the fossil record. Here, we introduce the Virtual Common Garden (VCG) to investigate phenotypic evolution in natural history collections while controlling for phenotypic plasticity in response to local growing conditions. Reconstructing 150 years of evolution in Lythrum salicaria (purple loosestrife) as it invaded North America, we analyze phenology measurements of 3,429 herbarium records, reconstruct growing conditions from more than 12 million local temperature records, and validate predictions across three common gardens spanning 10 degrees of latitude. We find that phenology clines have evolved along parallel climatic gradients, repeatedly throughout the range, during the first century of evolution. Thereafter, the rate of microevolution stalls, recapitulating macroevolutionary stasis observed in the fossil record. Our study demonstrates that preserved specimens are a critical resource for investigating limits to evolution in natural populations. Our results show how natural selection and trade-offs measured in field studies predict adaptive divergence observable in herbarium specimens over 15 decades at a continental scale.


title: Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation

abstract: The FtsLB complex is a key regulator of bacterial cell division, existing in either an off or on state which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a "detuned" structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.

category: biochemistry


title: Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation in cell division


title: The coiled coil of FtsLB is a structurally detuned element critical for modulating activation in bacterial cell division


title: The coiled-coil domain of E. coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division


title: Stem cell quiescence requires PRC2/PRC1-mediated mitochondrial checkpoint

abstract: Both normal and tumorous stem cells can arrest cell division, avoid apoptosis, and then regenerate lost daughter cells following acute genotoxic insult. This protective, reversible proliferative arrest, known as "quiescence," is still poorly understood. Here, we show that mTOR-regulated mitophagy is required for radiation insult-induced quiescence in Drosophila germline stem cells (GSCs). In GSCs, depletion of mito-fission (Drp1) or mitophagy (Pink1 and Parkin) eliminates entry into quiescence, while depletion of mitochondrial biogenesis (PGC1) or fusion (Mfn2) eliminates exit from quiescence. We also find that mitophagy-dependent quiescence is under epigenetic control; knockdown of Jarid2 (PRC2) or Pc or Sce (PRC1) stabilizes the mitochondria and locks GSCs out of quiescence, while knockdown of PRC2-specific demethylase, Utx, prevents re-accumulation of the mitochondria and locks GSCs in quiescence. These data suggest that mitochondrial number coordinates reversible quiescence. We further identify that the mechanism of quiescence in both GSCs and human induced pluripotent stem cells (iPSCs) relies on mitophagy to deplete the mitochondrial pool of CycE and limit cell cycle progression. This alternative method of G1/S regulation may present new opportunities for therapeutic purposes.


title: Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

abstract: Counting of numerous compartmentalized enzymatic reactions underlies quantitative and high sensitivity immunodiagnostic assays. However, digital enzyme-linked immunosorbent assays (ELISA) require specialized instruments which have slowed adoption in research and clinical labs. We present a lab-on-a-particle solution to digital counting of thousands of single enzymatic reactions. Hydrogel particles are used to bind enzymes and template the formation of droplets that compartmentalize reactions with simple pipetting steps. These hydrogel particles can be made at a high throughput, stored, and used during the assay to create ~500,000 compartments within 2 minutes. These particles can also be dried and rehydrated with sample, amplifying the sensitivity of the assay by driving affinity interactions on the hydrogel surface. We demonstrate digital counting of {beta}-galactosidase enzyme at a femtomolar detection limit with a dynamic range of 3 orders of magnitude using standard benchtop equipment and experiment techniques. This approach can faciliate the development of digital ELISAs with reduced need for specialized microfluidic devices, instruments, or imaging systems.

category: bioengineering


title: Dynamic conformational states of apo and cabozantinib bound TAM kinases to differentiate active-inactive kinetic models

abstract: Tyro3, Axl, Mer (TAM) receptor tyrosine kinases (RTKs) are overexpressed in several human cancers. Cabozantinib, a small molecule inhibitor constrains the activity of TAM kinases at nanomolar concentrations. The dynamic active and inactive conformations of kinases play a crucial role in inhibitor binding and the activation of intracellular downstream signalling pathways. The all atom molecular dynamics (MD) simulations at microsecond timescale and longer provide robust insights into the structural details of conformational alterations of proteins due to their role cellular metabolic activities and signaling pathways. In this current study we report microsecond molecular dynamics (MD) simulations of apo, cabozantinib complexed active and inactive TAM RTKs and analysed the post-MD trajectories using the principal component analysis (PCA). Markov State Models (MSM) and transition pathways from Perron-cluster cluster analysis. For consensus, the 1{micro}s atomistic simulations with enhanced computational algorithms indicated us to treat tyrosine kinase family by overwhelming dynamic states existence when bound to kinase inhibitors. The dynamic mechanistic pathways intrinsic to the kinase activity and protein conformational landscape in the TAM kinases are revealed due to the alterations in the P-loop, C-helix, activation loop and F-helix that result in breaking the regulatory and catalytic spines. We deciphered the long lived kinetic transition states of distinct active and inactive structural models from MD simulations trajectories of TAM RTKs bound inhibitor complex that have not been revealed so far.

category: biophysics