title: Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM

abstract:
The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the worlds highest time resolutions for single fluorescent-molecule imaging of 33 (100) {micro}s (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640x640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research.

SummaryAn ultrafast camera developed by Fujiwara et al. allows single fluorescent-molecule imaging every 33 s with a localization precision of 34 nm (every 100 s; 20 nm), and enables ultrafast PALM imaging of whole live cells.


title: Cyclic uniaxial mechanical load enhances chondrogenesis through entraining the molecular circadian clock

abstract:
ObjectiveThe biomechanical environment plays a key role in regulating cartilage formation, but current understanding of mechanotransduction pathways in chondrogenic cells is still incomplete. Amongst the combination of external factors that control chondrogenesis are temporal cues that are governed by the cell-autonomous circadian clock. However, mechanical stimulation has not yet directly been proven to modulate chondrogenesis via entraining the circadian clock in chondroprogenitor cells.

DesignThe purpose of this study was to establish whether mechanical stimuli entrain the core clock in chondrogenic cells, and whether augmented chondrogenesis caused by mechanical loading was at least partially mediated by the synchronised, rhythmic expression of the core circadian clock genes, chondrogenic transcription factors, and cartilage matrix constituents.

ResultsWe report here, for the first time, that cyclic uniaxial mechanical load applied for 1 hour for a period of 6 days entrains the molecular clockwork in chondroprogenitor cells during chondrogenesis in limb bud-derived micromass cultures. In addition to the several core clock genes, the chondrogenic markers SOX9, ACAN, and COL2A1 also followed a robust sinusoidal rhythmic expression pattern. These rhythmic conditions significantly enhanced cartilage matrix production and upregulated marker gene expression. The observed chondrogenesis-promoting effect of the mechanical environment was at least partially attributable to its entraining effect on the molecular clockwork, as co-application of the small molecule clock modulator longdaysin attenuated the stimulatory effects of mechanical load.

ConclusionsResults from this study suggest that an optimal biomechanical environment enhances tissue homeostasis and histogenesis during early chondrogenesis through entraining the molecular clockwork.

category: cell biology


title: Focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion

abstract:
Using the ultrafast camera system and new theories for hop diffusion described in the companion paper, we for the first time demonstrated that membrane molecules undergo hop diffusion among the compartments in the bulk basal plasma membrane (PM), with virtually the same compartment sizes (108 nm) as those in the bulk apical PM and the same dwell lifetimes within a compartment (10 and 24 ms for the phospholipid and transferrin receptor, respectively), suggesting that the basic structures and molecular dynamics are very similar in the bulk regions of the apical and basal PMs. Ultrafast PALM and single-molecule imaging revealed that the focal adhesion (FA) is mostly a fluid membrane, partitioned into [~]74-nm compartments where transferrin receptor and {beta}3 integrin undergo hop diffusion, and that the FA membrane is sparsely dotted with 51-nm diameter paxillin islands, where many other FA proteins probably assemble (compartmentalized archipelago model). {beta}3 integrin intermittently associates with the paxillin islands, dynamically linking them to the extracellular matrix.

SummaryAn ultrafast camera with single fluorescent-molecule sensitivities developed by Fujiwara et al. reveals that the focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion, and integrin {beta}3 molecules become temporarily immobilized at the islands.


title: A novel role for the extraembryonic area opaca in positioning the primitive streak of the early chick embryo

abstract:
Classical studies have established that the marginal zone, a ring of extraembryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extraembryonic region (area opaca) was only thought to have nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region: first, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extraembryonic tissues in early development.

Summary statementTwo adjacent extraembryonic tissues, the area opaca and the marginal zone, interact to influence the polarity of the early chick embryo.

category: developmental biology


title: Notch signaling determines cell-fate specification of the two main types of vomeronasal neurons of rodents.

abstract:
The ability of terrestrial vertebrates to find food, mating partners and to avoid predators heavily relies on the detection of chemosensory information from the environment. The olfactory system of most vertebrate species comprises two distinct chemosensory systems usually referred to as the main and the accessory olfactory system. Olfactory sensory neurons of the main olfactory epithelium detect and transmit odor information to main olfactory bulb (MOB), while the chemosensory neurons of the vomeronasal organ detect semiochemicals responsible for social and sexual behaviors and transmit information to the accessory olfactory bulb (AOB). The vomeronasal sensory epithelium (VNE) of most mammalian species contains uniform vomeronasal (VN) system with vomeronasal sensory neurons (VSNs) expressing vomeronasal receptors of the V1R family. However, rodents and some marsupials have developed a more complex binary VN system, where VNO containing a second main type of VSNs expressing vomeronasal receptors of the V2R family is identified. In mice, V1R and V2R VSNs form from a common pool of progenitors but have distinct differentiation programs. As they mature, they segregate in different regions of the VNE and connect with different parts of the AOB. How these two main types of VSNs are formed has never been addressed. In this study, using single cell RNA sequencing data, we identified differential expression of Notch1 receptor and Dll4 ligand among the neuronal precursors at the VSN dichotomy. We further demonstrated with loss of function (LOF) and gain of function (GOF) studies that Dll4-Notch1 signaling plays a crucial role in triggering the binary dichotomy between the two main types of VSNs in mice.

Graphical Abstract

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title: Anatomic development of the upper airway during the first five years of life: A three-dimensional imaging study

abstract:
PurposeNormative data on the growth and development of the upper airway across the sexes is needed for the diagnosis and treatment of congenital and acquired respiratory anomalies and to gain insight on developmental changes in speech acoustics and disorders with craniofacial anomalies.

MethodsThe growth of the upper airway in children ages birth-to-five years, as compared to adults, was quantified using an imaging database with computed tomography studies from typically developing individuals. Methodological criteria for scan inclusion and airway measurements included: head position, histogram-based airway segmentation, anatomic landmark placement, and development of a semi-automatic centerline for data extraction. A comprehensive set of 2D and 3D supra- and sub-glottal measurements from the choanae to tracheal opening were obtained including: naso-oro-laryngo-pharynx subregion volume and length, each subregions superior and inferior cross-sectional-area, and antero-posterior and transverse/width distances.

ResultsGrowth of the upper airway during the first five years of life was more pronounced in the vertical and transverse/lateral dimensions than in the antero-posterior dimension. By age five years, females have larger pharyngeal measurement than males. Prepubertal sex-differences were identified in the subglottal region.

ConclusionsOur findings demonstrate the importance of studying the growth of the upper airway in 3D. As the lumen length increases, its shape changes, becoming increasingly elliptical during the first five years of life. This study also emphasizes the importance of methodological considerations for both image acquisition and data extraction, as well as the use of consistent anatomic structures in defining pharyngeal regions.


title: Multi-model Order ICA: A Data-driven Method for Evaluating Brain Functional Network Connectivity Within and Between Multiple Spatial Scales

abstract:
BackgroundWhile functional connectivity is widely studied, there has been little work studying functional connectivity at different spatial scales. Likewise, the relationship of functional connectivity between spatial scales is unknown.

MethodsWe proposed an independent component analysis (ICA) - based approach to capture information at multiple model orders (component numbers) and to evaluate functional network connectivity (FNC) both within and between model orders. We evaluated the approach by studying group differences in the context of a study of resting fMRI (rsfMRI) data collected from schizophrenia (SZ) individuals and healthy controls (HC). The predictive ability of FNC at multiple spatial scales was assessed using support vector machine (SVM)-based classification.

ResultsIn addition to consistent predictive patterns at both multiple-model orders and single model orders, unique predictive information was seen at multiple-model orders and in the interaction between model orders. We observed that the FNC between model order 25 and 50 maintained the highest predictive information between HC and SZ. Results highlighted the predictive ability of the somatomotor and visual domains both within and between model orders compared to other functional domains. Also, subcortical-somatomotor, temporal-somatomotor, and temporal-subcortical FNCs had relatively high weights in predicting SZ.

ConclusionsIn sum, multi-model order ICA provides a more comprehensive way to study FNC, produces meaningful and interesting results which are applicable to future studies. We shared the spatial templates from this work at different model orders to provide a reference for the community, which can be leveraged in regression-based or fully automated (spatially constrained) ICA approaches.

Impact StatementMulti-model order ICA provides a comprehensive way to study brain functional network connectivity within and between multiple spatial scales, highlighting findings that would have been ignored in single model order analysis. This work expands upon and adds to the relatively new literature on resting fMRI-based classification and prediction. Results highlighted the differentiating power of specific intrinsic connectivity networks on classifying brain disorders of schizophrenia patients and healthy participants, at different spatial scales. The spatial templates from this work provide a reference for the community, which can be leveraged in regression-based or fully automated ICA approaches.

category: neuroscience


title: Fruit flies subjected to TBI exhibit genotype-dependent changes in seizure incidence and duration

abstract: Traumatic brain injury (TBI) is common and often debilitating. One complication following TBI is post-traumatic seizures (PTS). However, there is a poor understanding of PTS, in part, because it is challenging to model. We sought to develop a fly model of PTS. We used a high-impact trauma (HIT) device to inflict TBI and followed this with behavioral, bang-testing methods to assess seizure behavior. Our results showed PTS incidence was sensitive to genetic background. We also found seizure duration was most pronounced at 30 minutes after injury. Our findings support the efficacy of our fly model for coarse screening of seizure behavior. We expect this model will be useful in first-pass screens intended to identify modifiers of seizure risk following TBI.


title: Effect of channel assembly (KCNQ1 or KCNQ1 + KCNE1) on the response of zebrafish IKs to IKs inhibitors and activators

abstract: In cardiac myocytes, the slow component of the delayed rectifier K+ current (IKs) ensures repolarization of action potential during beta-adrenergic activation or when other repolarizing K+ currents fail. As a key factor of cardiac repolarization IKs should be present in model species used for cardiovascular drug screening, preferably with pharmacological characteristics similar to those of the human IKs. To this end, we investigated the effects of inhibitors and activators of the IKs on KCNQ1 and KCNQ1+KCNE1 channels of the zebrafish, an important model species, in Chinese hamster ovary cells. Inhibitors of IKs, chromanol 293B and HMR-1556, inhibited zebrafish IKs channels with approximately similar potency as that of mammalian IKs. Chromanol 293B concentration for half-maximal inhibition (IC50) of zebrafish IKs was at 13.1{+/-}5.8 and 13.4{+/-}2.8 M for KCNQ1 and KCNQ1+KCNE1 channels, respectively. HMR-1556 was a more potent inhibitor of zebrafish IKs with IC50=0.1{+/-}0.1 M and 1.5{+/-}0.8 M for KCNQ1 and KCNQ1+KCNE1 channels, respectively. R-L3 and mefenamic acid, generally identified as IKs activators, both inhibited zebrafish IKs. R-L3 almost completely inhibited zebrafish IKs generated by KCNQ1 and KCNQ1+KCNE1 channels with similar affinity (IC50 1.1{+/-}0.4 and 1.0{+/-}0.4 M, respectively). Mefenamic acid partially blocked zebrafish KCNQ1 (IC50=9.5{+/-}4.8 M) and completely blocked KCNQ1+KCNE1 channels (IC50=3.3{+/-}1.8 M). Although zebrafish IKs responds to IKs inhibitors in the same way as mammalian IKs, its response to activators is atypical, probably due to the differences in the binding domain of KCNE1 to KCNQ1. Therefore, care must be taken when translating the results from zebrafish to humans.

category: physiology


title: Deep autoencoder enables interpretable tissue-adaptive deconvolution and cell-type-specific gene analysis

abstract: Single-cell RNA-sequencing (RNA-seq) has become a powerful tool to study biologically significant characteristics at explicitly high resolution. However, its application on emerging data is currently limited by its intrinsic techniques. Though many methods have been proposed to analyze bulk data using single-cell profile as a reference, they are limited on the interpretability, processing speed, and data size requirement. Here, we introduce Tissue-AdaPtive autoEncoder (TAPE), a deep learning method connecting bulk RNA-seq and single-cell RNA-seq, to achieve precise prediction in short time. By constructing an interpretable decoder and training under a unique scheme, TAPE can predict cell-type fractions and cell-type-specific gene expression tissue-adaptively. Compared with existing methods on several benchmarking datasets, TAPE is more accurate (up to 40% improvement on the real bulk data) and faster. Thus, it is sensitive enough to provide biologically meaningful predictions. For example, only TAPE can predict the tendency of increasing monocytes-to-lymphocytes ratio in COVID-19 patients from mild to serious symptoms, of which estimated indices are consistent with laboratory data. More importantly, through the analysis of clinical data, TAPE shows its ability to predict cell-type-specific gene expression profiles with biological significance. Combining with single-sample gene set enrichment analysis (ssGSEA), TAPE also provides valuable clues to investigate the immune response in different virus-infected patients. We believe that TAPE will enable and accelerate the precise analysis of high-throughput clinical data in a wide range.

category: bioinformatics


title: Deep autoencoder for interpretable tissue-adaptive deconvolution and cell-type-specific gene analysis


title: Streptomyces and Bacillus species utilize volatile organic compounds to impact Fusarium oxysporum f.sp. vasinfectum race 4 (Fov4) virulence and suppress Fusarium wilt in Pima cotton

abstract: Emergence of a highly virulent Fusarium oxysporum f.sp. vasinfectum race 4 (Fov4) with aggressiveness towards Pima cotton (Gossypium barbadense) has raised significant concern for cotton producers while revealing challenges in soil-borne cotton disease management strategies which rely heavily on crop resistance and chemical controls. An alternative management approach uses antagonistic bacteria as biocontrol agents against Fov4. Initial studies showed a unique combination of bacteria Bacillus Rz141 and Streptomyces HC658 isolates displayed a mutualistic relationship capable of altering Fov4 growth. Notably, experimental design placed Fov4 between each isolate preventing direct physical contact of bacterial colonies. These observations led us to hypothesize that bacterial volatile organic compounds (VOCs) impact the growth and virulence of Fov4. Ensuring physical separation, I-plate cultures showed Rz141 had a VOC inhibition of 24%. Similarly, physically separated cultures of Rz141 and HC658 showed slight increase in VOC inhibition, 26% with some loss of Fov4 pigmentation. Pathogenicity assays where Fov4-infected Pima cotton was exposed to VOCs from physically separated Rz141 and HC658 showed VOCs can suppress Fov4 infection and reduce tissue darkening. Our results provide evidence that rhizosphere bacteria can use VOCs as a communication tool impacting fungal physiology and virulence, and ultimately Fov4-cotton interactions without direct physical contact.

category: microbiology


title: Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction

abstract: Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to {gamma}-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.2 [A] and further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions.


title: Total parenteral nutrition drives glucose metabolism disorders by modulating gut microbiota and its metabolites

abstract: The occurrence of glucose metabolism disorders is a potentially fatal complication of total parenteral nutrition (TPN). However, the mechanisms of TPN-associated glucose metabolism disorders remain unclarified. Given that the glucose metabolism was related to gut microbiome and TPN could induce the gut microbiota dysbiosis, we hypothesized that gut microbiota and its metabolites played the important roles in TPN-associated glucose metabolism disorders. By performing a cohort study of 256 type 2 IF patients given PN, we found that H-PN (PN>80%) patients exhibited insulin resistance and a higher risk of complications. Then, TPN and microbiome transfer mice model showed that TPN promoted glucose metabolism disorders by inducing gut microbiota dysbiosis; 16S rRNA sequencing demonstrated that the abundance of Lactobacillaceae was decreased in mice model and negatively correlated with HOMA-IR and lipopolysaccharide level in TPN patients. Untargeted metabolomics found that indole-3-acetic acid (IAA) was decreased in TPN mice, and the serum level was also decreased in H-PN patients. Furthermore, GLP-1 secretion regulated by IAA and aryl hydrocarbon receptor was reduced in TPN mice and patients; IAA or liraglutide completely prevented glucose metabolism disorders in TPN mice. In conclusion, TPN drives glucose metabolism disorders by inducing alteration of gut microbiota and its metabolites.


title: Artificial intelligence reveals nuclear pore complexity

abstract:
Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport. Their intricate 120 MDa architecture remains incompletely understood. Here, we report a near-complete structural model of the human NPC scaffold with explicit membrane and in multiple conformational states. We combined AI-based structure prediction with in situ and in cellulo cryo-electron tomography and integrative modeling. We show that linker Nups spatially organize the scaffold within and across subcomplexes to establish the higher-order structure. Microsecond-long molecular dynamics simulations suggest that the scaffold is not required to stabilize the inner and outer nuclear membrane fusion, but rather widens the central pore. Our work exemplifies how AI-based modeling can be integrated with in situ structural biology to understand subcellular architecture across spatial organization levels.

One sentence summaryAn AI-based, dynamic model of the human nuclear pore complex reveals how the protein scaffold and the nuclear envelope are coupled inside cells.

category: molecular biology


title: Conditional depletion reveals temporal requirements for the oscillating transcription factor NHR-23/NR1F1 in C. elegans larval progression

abstract:
Nematode molting is a remarkable process where the animals must essentially build a new epidermis underneath the old skin and then rapidly shed the old skin. The study of molting provides a gateway into the developmental program of many core cellular and physiological processes, such as oscillatory gene expression, coordinated intracellular trafficking, steroid hormone signaling, developmental timing, and extracellular remodeling. The nuclear hormone receptor NHR-23/NR1F1 is an important regulator of molting. Imaging and western blot time-courses revealed oscillatory NHR-23::GFP expression in the epithelium that closely followed the reported mRNA expression. Timed depletion experiments using the auxin-inducible degron system revealed that NHR-23/NR1F1 depletion early in a given larval stage caused animals to arrest with only weak molting defects, whereas later depletion resulted in highly penetrant severe molting and morphological defects. This larval arrest was independent of insulin signaling. Despite the weakly penetrant molting defects following early NHR-23/NR1F1 depletion, the epidermal barrier was defective suggesting that NHR-23/NR1F1 is necessary for establishing or maintaining this barrier. NHR-23/NR1F1 coordinates the expression of factors involved in molting, lipid transport/metabolism, and remodeling of the apical extracellular matrix. We propose that NHR-23/NR1F1 is a regulator in a recently discovered large-scale gene oscillatory network coordinating rhythmic skin regeneration.

Summary StatementThis work provides insight into how a recently discovered large-scale gene expression oscillator promotes rhythmic skin regeneration in C. elegans.


title: Ultra-deep sequencing reveals no evidence of oncogenic mutations or enrichment by ex vivo CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

abstract: As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.

category: genetics


title: Syrbactin-class dual constitutive- and immuno-proteasome inhibitor TIR-199 impedes myeloma-mediated bone degeneration in vivo.

abstract: Proteasome-addicted neoplastic malignancies present a considerable refractory and relapsed phenotype with patients exhibiting drug-resistance and high mortality rates. To counter this global problem, novel proteasome-based therapies are being developed. In the current study we extensively characterize TIR-199, a syrbactin-class proteasome inhibitor derived from a plant virulence factor of bacterium Pseudomonas syringae pv syringae. We report that TIR-199 is a potent constitutive and immunoproteasome inhibitor, capable of inducing cell death in multiple myeloma, triple-negative breast cancer and non-small cell lung cancer lines, effectively inhibit proteasome in primary myeloma cells of refractory patients, and bypass the PSMB5 A49T+A50V bortezomib-resistant mutant. TIR-199 treatment leads to accumulation of canonical proteasome substrates in cells, it is specific, and does not inhibit 50 other enzymes tested in vitro. The drug exhibits synergistic cytotoxicity in combination with proteasome-activating-kinase DYRK2 inhibitor LDN192960. Furthermore, low dose TIR-199 exhibits in vivo activity in delaying myeloma-mediated bone degeneration in a mouse xenograft model. Together, our data indicates that proteasome inhibitor TIR-199 could indeed be a next generation drug within the repertoire of proteasome-based therapeutics with a potential to target relapsed and refractory proteasome-addicted neoplasia.

category: biochemistry


title: An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

abstract: Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidAs chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidAs chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N- chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCls chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.


title: Efficient Inference of Sensitivity to Electrical Stimulation from Intrinsic Electrical Properties of Primate Retinal Ganglion Cells

abstract: High-fidelity sensory neural implants must be calibrated to precisely activate specific cells via extracellular stimulation. However, collecting and analyzing the required electrical stimulation data may be difficult in the clinic. A potential solution is to infer stimulation sensitivity from intrinsic electrical properties. Here, this inference was tested using large-scale high-density stimulation and recording from macaque retinal ganglion cells ex vivo. Electrodes recording larger spikes exhibited lower activation thresholds, with distinct trends for somas and axons, and consistent trends across cells and retinas. Thresholds for somatic electrodes increased with distance from the axon initial segment. Responses were inversely related to thresholds, and exhibited a steeper dependence on injected current for axonal than somatic electrodes. Dendritic electrodes were largely ineffective for eliciting spikes. Biophysical simulations qualitatively reproduced these findings. Inference of stimulation sensitivity was employed in simulated visual reconstruction, revealing that the approach could improve the function of future high-fidelity retinal implants.


title: Nostoc talks back: Temporal patterns of differential gene expression during establishment of the Anthoceros-Nostoc symbiosis

abstract:
Endosymbiotic association between hornworts and dinitrogen-fixing cyanobacteria form when the plant is limited for combined nitrogen (N). We generated RNA-Seq data to examine the temporal gene expression patterns during culture of N-starved Anthoceros punctatus in the absence and presence of the symbiotically competent cyanobacterium Nostoc punctiforme. Symbiotic nitrogenase activity commenced within 5 days of coculture reaching a maximal by 14 days. In symbiont-free gametophytes, chlorophyll content, chlorophyll fluorescence characteristics and transcription of genes encoding light harvesting and reaction center proteins, as well as the small subunit of ribulose-bisphosphate-carboxylase/oxygenase, were downregulated. The downregulation was complemented in a temporal pattern corresponding to the N. punctiforme provision of N2-derived ammonium. The impairment and complementation of photosynthesis was the most distinctive response of A. punctatus to N-starvation. Increases in transcription of ammonium and nitrate transporters and their N. punctiforme-dependent complementation was also observed. The temporal patterns of differential gene expression indicated N. punctiforme transmits signals to A. punctatus both prior to, and after its provision of fixed N. This is the only known temporal transcriptomic study during establishment of a symbiotic nitrogen-fixing association in this monophyletic evolutionary lineage of land plants.

HighlightsTemporal RNA-Seq analysis revealed how symbiotic cyanobacteria impact plant partners global gene expression and elucidated the nature of bidirectional communications between the partners


title: Diversity analysis of soil microbial population abundance before and after planting JunCao "Oasis No.1" in saline-alkali soil

abstract: In order to explore the difference of soil microbial population structure and abundance before and after planting JunCao "Oasis No. 1" in saline-alkali soil, verify the improvement effect of JunCao "Oasis No. 1" on microbial population structure and abundance in saline-alkali soil. Samples were collected from the blank saline area with and without JunCao "Oasis NO.1" and no plant growth on the surface, respectively, as Experimental group soil samples (S.Y.1-S.Y.8) and Blank group soil samples (K.B.1-K.B.8).16sDNA high-throughput sequencing technology was used for sequencing analysis respectively, and the diversity of microbial population abundance between them was compared and analyzed.The results showed that the diversity of microbial population abundance in the experimental group was significantly higher than that in the blank group, and the diversity of microbial population abundance in the experimental group was significantly different from that in the blank group, indicating that the composition of microbial population in the experimental group was significantly different from that in the blank group. In the OTU cluster analysis, the number of OTU clusters in the Experimental group soil samples (S.Y.1-S.Y.8) was significantly higher than that in the Blank group soil samples (K.B.1-K.B.8). In the sample complexity analysis of -diversity analysis, the richness and diversity of microbial population in soil samples of Experimental group (S.Y.1-S.Y.8) were significantly higher than that in soil samples of Blank group (K.B.1-K.B.8), which was clearly reflected in the Species accumulation boxplot and Graph of species diversity. In the {beta}-diversity analysis, PcoA, PCA and NMDS analysis methods were used to analyze the difference of microbial population diversity between Experimental soil samples (S.Y.1-S.Y.8) and Blank soil samples (K.B.1-K.B.8). The results showed that the diversity of microbial population in Experimental soil sample (S.Y.1-S.Y.8) was significantly different from that in Blank soil sample (K.B.1-K.B.8). In this paper, 16sDNA high-throughput sequencing technology was used to analyze the diversity of microbial population abundance between Blank soil samples and Experimental soil samples, and it was proved that JunCao "Oasis No. 1" had good saline-alkali soil improvement characteristics. It can effectively increase the abundance of microbial population in saline-alkali soil, so as to restore the microbial population ecosystem in saline-alkali soil, which has important application value in soil saline-alkali control.


title: DNA-dependent binding of nargenicin to DnaE1 inhibits replication in Mycobacterium tuberculosis

abstract: Natural products provide a rich source of potential antimicrobials for use in treating infectious diseases for which drug resistance has emerged. Foremost among these is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.


title: The transcription factor IscR promotes Yersinia type III secretion system activity by antagonizing the repressive H-NS-YmoA histone-like protein complex

abstract:
The type III secretion system (T3SS) is a appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive YmoA/H-NS histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. Although IscR has been shown to bind the lcrF promoter and is required for in vivo expression of lcrF, in this study we show IscR alone fails to enhance lcrF transcription in vitro. Rather, we find that in a ymoA mutant, IscR is no longer required for LcrF expression or T3SS activity. Additionally, a mutation in YmoA that prevents H-NS binding (ymoAD43N) rescues the T3SS defect of a {Delta}iscR mutant, suggesting that a YmoA/H-NS complex is needed for this repressive activity. Furthermore, chromatin immunoprecipitation analysis revealed that H-NS is enriched at the lcrF promoter at environmental temperatures, while IscR is enriched at this promoter at mammalian body temperature under aerobic conditions. Importantly, CRISPRi knockdown of H-NS leads to increased lcrF transcription. Collectively, our data suggest that as IscR levels rise with iron limitation and oxidative stress, conditions Yersinia experiences during extraintestinal infection, IscR antagonizes YmoA/H-NS-mediated repression of lcrF transcription to drive T3SS activity and manipulate host defense mechanisms.

Author SummaryFacultative pathogens must silence virulence gene expression during growth in the environment, while retaining the ability to upregulate these genes upon infection of a host. H-NS is an architectural DNA binding protein proposed to silence horizontally acquired genes, regulating virulence genes in a number of pathogens. Indeed, H-NS was predicted to regulate plasmid-encoded virulence genes in pathogenic Yersinia. However, Yersinia H-NS is reported to be essential, complicating testing of this model. We used chromatin immunoprecipitation and inducible CRISPRi knockdown to show that H-NS binds to the promoter of a critical plasmid-encoded virulence gene, silencing its expression. Importantly, under conditions that mimic Yersinia infection of a mammalian host, the transcriptional regulator IscR displaces H-NS to drive virulence factor expression.


title: An evolutionarily conserved stop codon enrichment at the 5' ends of mammalian piRNAs

abstract: PIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5 ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. We examined murine piRNAs and revealed a strong and specific enrichment of three sequences (UAA, UAG, UGA)-- corresponding to stop codons--at piRNA 5 ends. Stop codon sequences were also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses revealed that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5 end stop codons. This observation was conserved across mammals and possibly further. Our work provides new insights into Zuc-dependent cleavage and may point to a previously unrecognized connection between piRNA biogenesis and the translational machinery.


title: Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

abstract:
Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. Here, we report human induced pluripotent stem cell derived kidney organoids containing a podocyte population that heads towards adult podocytes and were superior compared to 2D counterparts, based on scRNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Summary StatementKidney organoid podocytes allow personalized nephrotic sydrome modeling,


title: The molecular chaperone DNAJB6 provides surveillance of FG-Nups and is required for interphase nuclear pore complex biogenesis

abstract: Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage/crossing of biomolecules. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separate1,2 and aggregate when isolated3. It has remained largely unclear how FG-Nups are protected from making inappropriate interactions during NPC biogenesis. We found that DNAJB6, a molecular chaperone of the heat shock protein network, formed foci next to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Reversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and they could be identified as herniations at the nuclear envelope (NE). Immunoelectron tomography showed that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Interestingly, loss of DNAJB6 results in annulate lamellae, which are structures containing partly assembled NPCs associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG-region of several FG-Nups in cells and in vitro. Together, our data show that DNAJB6 provides quality control during NPC biogenesis and is the first molecular chaperone that is involved in the surveillance of native intrinsically disordered proteins, including FG-Nups.


title: Evaluation of 6 MALDI-Matrices for 10 μm lipid imaging and on-tissue MSn with AP-MALDI-Orbitrap

abstract:
Mass spectrometry imaging (MSI) is a technique uniquely suited to localize and identify lipids in a tissue sample. Using an AP-MALDI UHR source coupled to an Orbitrap Elite, numerous lipid locations and structures can be determined in high mass resolution spectra and at cellular spatial resolution, but careful sample preparation is necessary. We tested 11 protocols on serial brain sections for the commonly used MALDI matrices, CHCA, Norharmane, DHB, DHAP, THAP, and DAN, in combination with tissue washing and matrix additives, to determine the lipid coverage, signal intensity, and spatial resolution achievable with AP-MALDI. In positive ion mode, the most lipids could be detected with CHCA and THAP, while THAP and DAN without additional treatment offered the best signal intensities. In negative ion mode, DAN showed the best lipid coverage and DHAP performed superior for Gangliosides. DHB produced intense cholesterol signals in the white matter. 155 lipids were assigned in positive (THAP), 137 in negative ion mode (DAN) and 76 lipids were identified using on tissue tandem-MS. The spatial resolution achievable with DAN was 10 m, confirmed with on tissue line-scans. This enabled the association of lipid species to single neurons in AP-MALDI images. The results show that the performance of AP-MALDI is comparable to vacuum MALDI techniques for lipid imaging.

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title: Investigation of the futalosine pathway for menaquinone biosynthesis as a novel target in the inhibition of Chlamydia trachomatis infection

abstract: Chlamydia trachomatis, an obligate intracellular bacterium with limited metabolic capabilities, possesses the futalosine pathway for menaquinone biosynthesis. Futalosine pathway enzymes have promise as narrow spectrum targets, but the activity and essentiality of chlamydial menaquinone biosynthesis have yet to be established. In this work, menaquinone-7 (MK-7) was identified as a C. trachomatis-produced quinone through LC-MS/MS. An immunofluorescence-based assay revealed that treatment of C. trachomatis-infected HeLa cells with futalosine pathway inhibitor docosahexaenoic acid (DHA) reduced inclusion number, inclusion size, and infectious progeny. Supplementation with MK-7 nanoparticles rescued the effect of DHA on inclusion number, indicating that the futalosine pathway is a target of DHA in this system. These results open the door for menaquinone biosynthesis inhibitors to be pursued in antichlamydial development.


title: DeepMicrobeFinder sorts metagenomes into prokaryotes, eukaryotes and viruses, with marine applications

abstract: Sequence classification is valuable for reducing the complexity of metagenomes and providing a fundamental understanding of the composition of metagenomic samples. Binary metagenomic classifiers offer an insufficient solution because metagenomes of most natural environments are typically derived from multiple sequence sources including prokaryotes, eukaryotes and the viruses of both. Here we introduce a deep-learning based (not reference-based) sequence classifier, DeepMicrobeFinder, that classifies metagenomic contigs into five sequence classes, e.g., viruses infecting prokaryotic or eukaryotic hosts, eukaryotic or prokaryotic chromosomes, and prokaryotic plasmids. At different sequence lengths, DeepMicrobeFinder achieved area under the receiver operating characteristic curve (AUC) scores >0.9 for most sequence classes, the exception being distinguishing prokaryotic chromosomes from plasmids. By benchmarking on 20 test datasets with variable sequence class composition, we showed that DeepMicrobeFinder obtained average accuracy scores of 0.94, 0.87, and 0.92 for eukaryotic, plasmid and viral contig classification respectively, which were significantly higher than the other state-of-the-art individual predictors. Using a 1-300 m daily time-series metagenomic dataset sampled from coastal Southern California as a case study, we showed that metagenomic read proportions recruited by eukaryotic contigs could be doubled with DeepMicrobeFinders classification compared to the counterparts of other reference-based classifiers. In addition, a positive correlation could be observed between eukaryotic read proportions and potential prokaryotic community growth rates, suggesting an enrichment of fast-growing copiotrophs with increased eukaryotic particles. With its inclusive modeling and unprecedented performance, we expect DeepMicrobeFinder will be a useful addition to the toolbox of microbial ecologists, and will promote metagenomic studies of under-appreciated sequence types.


title: Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

abstract:
Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis.

ImportanceMCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here we provide evidence of a potential mechanism inS. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

category: synthetic biology


title: The structure of a native orthobunyavirus ribonucleoprotein reveals a key role for viral RNA in maintaining its helical architecture

abstract:
The Bunyavirales order of RNA viruses comprises emerging pathogens for which approved preventative or therapeutic measures for human use are not available. The genome of all Bunyavirales consists of negative-sense RNA segments wrapped by the virus-encoded nucleocapsid protein (NP) to form ribonucleoproteins (RNPs). RNPs represent the active template for RNA synthesis and the form in which the genome is packaged into virions, functions that require inherent flexibility. We present a pseudo-atomic model of a native RNP purified from Bunyamwera virus (BUNV), the prototypical Bunyavirales member, based on a cryo-electron microscopy (cryo-EM) average at 13 [A] resolution with subsequent fitting of the BUNV NP crystal structure by molecular dynamics. We show the BUNV RNP possesses relaxed helical architecture, with successive helical turns separated by [~]18 [A]. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms, with no helix-stabilizing interactions along the longitudinal axis. Instead, EM analysis of RNase-treated RNPs suggests their chain integrity is dependent on the encapsidated genomic RNA, thus providing the molecular basis for RNP flexibility. Overall, this work will assist in designing anti-viral compounds targeting the RNP and inform studies on bunyaviral RNP assembly, packaging and RNA replication.

SignificanceBunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for human use do not exist. The bunyavirus genome does not exist as naked RNA; instead it is wrapped up by the nucleoprotein (NP) to form a ribonucleoprotein (RNP). Using the prototypical bunyavirus, Bunyamwera virus, we determined the 3D structure of the native RNP, revealing a helical architecture with NP molecules linked by lateral contacts only, with no helix-stabilizing longitudinal contacts. Instead, the RNA genome itself plays a role in maintaining the helical architecture, allowing a high degree of flexibility that is critical for several stages of the virus replication cycle, such as segment circularization and genome packaging into virions.


title: Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

abstract: {beta}-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative {beta}-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 {degrees}C towards 4-Nitrophenyl-{beta}-galactofuranoside (pNP-{beta}-Galf), with a Km of 17.9 {+/-} 1.9 mM and Vmax of 70.6 {+/-} 5.3 mol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.


title: Relationship between systolic blood pressure and decision-making during emotional processing

abstract:
ObjectiveEmotional states are expressed in body and mind through subjective experience of physiological changes. In previous work, subliminal priming of anger prior to lexical decisions increased systolic blood pressure (SBP). This increase predicted the slowing of response times (RT), suggesting that baroreflex-related autonomic changes and their interoceptive (feedback) representations, influence cognition. Alexithymia is a subclinical affective dysfunction characterized by difficulty in identifying emotions. Atypical autonomic and interoceptive profiles are observed in alexithymia. Therefore, we sought to identify mechanisms through which SBP fluctuations during emotional processing might influence decision-making, including whether alexithymia contributes to this relationship.

MethodsThirty-two male participants performed an affect priming paradigm and completed the Toronto Alexithymia Scale. Emotional faces were briefly presented (20ms) prior a short-term memory task. RT, accuracy and SBP were recorded on a trial-by-trial basis. Generalized mixed-effects linear models were used to evaluate the impact of emotion, physiological changes, alexithymia score, and their interactions, on performances.

ResultsA main effect of emotion was observed on accuracy. Participants were more accurate on trials with anger primes, compared to neutral priming. Greater accuracy was related to increased SBP. An interaction between SBP and emotion was observed on RT: Increased SBP was associated with RT prolongation in the anger priming condition, yet this relationship was absent under the sadness priming. Alexithymia did not significantly moderate the above relationships.

ConclusionsOur data suggest that peripheral autonomic responses during affective challenges guide cognitive processes. We discuss our findings in the theoretical framework proposed by Lacey and Lacey (1970).


title: Investigating Biases Associated with Dietary Starch Incorporation and Retention with an Oral Biofilm Model

abstract:
Dental calculus has proven to contain a wealth of information on the dietary habits of past populations. These insights have, to a large extent, been obtained by the extraction and identification of starch granules contained within the mineralised dental plaque from a wide range of regions and time periods. The scope of previous studies have been limited to microfossil extraction and identification to reconstruct dietary preferences from the archaeological record, and few studies have attempted to address the biases of starch retention in dental calculus. Those that have considered this problem have been limited to in vivo studies on modern humans and non-human primates. Here, we present a multispecies oral biofilm model, which allows experimental research on starch incorporation and retention to be conducted on in vitro dental calculus in a controlled laboratory setting. The biofilms were exposed to treatment solutions with known quantities of dietary starches (wheat and potato) during the 25-day growth period. After this, the starch granules were extracted from the mature biofilm (by dissolution in EDTA), and counted. We show that the granule counts extracted from the model dental calculus represented a low proportion (ranging from 0.06% to 0.16%) of the total number of granules exposed to the biofilms throughout the experiment. Additionally, we found that the ratios of granule sizes from the extracted starch granules differed from the original treatment solutions, with large granules (>20 m) consistently being under-represented. We also found a correlation between the absolute granule counts and dry-weight of the biofilm (r = 0.66, 90%CI[0.46,0.79]), as well as between the concentration (count per mg) of granules and dry-weight (r = 0.30, 90%CI[0.06,0.51]).

Our results reinforce previous in vivo studies suggesting that dental calculus presents a very small, and partly biased picture of the original dietary intake of starches, with an over-representation of plants producing granules smaller than 20 m in size. The experimental model presented here is well-suited to address the need for further validation of methods and biases associated with dietary research on dental calculus.


title: SpatialDE2: Fast and localized variance component analysis of spatial transcriptomics

abstract: Spatial transcriptomics is now a mature technology, allowing to assay gene expression changes in the histological context of complex tissues. A canonical analysis workflow starts with the identification of tissue zones that share similar expression profiles, followed by the detection of highly variable or spatially variable genes. Rapid increases in the scale and complexity of spatial transcriptomic datasets demand that these analysis steps are conducted in a consistent and integrated manner, a requirement that is not met by current methods. To address this, we here present SpatialDE2, which unifies the mapping of tissue zones and spatial variable gene detection as integrated software framework, while at the same time advancing current algorithms for both of these steps. Formulated in a Bayesian framework, the model accounts for the Poisson count noise, while simultaneously offering superior computational speed compared to previous methods. We validate SpatialDE2 using simulated data and illustrate its utility in the context of two real-world applications to the spatial transcriptomics profiles of the mouse brain and human endometrium.


title: MLF2 modulates phase separated nuclear envelope condensates that provoke dual proteotoxicity

abstract: DYT1 dystonia is a highly debilitating neurological movement disorder arising from mutation in the AAA+ ATPase TorsinA. The hallmark of Torsin dysfunction is nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis. Whether blebs actively contribute to disease manifestation is presently unknown. We report that FG-nucleoporins in the bleb lumen undergo phase separation and contribute to DYT1 dystonia by provoking two proteotoxic insults. Short-lived ubiquitinated proteins that are normally rapidly degraded in healthy cells partition into the bleb lumen and become stabilized. Additionally, blebs selectively sequester a chaperone network composed of HSP70s and HSP40s. The composition of this chaperone network is altered by the bleb component MLF2. We further demonstrate that MLF2 is a catalyst of phase separation that suppresses the ectopic accumulation of FG-nucleoporins and modulates the selective properties and size of condensates in vitro. Our studies identify unprecedented, dual mechanisms of proteotoxicity in the context of liquid-liquid phase separation with direct implications for our understanding of disease etiology and treatment.


title: Robust enhancer-gene regulation identified by single-cell transcriptomes and epigenomes

abstract: Integrating single-cell transcriptomes and epigenomes across diverse cell types can link genes with the cis-regulatory elements (CREs) that control expression. Gene co-expression across cell types confounds simple correlation-based analysis and results in high false prediction rates. We developed a procedure that controls for co-expression between genes and integrates multiple molecular modalities, and used it to identify >10,000 gene-CRE pairs that contribute to gene expression programs in different cell types in the mouse brain.


title: Cell region fingerprints enable highly precise single-cell tracking and lineage reconstruction

abstract: Experimental studies of cell growth, inheritance, and their associated processes by microscopy require accurate single-cell observations of sufficient duration to reconstruct the genealogy. However, cell tracking--assigning identical cells on consecutive images to a track--is often challenging due to imperfect segmentation, moving cells, or focus drift, resulting in laborious manual verification. Here, we propose fingerprints to identify problematic assignments rapidly. A fingerprint distance measures the similarity between cells in two consecutive images by comparing the structural information contained in the low frequencies of a Fourier transform. We show that it is broadly applicable across cell types and image modalities, provided the image has sufficient structural information. Our tracker (TracX) uses the concept to reject unlikely assignments, thereby substantially increasing tracking performance on published and newly generated long-term data sets from various species. For S. cerevisiae, we propose a comprehensive model for cell size control at the single-cell and population level centered on the Whi5 regulator. It demonstrates how highly precise tracking can help uncover previously undescribed single-cell biology.

category: systems biology


title: Left angular gyrus disconnection impairs multiplication fact retrieval

abstract:
Arithmetic fact retrieval has been suggested to recruit a left-lateralized network comprising perisylvian language areas, parietal areas such as the angular gyrus (AG), and subcortical structures such as the hippocampus. However, the underlying white matter connectivity of these areas has not been evaluated systematically so far.

Using simple multiplication problems, we evaluated how disconnections in parietal brain areas affected arithmetic fact retrieval following stroke. We derived disconnectivity measures by jointly considering data from n=73 patients with acute unilateral lesions in either hemisphere and a white-matter tractography atlas (HCP-842) using the Lesion Quantification Toolbox (LQT). Whole-brain voxel-based analysis indicated a left-hemispheric cluster of white matter fibers connecting the AG and superior temporal areas to be associated with a fact retrieval deficit. Subsequent analyses of direct grey-to-grey matter disconnections revealed that disconnections of additional left-hemispheric areas (e.g., between the superior temporal gyrus and parietal areas) were significantly associated with the observed fact retrieval deficit.

Results imply that disconnections of parietal areas (i.e., the AG) with language-related areas (i.e., superior and middle temporal gyri) seem specifically detrimental to arithmetic fact retrieval. This suggests that arithmetic fact retrieval recruits a widespread left-hemispheric network and emphasizes the relevance of white matter connectivity for number processing.


title: Social experience dependent plasticity of mouse song selectivity without that of song components

abstract: Syllable sequences in male mouse ultrasonic-vocalizations (USVs), "songs", contain structure -quantified through predictability, like birdsong and aspects of speech. Apparent USV innateness and lack of learnability, discount mouse USVs for modelling speech-like social communication and its deficits. Informative contextual natural sequences (SN) were theoretically extracted and they were preferred by female mice. Primary auditory cortex (A1) supragranular neurons show differential selectivity to the same syllables in SN and random sequences (SR). Excitatory neurons (EXNs) in females showed increases in selectivity to whole SNs over SRs based on extent of social exposure with male, but syllable selectivity remained unchanged. Thus mouse A1 single neurons adaptively represent entire order of acoustic units without altering selectivity of individual units, fundamental to speech perception. Additionally, observed plasticity was replicated with silencing of somatostatin positive neurons, which had plastic effects opposite to EXNs, thus pointing out possible pathways involved in perception of sound sequences.


title: Activation of Npas1-neurons in the Ventral Pallidum Mediates Stress Susceptibility

abstract:
BackgroundAltered activity of the ventral pallidum (VP) underlies disrupted motivation after stress exposure. The VP is a very heterogeneous structure comprised of many different neuron types with distinct electrophysiological properties and projections. Neuronal PAS 1-positive (Npas1+) VP neurons are thought to send projections to brain regions critical for stress response. In this study, we evaluated how activity of VP Npas1+ neurons affect emotional behaviors and responses to social stress.

MethodsWe used a chemogenetic approach to manipulate VP Npas1+ neurons during social defeat stress (SDS) and behavioral tasks related to anxiety and motivation in Npas1-Cre mice. We employed a similar approach in females using the chronic witness defeat stress (CWDS). Finally, to characterize VP Npas1+ neuron circuitry and molecular identity we evaluated the projection targets of the VP Npas1+ neurons and performed RNA-seq on ribosome-associated mRNA from VP Npas1+ neurons.

ResultsChemogenetic activation of VP Npas1+ neurons increased susceptibility to a subthreshold (S)SDS and anxiety-like behavior in the elevated plus maze and open field. Inhibition of VP Npas1+ neurons enhanced resilience to chronic (C)SDS and CWDS. We identified VP Npas1+ projections to the nucleus accumbens (NAc), ventral tegmental area (VTA), medial and lateral habenula (LHb), lateral hypothalamus (LH), thalamus, medial and lateral septum, and periaqueductal gray area. VP Npas1+ neurons displayed distinct transcriptomes representing distinct biological processes.

ConclusionsActivity, of VP Npas1+ neurons, modulates susceptibility to social stressors and anxiety-like behavior. These outcomes could be related to their projections to brain regions that modulate reward and aversion.


title: Ormyrus labotus Walker (Hymenoptera: Ormyridae): another generalist that should not be a generalist is not a generalist

abstract: Several recent reappraisals of supposed generalist parasite species have revealed hidden complexes of species, each with considerably narrower host ranges. Parasitic wasps that attack gall-forming insects on plants have life history strategies that are thought to promote specialization, and though many species are indeed highly specialized, others have been described as generalist parasites. Ormyrus labotus Walker (Hymenoptera: Ormyridae) is one such apparent generalist, with rearing records spanning more than 65 host galls associated with a diverse set of oak tree species and plant tissues. We pair a molecular approach with morphology, host ecology, and phenological data from across a wide geographic sample to test the hypothesis that this supposed generalist is actually a complex of several more specialized species. We find 16-18 putative species within the morphological species O. labotus, each reared from only 1-6 host gall types, though we identify no single unifying axis of specialization. We also find cryptic habitat specialists within two other named Ormyrus species. Our study suggests that caution should be applied when considering host ranges of parasitic insects described solely by morphological traits, particularly given their importance as biocontrol organisms and their role in biodiversity and evolutionary studies.

category: evolutionary biology


title: Sign inversion in selection on ploidy

abstract: Ploidy - the number of homologous chromosome sets in a cell - is remarkably variable across the natural world, yet the evolutionary processes that have resulted in such diversity remain poorly understood. Here we use stochastic agent-based simulations to model ploidy evolution under the influence of indirect selection, i.e., selection mediated solely by statistical associations with fitness-affecting mutations. We find that in non-equilibrium asexual populations, the sign of selection on ploidy can change with population size - a phenomenon we have previously termed sign inversion. In large populations, ploidy dynamics are dominated by indirect effects of selection on beneficial mutations, which favors haploids over diploids. However, as population size declines, selection for beneficial mutations is neutralized by random genetic drift before drift can overwhelm selection against the cost of the deleterious mutational load. As a result, in small populations indirect selection is dominated by the cost of the deleterious load, which favors diploids over haploids. Our work adds to the growing body of evidence challenging established evolutionary theory that population size can affect only the efficiency, but not the sign, of natural selection.


title: A cautionary tale on proper use of branch-site models to detect convergent positive selection

abstract: Comparative genomics has become a powerful tool to elucidate genotype-phenotype relationships, particularly through the study of convergently acquired phenotypes. By identifying genes under positive selection specifically on branches with the convergent phenotype we can potentially link genes to that phenotype. Such gene scans are often done using branch-site codon models. However, we have observed a recent troubling trend of misinterpretation of branch-site models in which phylogeny-wide positive selection is not distinguished from positive selection specific to convergent branches. Here, we use simulations and real data to demonstrate how failing to discern between these two cases leads to false inferences of positive selection associated with a convergent trait. We then present a " drop-out" test solution to distinguish the two cases and thereby truly capture positive selection events associated with convergent phenotypes, thus allowing for further insights into both genetic and phenotypic evolution.


title: Ribosomal protein database profiling lends clarity to ribosomal protein evolution and mass distribution

abstract:
Existence of theoretical ribosomal protein mass fingerprint as well as utility of ribosomal protein as biomarkers in mass spectrometry microbial identification suggests phylogenetic significance for this class of proteins. To serve the above two functions, facile means of identifying and extracting important attributes of ribosomal proteins from proteome data file of microbial species must be found. Additionally, there is a need to calculate important properties of ribosomal proteins such as molecular weight and nucleotide sequence based on amino acid sequence information from FASTA proteome file. This work sought to support the above endeavour through developing a MATLAB software that extracts the amino acid sequence information of all ribosomal proteins from the FASTA proteome datafile of a microbial species downloaded from UniProt. Built-in functions in MATLAB are subsequently employed to calculate important properties of extracted ribosomal proteins such as number of amino acid residue, molecular weight and nucleotide sequence. All information above are output, as a database, to an Excel file for ease of storage and retrieval. Data available from the analysis of an Escherichia coli K-12 proteome revealed that the bacterium possess a total of 59 ribosomal proteins distributed between the large and small ribosome subunits. The ribosomal protein ranges in sequence length from 38 (50S ribosomal protein L36) to 557 (30S ribosomal protein S1). In terms of molecular weight distribution, the profiled ribosomal proteins range in weight from 4364.305 Da (50S ribosomal protein L36) to 61157.66 Da (30S ribosomal protein S1). More important, analysis of the distribution of the molecular weight of different ribosomal proteins in E. coli reveals a smooth curve that suggests strong co-evolution of ribosomal protein sequence and mass given the tight constraints that a functional ribosome presents. Finally, cluster analysis reveals a preponderance of small ribosomal proteins compared to larger ones, which remains to be a mystery to evolutionary biologists. Overall, the information encapsulated in the ribosomal protein database should find use in gaining a better appreciation for the molecular weight distribution of ribosomal proteins in a species, as well as delivering information for using ribosomal protein biomarkers in identifying particular microbial species in mass spectrometry microbial identification.

Subject areasbiochemistry, microbiology, bioinformatics, biotechnology, molecular biology,

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/465821v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@17b8205org.highwire.dtl.DTLVardef@195517eorg.highwire.dtl.DTLVardef@1940a0org.highwire.dtl.DTLVardef@1e1ea47_HPS_FORMAT_FIGEXP  M_FIG C_FIG Short descriptionSmooth function describes the distribution of molecular weight of ribosomal proteins in Escherichia coli, which suggests strong co-evolution pressure that fine-tunes the molecular weight of individual proteins with the constraint coming from the overall structure of the ribosome that needs to deliver a consistent function to the living cell. Multitude of ribosomal proteins with different roles in the ribosomal proteins under tight co-evolution pressure likely engender the observed smooth curve in the above plot.

O_TEXTBOXHighlightsO_LIAutomated MATLAB software for extracting ribosomal protein sequence from FASTA proteome file of a microbial species was developed.
C_LIO_LIIn-built functions were used to calculate nucleotide sequence, number of residues and molecular weight of each of the extracted ribosomal protein
C_LIO_LIAll extracted and calculated information were encapsulated as a ribosomal protein database for output to an Excel file for ease of storage and retrieval.
C_LIO_LIInformation in database could be useful for theoretical ribosomal protein mass fingerprint or help assign ribosomal protein biomarker peaks in matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) microbial identification.
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title: Extensive sequence divergence of non-coding regions between Aspergillus fumigatus, a major fungal pathogen of humans, and its relatives

abstract: Invasive aspergillosis is a deadly fungal disease; more than 400,000 patients are infected worldwide each year and the mortality rate can be as high as 50-95%. Of the [~]450 species in the genus Aspergillus only a few are known to be clinically relevant, with the major pathogen Aspergillus fumigatus being responsible for [~]50% of all invasive mold infections. Genomic comparisons of A. fumigatus to other Aspergillus species have historically focused on protein-coding regions. However, most A. fumigatus genes, including those that modulate its virulence, are also present in non-pathogenic close relatives of A. fumigatus. Our hypothesis is that differential gene regulation - mediated through the non-coding regions upstream of genes transcription start sites - contributes to A. fumigatus pathogenicity. To begin testing this, we compared non-coding regions up to 500 base pairs upstream of the first codon of single-copy orthologous genes from the two A. fumigatus reference strains Af293 and A1163 and eight closely related Aspergillus section Fumigati species. We found that non-coding regions showed extensive sequence variation and lack of homology across species. By examining the evolutionary rates of both protein-coding and non-coding regions in a subset of orthologous genes with highly conserved non-coding regions across the phylogeny, we identified 418 genes, including 25 genes known to modulate A. fumigatus virulence, whose non-coding regions exhibit a different rate of evolution in A. fumigatus. Examination of sequence alignments of these non-coding regions revealed numerous instances of insertions, deletions, and other types of mutations of at least a few nucleotides in A. fumigatus compared to its close relatives. These results show that closely related Aspergillus species that vary greatly in their pathogenicity exhibit extensive non-coding sequence variation and identify numerous changes in non-coding regions of A. fumigatus genes known to contribute to virulence.


title: A thermodynamic model of bacterial transcription

abstract: Transcriptional pausing is highly regulated by the template DNA and nascent transcript sequences. Here, we propose a thermodynamic model of transcriptional pausing, based on the thermal energy of transcription bubbles and nascent RNA structures, to describe the kinetics of the reaction pathways between active translocation, intermediate, backtracked, and hairpin-stabilized pauses. The model readily predicts experimentally detected pauses in high-resolution optical tweezers measurements of transcription. Unlike other models, it also predicts the effect of tension and the GreA transcription factor on pausing.

category: biophysics


title: Hoop-like role of the cytosolic interface helix in Vibrio PomA, an ion-conducting membrane protein, in the bacterial flagellar motor

abstract:
Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The three-dimensional structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66, and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

ImportanceBacteria have a motor embedded in the membrane to rotate flagella as screw for swimming. The motor is composed rotor and stator complexes. The interaction between the rotor and stator converts the electrochemical potential gradient across the membrane into motor torque. The stator functions as an ion channel and is composed of two membrane proteins, MotA and MotB for proton or PomA and PomB for sodium ion. Based on the structural data of stator, we systematically introduce the proline replacement mutations and found that the cytosolic interface (CI) helix which is located parallel to the inner membrane between the second and third transmembrane (TM) segments, performs a hoop-like function in securing the stability of the stator complex and the ion conduction pathway. The results of this study provide novel insights into the energy conversion mechanism of the flagellar motor and the general mechanism of the ion channel function.


title: DEAD-box RNA helicase 18 disrupts IRF3-binding to the interferon-β promoter

abstract: The production of type I interferons (IFN-/{beta}) requires strict control to avoid excessive activation during viral infections. The binding of interferon regulatory factor 3 (IRF3) to the IFN-{beta} promoter region in the nucleus is essential for IFN-{beta} transcription; however, whether nuclear factors have important negative-regulatory roles in this process is largely unknown. By screening for IRF3-interacting partners in the nucleus, we identified DEAD-box RNA helicase 18 (DDX18) as an important negative regulator of intranuclear IRF3. Overexpression of DDX18 suppressed virus- and IRF3-induced IFN-{beta} production, whereas knockdown of DDX18 expression or knockout of the DDX18 gene had opposite effects. Mechanistically, DDX18 interacts with IRF3 and decreases the binding of IRF3 to the IFN-{beta} promoter after viral infection. DDX18 knockdown mice (Ddx18+/-) further demonstrated that DDX18 suppressed antiviral innate immunity in vivo. Thus, despite many members of the DDX family act as important positive regulators in the cytoplasm, DDX18 plays a unique "braking" role in balancing virus-induced type I IFN production.

category: immunology


title: A hyper-immunogenic and slow-growing fungal strain induces a murine granulomatous response to cryptococcal infection

abstract: Many successful pathogens cause latent infections, remaining dormant within the host for years but retaining the ability to reactivate to cause symptomatic disease. The human opportunistic pathogen Cryptococcus neoformans is a ubiquitous yeast that establishes latent pulmonary infections in immunocompetent individuals upon fungal inhalation from the environment. These latent infections are frequently characterized by granulomas, or foci of chronic inflammation, that contain dormant cryptococcal cells. Immunosuppression causes these granulomas to break down and release viable fungal cells that proliferate, disseminate, and eventually cause lethal cryptococcosis. This course of C. neoformans dormancy and reactivation is understudied due to limited models, as chronic pulmonary granulomas do not typically form in most mouse models of cryptococcal infection. Here, we report that a previously characterized Cryptococcus-specific gene which is required for host-induced cell wall remodeling, MAR1, inhibits murine granuloma formation. Specifically, the mar1{Delta} loss-of-function mutant strain induces mature pulmonary granulomas at sites of infection dormancy in mice. Our data suggest that the combination of reduced fungal burden and increased immunogenicity of the mar1{Delta} mutant strain stimulates a host immune response that contains viable fungi within granulomas. Furthermore, we find that the mar1{Delta} mutant strain has slow growth and hypoxia resistance phenotypes, which may enable fungal persistence within pulmonary granulomas. Together with the conventional primary murine infection model, latent murine infection models will advance our understanding of cryptococcal disease progression and define fungal features important for persistence in the human host.


title: TRF1 poly(ADP-ribosyl)ation by PARP1 allows proper telomere replication through helicase recruitment in non-ALT cells

abstract: Telomeres are nucleoprotein structures at eukaryotic chromosome termini. Their stability is preserved by a six-protein complex named shelterin. Among these, TRF1 binds telomere duplex and assists DNA replication with mechanisms only partly clarified. Poly (ADP-ribose) polymerase 1 (PARP1) is a chromatin associated enzyme which adds poly (ADP-ribose) polymers (PARs) to acceptor proteins by covalent hetero-modification. Here we found that TRF1 is covalently PARylated by PARP1 during DNA synthesis. PARP1 downregulation perturbs bromodeoxyuridine incorporation at telomeres in S-phase, triggering replication-dependent DNA damage and telomere fragility. PARylated TRF1 recruits WRN and BLM helicases in S-phase in a PARP1-dependent manner, probably through non-covalent PAR binding to solve secondary structures during telomere replication. ALT telomeres are less affected by PARP1 downregulation and are less sensitive to PARP inhibitors. This work unveils an unprecedented role for PARP1 as a "surveillant" of telomere replication, in absence of exogenous DNA insults, which orchestrates protein dynamics at proceeding replication fork.


title: (--)-Epicatechin alters reactive oxygen and nitrogen species production independent of mitochondrial respiration in human vascular endothelial cells

abstract:
IntroductionVascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction and altered cell signalling. (-)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPIs effects remain unclear.

Objective(s)Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function.

MethodsHUVECs were treated with EPI (0-20 {micro}M) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting.

ResultsMitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 {micro}M EPI treatment vs. CTRL (P<0.001). With AA, only 10 {micro}M EPI increased mitochondrial superoxide production vs. CTRL (25%, P<0.001). NO bioavailability was increased by 45% with 10 {micro}M EPI vs. CTRL (P=0.010). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 {micro}M EPI over 48 h vs. CTRL (P=0.015 and P=0.001, respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P=0.035 and P=0.011).

Conclusion(s)EPI dose dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.


title: Auditory Brainstem Responses in the C57BL/6J Fragile X Syndrome Knockout Mouse Model

abstract: Sensory hypersensitivity, especially in the auditory system, is a common symptom in Fragile X Syndrome (FXS), the most common monogenic form of intellectual disability. However, linking phenotypes across genetic background strains of mouse models has been a challenge and could underly some of the issues with translatability of drug studies to the human condition. This study is the first to characterize the auditory brainstem response (ABR), a minimally invasive physiological readout of early auditory processing that is also used in humans, in a commonly used mouse background strain model of FXS, C57BL/6J. We measured morphological features of pinna and head and used ABR to measure hearing range, monaural and binaural auditory responses in hemizygous males, homozygous females and heterozygous females compared to wildtype mice. Consistent with previous work we showed no difference in morphological parameters across genotypes or sexes. Male FXS mice had increased threshold for high frequency hearing at 64 kHz compared to wildtype males, while females had no difference in hearing range between genotypes. In contrast, female homozygous FXS mice had decreased amplitude of wave IV of the monaural ABR, while there was no difference in males for amplitudes and no change in latency of ABR waveforms across sexes and genotypes. Lastly, FXS males had increased latency of the binaural interaction component (BIC) at 0 ITD compared to wildtype males. These findings further clarify auditory brainstem processing in FXS by adding more information across genetic background strains allowing for a better understanding of shared phenotypes.


title: Impact of protein conformational diversity on AlphaFold predictions

abstract: After the outstanding breakthrough of AlphaFold in predicting protein 3D models, new questions appeared and remain unanswered. The ensemble nature of proteins, for example, challenges the structural prediction methods because the models should represent a set of conformers instead of single structures. The evolutionary and structural features captured by effective deep learning techniques may unveil the information to generate several diverse conformations from a single sequence. Here we address the performance of AlphaFold2 predictions under this ensemble paradigm. Using a curated collection of apo-holo conformations, we found that AlphaFold2 predicts the holo form of a protein in 70% of the cases, being unable to reproduce the observed conformational diversity with an equivalent error than in the estimation of a single conformation. More importantly, we found that AlphaFold2s performance worsens with the increasing conformational diversity of the studied protein. This impairment is related to the heterogeneity in the degree of conformational diversity found between different members of the homologous family of the protein under study. Finally, we found that main-chain flexibility associated with apo-holo pairs of conformers negatively correlates with the predicted local model quality score plDDT, indicating that plDDT values in a single 3D model could be used to infer local conformational changes linked to ligand binding transitions.


title: QTL analysis of vegetative phase change in natural accessions of Arabidopsis thaliana

abstract: Shoot development in plants is divided into two phases, a vegetative phase and a reproductive phase. Vegetative growth also has two distinct juvenile and adult phases, the transition between which is termed vegetative phase change. To understand how this developmental transition is regulated in natural populations of plants, we grew a group of 70 accessions of Arabidopsis thaliana and measured the appearance of traits associated with vegetative and reproductive phase change. We found that these transitions were uncorrelated, implying they are regulated by different mechanisms. Furthermore, an analysis of accessions from Central Asia revealed that precocious changes in leaf shape poorly correlated with the timing of abaxial trichome production (an adult trait) and with variation in the level of miR156 (a key regulator of vegetative phase change). This suggests the timing of vegetative phase change is regulated by more than one mechanism. To identify the genes responsible for the precocious vegetative phenotype of these accessions, we used a set of recombinant inbred lines derived from a cross between the standard lab strain, Col-0, and one of these accessions, Shakdara. We identified eight quantitative trait loci involved in the vegetative phase change, some of which regulated different components of leaf development. All of these loci were distinct from those that regulate flowering time. These data provide the foundation for future studies to identify the loci and the regulatory networks responsible for natural variation in the timing of vegetative phase change in A. thaliana.

category: plant biology


title: NON-SPECIFIC EFFECTS OF A CINNAMATE-4-HYDROXYLASE INHIBITOR ON AUXIN HOMEOSTASIS

abstract: Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although being a powerful tool, off-target effects of these inhibitors are easily overlooked in a complex biological setting. Here we illustrate the implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is often used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the auxin catabolite indole-3-acetic acid (IAA)-Asp. By competing for the same enzyme, PA interferes with auxin conjugation, resulting in an increase in cellular auxin concentrations. These increased auxin levels likely further contribute to an increase in adventitious rooting previously observed upon PA-treatment. Despite the focus on GH3.6 in this report, PA is conjugated by an array of enzymes and their subsequent reduced activity on native substrates could potentially affect a whole set of physiological processes in the plant. We conclude that surrogate occupation of the endogenous conjugation machinery in the plant by exogenous compounds is likely a more general phenomenon that is rarely considered in pharmacological studies. Our results hereby provide an important basis for future reference in studies using chemical inhibitors.


title: Non-specific effects of a CINNAMATE-4-HYDROXYLASE inhibitor on auxin homeostasis