title: Separate physiological roles of specific and non-specific DNA binding of HU protein in Escherichia coli

abstract: Conserved in bacteria, the histone-like protein HU is crucial for genome organization and expression of many genes. It binds DNA regardless of the sequence and exhibits two binding affinities in vitro, low-affinity to any B-DNA (non-specific) and high-affinity to DNA with distortions like kinks and cruciforms (structure-specific), but the physiological relevance of the two binding modes needed further investigation. We validated and defined the three conserved lysine residues, K3, K18, and K83, in Escherichia coli HU as critical amino acid residues for both non-specific and structure-specific binding and the conserved proline residue P63 additionally for only the structure-specific binding. By mutating these residues in vivo, we showed that two DNA binding modes of HU play separate physiological roles. The DNA structure-specific binding, occurring at specific sites in the E. coli genome, promotes higher-order DNA structure formation, regulating the expression of many genes, including those involved in chromosome maintenance and segregation. The non-specific binding participates in numerous associations of HU with the chromosomal DNA, dictating chromosome structure and organization. Our findings underscore the importance of DNA structure in transcription regulation and promiscuous DNA-protein interactions in a dynamic organization of a bacterial genome.


title: Alzheimer's disease-associated P460L mutation in ephrin receptor type A1 (EphA1) leads to dysregulated Rho-GTPase signaling

abstract: Recently, late onset AD (LOAD) genome-wide association studies identified EphA1, a member of receptor tyrosine kinase family (RTK) as a disease associated loci. In the follow-up study where 3 independent LOAD cohorts were performed, a P460L coding mutation in EphA1 loci showed a significant association with LOAD. However, the role of EphA1 and P460L mutant EphA1 in AD is not fully understood. We have characterized this mutation biophysically and biochemically. Our structural in silico model and in vitro biochemical analysis demonstrate that EphA1-P460L mutation makes the receptor constitutively active suggesting a gain-of-toxic function leading to chronic EphA1 signaling in the brain. Moreover, we report that the EphA1 P460L variant triggers Rho-GTPase signaling dysregulation that could potentially contribute to spine morphology abnormalities and synaptic dysfunction observed in AD pathology.

category: molecular biology


title: Structural basis for the context-specific action of classic peptidyl transferase inhibitors.

abstract: Ribosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.


title: Profiling a single-stranded DNA region within an rDNA segment that is a loading site for bacterial condensin

abstract: Bacterial condensin preferentially loads to single-stranded DNA (ssDNA) in vitro and loads onto rDNA in vivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the topological loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines via uracils (CT-conversion) at locally melted DNA of a bacterial genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the PCR-amplified rDNA segment to obtain the actual CT-conversion rate. In principle, the CT-conversion rate is an accurate guide to detect the formation of the ssDNA segment. We expected that an increment of the CT-conversion rate would reflect a trend toward ssDNA accumulation at a given site within the rDNA. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA that affect the bacterial-condensin loading hindered the ssDNA formation only at the 100-500 bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the bacterial condensin-loading site and suggest the mechanism of condensin loading onto rDNA.


title: Single-cell RNA sequencing-based characterization of resident lung mesenchymal stromal cells in bronchopulmonary dysplasia

abstract: Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a+ L-MSCs in the lungs of normal and O2-exposed neonatal mice (a well-established model to mimic BPD) at three developmental timepoints (postnatal days 3, 7 and 14). Hyperoxia exposure increased the number, and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15,000 Ly6a+ L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.

category: cell biology


title: Strategic decision support for long-term conservation management planning.

abstract:
Forward thinking conservation-planning can benefit from modeling future landscapes that result from multiple alternative management scenarios. However, long-term landscape modeling and downstream analyses of modeling results can lead to massive amounts of data that are difficult to assemble, analyze, and to report findings in a way that is easily accessible to decision makers. In this study, we developed a decision support process to evaluate modeled forest conditions resulting from five management scenarios, modeled across 100 years in Californias Lake Tahoe basin; to this end we drew upon a large and complex hierarchical dataset intended to evaluate landscape resilience. Trajectories of landscape characteristics used to inform an analysis of landscape resilience in the Lake Tahoe basin were modeled with the spatially explicit LANDIS-II vegetation simulator. Downstream modeling outputs of additional landscape characteristics were derived from the LANDIS-II outputs (e.g., wildlife conditions, water quality, effects of fire). The later modeling processes resulted in the generation of massive data sets with high dimensionality of landscape characteristics at both high spatial and temporal resolution. Ultimately, our analysis distilled hundreds of data inputs into trajectories of the performance of the five management scenarios over the 100-year time horizon of the modeling. We then evaluated each management scenario based on inter-year variability, and absolute and relative performance. We found that the management scenario that relied on prescribed fire, outperformed the other four management approaches. Both these results, and the process that led to them, provided decision makers with easy-to-understand results based on a rational, transparent, and repeatable decision support process.

One sentence descriptionWe present a novel approach to employ decision support tools for conservation over long time horizons.

category: ecology


title: Does habitat restoration disturb? A case study of a shallow urban water reservoir in western India using cladoceran zooplankton.

abstract: Anthropogenic stressors, including restoration activities, can have ecosystem wide impacts, reflecting in various biotic components, particularly the basal levels in the trophic webs. Functional traits link taxonomic diversity to ecosystem function, thereby enabling a better ecological assessment of ecosystem health. We studied the effects of restoration activities on the community structure and functional diversity of freshwater cladoceran zooplankton in an urban water reservoir. Samples were taken in the early and late phases of the restoration work. Cladoceran species community and functional composition was significantly different between the two phases. There was a considerable reduction in taxonomic richness, functional richness and redundancy in the late phase. Taxonomic beta diversity between the two phases was explained more by nestedness. Habitat degradation due to haphazard restoration measures such as destruction of littoral zone and arbitrary desilting in addition to the dumping of untreated sewage could have contributed to the decrease in species and functional richness within the reservoir.


title: The widespread nature of Pack-TYPE transposons reveals their importance for plant genome evolution

abstract: Pack-TYPE transposable elements (TEs) are a group of non-autonomous DNA transposons found in plants. These elements can efficiently capture and shuffle coding DNA across the host genome, accelerating the evolution of genes. Despite their relevance for plant genome plasticity, the detection and study of Pack-TYPE TEs are challenging due to the high similarity these elements have with genes. Here, we produced an automated annotation procedure designed to study Pack-TYPE elements and used it to successfully annotate and analyse more than 4000 new Pack-TYPE TEs in the rice and maize genomes. Our analysis indicates that Pack-TYPE TEs are an abundant and heterogeneous group of elements. We found that these elements are associated with all main superfamilies of Class II DNA transposons in plants and likely share a similar mechanism to capture new chromosomal DNA sequences. Furthermore, we report examples of the direct contribution of these TEs to coding genes, suggesting a generalised and extensive role of Pack-TYPE TEs in plant genome evolution.

category: genomics


title: Nanoscale surface topography reduces focal adhesions and cell stiffness by enhancing integrin endocytosis

abstract:
Both substrate stiffness and surface topography regulate cell behavior through mechanotransduction signaling pathways. Such intertwined effects suggest that engineered surface topographies might substitute or cancel the effects of substrate stiffness in biomedical applications. However, the mechanisms by which cells recognize topographical features are not fully understood. Here we demonstrate that the presence of nanotopography drastically alters cell behavior such that neurons and stem cells cultured on rigid glass substrates behave as if they were on soft hydrogels. We further show that rigid nanotopography resembles the effect of soft hydrogels in reducing cell stiffness and membrane tension as measured by atomic force microscopy. Finally, we demonstrate that nanotopography reduces focal adhesions and cell stiffness by enhancing the endocytosis and the subsequent removal of integrin receptors. This mechanistic understanding will support the rational design of nanotopography that directs cells on rigid materials to behave as if they were on soft ones.

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category: bioengineering


title: External fertilization is orchestrated by a pH-regulated soluble adenylyl cyclase controlling sperm motility and chemotaxis

abstract:
The reaction of CO2 with H2O to form HCO3- and H+ is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO3- sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO3-. Two amino-acid residues required for HCO3- binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca2+ response. Finally, the sAC and the voltage- and cAMP-activated Na+/H+ exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO3- or pHi, respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea.

Statement of significanceAdenylyl cyclases synthesize cAMP, a prominent cellular messenger. A bicarbonate-sensitive AC family member, soluble AC (sAC), is tied to the chemical equilibrium: H2O + CO2 {leftrightarrow} HCO3- (bicarbonate) + H+. The sAC is required for fertilization: Mammals lacking sAC are infertile and sperm immotile. We now identify a new sAC form in sperm of non-mammalian animals that reproduce in the sea. This novel sAC is activated at alkaline pH rather than bicarbonate. It controls sperm motility and chemotaxis. The switch from HCO3- to pH rests on substitution of two amino-acids, which represents an adaptation to aquatic environments low in bicarbonate. Acidification of aquatic habitats due to climate change may adversely affect sAC activity and, thereby, fertilization.

category: biophysics


title: Alpha peak frequency is linked to episodic memory impairment in pathological aging

abstract: The Free and Cued Selective Reminding Test (FCSRT) is a largely validated neuropsychological test for the identification of amnestic syndrome from the early stage of Alzheimers disease (AD). Previous electrophysiological data suggested a slowing down of the alpha rhythm in the AD-continuum as well as a key role of this rhythmic brain activity for episodic memory processes. This study therefore investigates the link between alpha brain activity and alterations in episodic memory as assessed by the FCSRT. For that purpose, 37 patients with altered FCSRT performance underwent a comprehensive neuropsychological assessment, supplemented by 18F-fluorodeoxyglucose positron emission tomography/structural magnetic resonance imaging (18FDG-PET/MR), and 10 minutes of resting-state magnetoencephalography (MEG). The individual alpha peak frequency (APF) in MEG resting-state data was positively correlated with patients encoding efficiency as well as with the efficacy of semantic cues in facilitating patients retrieval of previous stored word. The APF also correlated positively with patients hippocampal volume and their regional glucose consumption in the posterior cingulate cortex. Overall, this study demonstrates that alterations in the ability to learn and store new information for a relatively short-term period are related to a slowing down of alpha rhythmic activity, possibly due to altered interactions in the extended mnemonic system. As such, a decreased APF may be considered as an electrophysiological correlate of short-term episodic memory dysfunction accompanying pathological aging.

category: neuroscience


title: The Promise of Deep Learning for BCIs: Classification of Motor Imagery EEG using Convolutional Neural Network

abstract: Motor Imagery (MI) is a mental process by which an individual rehearses body movements without actually performing physical actions. Motor Imagery Brain-Computer Interfaces (MI-BCIs) are AI-driven systems that capture brain activity patterns associated with this mental process and convert them into commands for external devices. Traditionally, MI-BCIs operate on Machine Learning (ML) algorithms, which require extensive signal processing and feature engineering to extract changes in sensorimotor rhythms (SMR). However, in recent years, Deep Learning (DL) models have gained popularity for EEG classification as they provide a solution for automatic extraction of spatio-temporal features in the signals. In this study, EEG signals from 54 subjects who performed a MI task of left-or right-hand grasp was employed to compare the performance of two MI-BCI classifiers; a ML approach vs. a DL approach. In the ML approach, Common Spatial Patterns (CSP) was used for feature extraction and then Linear Discriminant Analysis (LDA) model was employed for binary classification of the MI task. In the DL approach, a Convolutional Neural Network (CNN) model was constructed on the raw EEG signals. The mean classification accuracies achieved by the CNN and CSP+LDA models were 69.42% and 52.56%, respectively. Further analysis showed that the DL approach improved the classification accuracy for all subjects within the range of 2.37 to 28.28% and that the improvement was significantly stronger for low performers. Our findings show promise for employment of DL models in future MI-BCI systems, particularly for BCI inefficient users who are unable to produce desired sensorimotor patterns for conventional ML approaches.


title: Representational similarity scores of digits in the sensorimotor cortex are associated with behavioral performance

abstract: Previous studies aimed to unravel a digit-specific somatotopic organization in the primary sensorimotor (SM1) cortex. It is, however, yet to be determined whether such digit somatotopy is associated with motor performance (i.e., effector selection) and digit enslaving (unintentional co-contraction of fingers) during different types of motor tasks. Here, we adopted multivariate representational similarity analysis, applied to high-field (7T) MRI data, to explore digit activation patterns in response to online finger tapping. Sixteen young adults (7 males, mean age: 24.4 years) underwent MRI, and additionally performed an offline choice reaction time task (CRTT) to assess effector selection. During both the finger tapping task (FTT) and the CRTT, force sensor data of all digits were acquired. This allowed us to assess digit enslaving (obtained from CRTT & FTT), as well as digit interference (i.e., erroneous effector selection; obtained from CRTT) and determine the correlation between these variables and digit representational similarity scores of SM1. Digit enslaving during finger tapping was associated with contralateral SM1 representational similarity scores of both hands. During the CRTT, digit enslaving of the right hand only was associated with representational similarity scores of left SM1. Additionally, right hand digit interference was associated with representational similarity scores of left S1. In conclusion, we demonstrate a cortical origin of digit enslaving, and uniquely reveal that effector selection performance is predicted by digit representations in the somatosensory cortex.


title: Analysis of human brain tissue derived from DBS surgery

abstract:
BackgroundTranscriptomic and proteomic profiling of human brain tissue is hindered by availability of fresh samples from living patients. Postmortem samples usually represent the advanced disease stage of the patient. Furthermore, the postmortem interval affects the observed transcriptomic and proteomic profiles. Therefore, access to fresh brain tissue samples from living patients is valuable resource to obtain information on metabolically intact tissue. The implantation of deep brain stimulation (DBS) electrodes into the human brain is a neurosurgical treatment for, e.g., movement disorders. Here, we describe an improved approach to collect brain tissue from surgical instruments used in implantation of DBS device for transcriptomics and proteomics analyses.

MethodsSamples were extracted from guide tubes and recording electrodes used in routine DBS implantation procedure that was carried out to treat patients with Parkinsons disease, genetic dystonia and tremor. RNA sequencing was carried out to tissue extracted from the recording microelectrodes and liquid chromatography-mass spectrometry was carried out to analyze tissue from guide tubes. To assess the performance of the current approach, obtained datasets were compared with previously published datasets representing brain tissue.

ResultsIn RNA sequencing, altogether 32,034 transcripts representing unique Ensembl gene identifiers were detected from eight samples representing both hemispheres of four patients. By using liquid chromatography-mass spectrometry, we identified 734 unique proteins from 31 samples collected from 14 patients. Comparison with previously published brain derived data indicated that both of our datasets reflected the expected brain tissue specific features. The datasets are available via BioStudies database (accession number S-BSST667).

ConclusionsSurgical instruments used in DBS installation retain enough brain material for protein and gene expression studies. Analysis of the datasets indicated that hemisphere-specific expression data can be obtained from individual patients without any sample pooling and without any modifications to the standard surgical protocol. Comparison with previously published datasets obtained with similar approach proved the robustness and reproducibility of the current improved protocol. This approach overcomes the issues that arise from using postmortem tissue, such as effect of postmortem interval, on proteomic and transcriptomic landscape of the brain and can be used for studying molecular aspects of DBS-treatable diseases.


title: Exosome TDP-43 profile in canine model of ALS: A preliminary study in developing surrogate biomarker

abstract:
ObjectiveBlood-based biomarkers provide a crucial information in progress of neurodegenerative diseases with minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in central nervous system.

MethodsExosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analysed by Western blotting method.

ResultsThe profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compare to control subjects.

ConclusionsSerum-derived exosomal biomolecules can serve as surrogate biomarkers in neuro degenerative diseases.

Key MessagesO_LIA canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis.
C_LIO_LISerum-derived exosomes contains Transactive Response DNA Binding Protein 43 (TDP-43), potential biomarker candidate.
C_LIO_LIThe levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited a similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.
C_LI


title: Exosomal TAR DNA binding protein 43 profile in canine model of amyotrophic lateral sclerosis: A preliminary study in developing blood-based biomarker for neurodegenerative diseases


title: Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

abstract: PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9-3.1 [A], as well as the complete E. faecalis 70S ribosome at 2.2-2.5 [A]. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15 [A] from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by [~]4 [A] out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.

category: microbiology


title: Progesterone receptor-A isoform interaction with RUNX transcription factors controls chromatin remodelling at promoters during ovulation

abstract: Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, acutely induced PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for PGRs specialised function in ovulation is poorly understood. To address this, we assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrated the unique action of PGR-A isoform in the ovary through a transcriptional complex involving physical interaction with RUNX and JUN/FOS transcription factors. The assembly of this unique complex directs targeting of PGR binding to proximal promoter regions and enables chromatin accessibility, leading to ovulatory gene induction. This PGR signalling mechanism is specific to ovulation and provides potential targets for infertility treatments as well as new contraceptives that block ovulation.


title: Characterization of a flexible AAV-DTR/DT mouse model of acute epithelial lung injury

abstract:
Background & aimRecurring epithelial injury and aberrant repair are considered as a major driver of idiopathic pulmonary fibrosis (IPF) leading to chronic inflammation, fibroblast activation and ultimately to scarring and stiffening of the lung. As decline of lung function is the first reported symptom by IPF patients and occurs once fibrosis is firmly established, animal models are required to study early disease-driving mechanisms.

MethodsWe developed a novel and flexible mouse model of acute epithelial injury based on adeno-associated virus (AAV) variant 6.2 mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial epithelial and alveolar epithelial type II cells can be observed.

ResultsDetailed characterization of the AAV-DTR/DT mouse model revealed increasing cell numbers in bronchoalveolar lavage (BAL; macrophages, neutrophils, and atypical cells) and elevation of apoptotic cells and infiltrated leukocytes in lung tissue, which were dependent of viral genome load and DT dose. Cytokine levels in BAL fluid showed different patterns dependent of viral genome load with IFN{gamma}, TNF, and IP-10 increasing and IL-5 and IL-6 decreasing, while lung function was not affected. Additionally, laser-capture microdissection-based proteomics of bronchial and alveolar epithelium showed upregulated immune and inflammatory response in all epithelial cell regions and extracellular matrix deposition in infiltrated alveoli, while proteins involved in pulmonary surfactant synthesis, alveolar fluid clearance and alveolar-capillary barrier were downregulated in the parenchyma.

ConclusionOur novel AAV-DTR/DT model resembles specific aspects of pulmonary diseases like IPF and acute respiratory distress syndrome.

Short summary for social mediaA novel and flexible mouse model of acute epithelial lung injury based on AAV-mediated expression of the human diphtheria toxin receptor followed by intratracheal instillation of diphtheria toxin resembles specific aspects of pulmonary diseases like IPF.


title: Endocytic vacuole formation by WASH-mediated endocytosis

abstract: Endocytosis of extracellular or plasma membrane material is a fundamental process. A variety of endocytic pathways exist, several of which are barely understood in terms of mechanistic execution and biological function. Importantly, some mechanisms have been identified and characterized by following virus internalization into cells. This includes a novel endocytic pathway exploited by human papillomavirus type 16 (HPV16). However, its cellular role and mechanism of endocytic vacuole formation remain unclear. Here, HPV16 was used as a tool to examine the mechanistic execution of vesicle formation by combining systematic perturbation of cellular processes with electron and video microscopy. Our results indicate cargo uptake by uncoated, inward-budding pits facilitated by the membrane bending retromer protein SNX2. Actin polymerization-driven vesicle scission is promoted by WASH, an actin regulator typically not found at the plasma membrane. Uncovering a novel role of WASH in endocytosis, we propose to term the new pathway WASH-mediated endocytosis (WASH-ME).


title: Tfap2b specifies an embryonic melanocyte stem cell population that retains adult multi-fate potential

abstract:
Melanocytes, our pigment producing cells, are replenished from multiple stem cell niches in adult tissues. Although pigmentation traits are known risk-factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs, and lack key lineage markers with which to identify McSCs and study their function. Here, we discover that Tfap2b, and a select set of its target genes, specifies an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and instead is essential for McSC-dependent melanocyte regeneration. Fate- mapping data reveal that tfap2b-expressing McSCs have multi-fate potential, and are the cell-of- origin for large patches of adult melanocytes, and two other pigment cell types, iridophores and xanthophores. Hence, Tfap2b confers McSC identity in early development, thereby distinguishing McSCs from other neural crest and pigment cell lineages, and retains multi-fate potential in the adult zebrafish.

HighlightsO_LITfap2b and its target genes specify McSCs with mixed pigment cell identities
C_LIO_LIFunctional dependence on Tfap2b for melanocyte regeneration from the McSC
C_LIO_LItfap2b specifies ErbB-dependent McSCs at the stem cell niche
C_LIO_LIFate mapping reveals Tfap2b-McSCs have multi-fate potential for adult pigment cells
C_LI

category: developmental biology


title: Expression and function of SLC38A5, an amino acid-coupled Na+/H+ exchanger, in triple-negative breast cancer and its relevance to macropinocytosis.

abstract:
Metabolic reprogramming in cancer cells necessitates increased amino acid uptake, which is accomplished by upregulation of specific amino acid transporters. Since amino acid transporters differ in substrate selectivity, mode of transport, and driving forces, not all tumors rely on any single amino acid transporter for this purpose. Here we report on the differential upregulation of the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The upregulation is evident in primary TNBC tumors, conventional TNBC cell lines, patient-derived xenograft TNBC cell lines, and a mouse model of spontaneous mammary tumor representing TNBC. The upregulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na+/H+ exchanger which transports Na+ and amino acids into cells coupled with H+ efflux. Since the traditional Na+/H+ exchanger is an established inducer of macropinocytosis, an endocytic process for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by amilorides, the well-known inhibitors of Na+/H+ exchanger, possibly related to the amino acid-dependent Na+/H+ exchange function of SLC38A5. The Cancer Genome Atlas database corroborates SLC38A5 upregulation in TNBC. This represents the first report on the selective expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid delivery but is still relevant to cancer cell nutrition.

Summary StatementSLC38A5 is an amino acid-coupled Na+/H+ exchanger that is upregulated in triple-negative breast cancer, and its function in cancer cells goes beyond amino acid delivery; it promotes macropinocytosis, a distinct form of endocytic process for cellular uptake of proteins and other nutrients present in extracellular fluid.

category: cancer biology


title: A new approach to measure forces at junction vertices in an epithelium

abstract:
The mechanical properties of cell-cell junctions are critical for the stability of an epithelium. Cell-cell junction ablation experiments are classically used as a readout for junctional mechanics. However, without the knowledge of the viscoelastic properties of the microenvironment of the ablated junction, tensile junctional forces cannot be measured. Here we combine laser ablation with intracellular microrheology and develop a model to measure tensile forces exerted on cell-cell junctions. We show that the overexpression of the proto-oncogene atypical Protein Kinase C iota (aPKCi) in a single cell within a normal epithelium induces a gradient of junctional tension with neighbouring cells. Our method allows us to demonstrate that junctions contacting the aPKCi-overexpressing cell display a mechanical asymmetry which correlates with the levels of E-cadherin and P-MLC2. Measuring intracellular viscoelasticity is crucial for accurate measurements of cell-cell junction mechanics in the context of development or cancer research.

TeaserWe combine cytoplasmic and junctional mechanics to detect asymmetry in forces exerted on cell-cell junctions.


title: A highly efficient reporter system for identifying and characterizing in vitro expanded hematopoietic stem cells

abstract: Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.


title: Lysosomal proteolysis of amyloid beta is impeded by fibrils grown in both acidic and neutral pH environments

abstract: Aggregation of amyloid-beta (A{beta}) into extracellular plaques is a well-known hallmark of Alzheimers disease (AD). Similarly, autophagic vacuoles, autophagosomes, and other residual bodies within dystrophic neurites, though more difficult to detect, are characteristic features of AD. To explore the potential intersection between these observations, we conducted experiments to assess whether A{beta} fibril formation disrupts lysosomal proteolysis. Fibrils constituted from either A{beta} 1-40 or A{beta} 1-42 were grown under both neutral and acidic pH. The extent of proteolysis by individual cathepsins (L, D, B, and H) was monitored by both thioflavin T fluorescence and liquid-chromatography combined with mass spectrometry. The results show that all A{beta} fibrils are resistant to cathepsin digestion, with significant amounts of undigested material remaining for samples of fibrils grown in both neutral and acidic pH. Further analysis revealed that the neutral-grown fibrils are proteolytically resistant throughout the sequence, while the acid-grown fibrils prevented digestion primarily in the C-terminal portion of the sequence. Fibrils grown from A{beta} 1-42 are generally more resistant to degradation compared to A{beta} 1-40. Overall, the results indicate that A{beta} fibrils formed in the neutral pH environments found in intracellular or extracellular spaces may pose the greatest difficulty for complete digestion by the lysosome, particularly when the fibrils are comprised of A{beta} 1-42.

category: biochemistry


title: Interactome analyses reveal profound proteasome structural and functional rearrangements throughout mammalian spermatogenesis

abstract: During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific 4s subunit replaces the 4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82-92%) during differentiation, presumably due to the shift from 4 to 4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S, and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between 4 and 4s. We propose that these differences induced by 4s incorporation result in significant changes in the way the s20S interacts with its partners, and dictate its role in germ cell differentiation.


title: Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis


title: N-terminal phosphorylation regulates the activity of Glycogen Synthase Kinase 3 from Plasmodium falciparum

abstract: As the decline of malaria cases stalled over the last five years, novel targets in Plasmodium falciparum are necessary for the development of new drugs. Glycogen Synthase Kinase (PfGSK3) has been identified as a potential target, since its selective inhibitors were shown to disrupt the parasites life cycle. Here, we show that PfGSK3 exhibits autophosphorylation, leading to an extensive phosphorylation both in vitro and in the parasite. In the uncanonical N-terminal region of the parasite enzyme, we identified several autophosphorylation sites that regulate the activity of PfGSK3. By combining molecular modeling with experimental small-angle X-ray scattering data, we show that increased PfGSK3 activity is promoted by conformational changes in the PfGSK3 N-terminus, triggered by N-terminal phosphorylation. Our work provides novel insights into the structure and regulation of the malarial PfGSK3.


title: A human iPSC derived bone marrow milieu identifies a novel target against niche-conferred leukaemia proliferation and treatment resistance

abstract:
Being able to clinically target the interaction of leukaemic cells with their microenvironment has remained a key therapeutic obstacle. Leukaemia cells re-program their microenvironment to provide support and protection from standard chemotherapy, molecularly targeted therapies as well as immunotherapy. To address this challenge, we have developed experimentally accessible human induced pluripotent stem cell engineered (iPSC) niches ex vivo to reveal insights into druggable cancer-niche interactions. We show that mesenchymal (iMSC) and vascular niche-like (iANG) cells support ex vivo proliferation of patient-derived leukaemia cells, impact dormancy and mediate therapy resistance. Mesenchymal stem cells protected both cycling and non- cycling blasts against dexamethasone treatment while vascular niche-like cells only protected dormant cells. Growth support and protection from dexamethasone induced- apoptosis was dependent on direct cell-cell contact and was mediated by N-cadherin (CDH2). To explore the therapeutic potential of disrupting this cell-cell interaction, we tested the CDH2 antagonist ADH-1 (previously in phase I / II for solid tumours) in a very aggressive patient-derived xenograft acute lymphoblastic leukaemia mouse model. ADH-1 showed high in vivo efficacy and combination therapy with ADH-1 and dexamethasone was superior compared with dexamethasone alone. These findings provide a proof-of-concept starting point to develop novel and potentially safer anti- cancer therapeutics that target niche-mediated cancer cell dependencies in haematological malignancies.

One Sentence SummaryCDH2 drives niche-dependent treatment resistance targetable via ADH-1, a low toxic potential candidate for future leukaemia clinical trials.


title: An ex vivo human bone marrow milieu identifies CDH2 as a driver of niche-mediated treatment resistance in leukaemia


title: Dexamethasone promotes breast cancer stem cells in obese and not lean mice

abstract: Obesity is highly prevalent in breast cancer patients and it is associated with increased recurrence and breast cancer specific mortality. Glucocorticoid (GC) use, in addition to obesity is associated with promoting breast cancer metastasis through activation of stemness-related pathways. Therefore, we utilised the synergetic allograft E0771 breast cancer model to investigate if treatment with GCs had differential effects on promoting cancer stem cells in lean and diet-induced obese mice. Indeed, both lean mice treated with dexamethasone and obese mice with no treatment had no effect on the ex vivo colony forming ability, mammosphere formation or ALDH bright subpopulation. However, treatment of obese mice with dexamethasone resulted in a significant increase in ex vivo colony formation, mammosphere formation, ALDH bright subpopulation and expression of pluripotency transcription factors. GC transcriptionally regulated genes were not altered in the dexamethasone treated groups compared to treatment controls. In summary, these results provide initial evidence that obesity presents a higher risk of GC induced cancer stemness via non-genomic GC signalling which is of potential translational significance.


title: Genome-wide ChIPseq analysis of AhR, COUP-TF, and HNF4 enrichment in TCDD-treated mouse liver

abstract: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4 and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 hrs after oral gavage with 30 {micro}g/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2 - 72 hrs) and dose-response (0.01 - 30 {micro}g/kg) datasets to assess putative AhR, HNF4 and COUP-TFII interactions associated with differential gene expression. TCDD treatment resulted in the genomic enrichment of 23,701, 11,688, and 9,547 binding regions for AhR, COUP-TFII and HNF4, respectively, throughout the genome. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4a to regions within liver-specific genes suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4 target and hepatocyte differentiation genes, involved increased AhR and HNF4 binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4.

category: pharmacology and toxicology


title: Estimates of persistent inward currents are reduced in upper limb motor units of older adults

abstract:
Aging is a natural process that causes alterations in the neuromuscular system, which contribute to weakness and reduced quality of life. Reduced firing rates of individual motor units (MUs) likely contribute to weakness, but the mechanisms underlying reduced firing rates are not clear. Persistent inward currents (PICs) are crucial for the initiation, gain control, and maintenance of motoneuron firing, and are directly proportional to the level of monoaminergic input. Since the concentration of monoamines (i.e. serotonin and norepinephrine) are reduced with age, we sought to determine if estimates of PICs are reduced in older (>60 years old) compared to younger adults (<35 years old). We decomposed MU spike trains from high-density surface electromyography over the biceps brachii and triceps brachii during isometric ramp contractions to 20% of maximum. Estimates of PICs (i.e. {Delta}F) were computed using the paired MU analysis technique. Regardless of the muscle, peak firing rates of older adults were reduced by ~1.6 pulses per second (pps) (P = 0.0292), and {Delta}F was reduced by ~1.9 pps (P < 0.0001), compared to younger adults. We further found that age predicted {Delta}F in older adults (P = 0.0261), resulting in a reduction of ~1pps per decade, but there was no relationship in younger adults (P = 0.9637). These findings suggest that PICs are reduced in older adults, and, further, age is a significant predictor of estimates of PICs in older adults. Reduced PIC magnitude represents one plausible mechanism for reduced firing rates and weakness in older individuals.

KEY POINTSO_LIPersistent inward currents play an important role in the neural control of human movement and are influenced by neuromodulation via monoamines originating in the brainstem.
C_LIO_LIDuring aging, motor unit firing rates are reduced, and there is deterioration of brainstem nuclei, which may reduce persistent inward currents in alpha motoneurons.
C_LIO_LIHere we show that estimates of persistent inward currents ({Delta}F) of both elbow flexor and extensor motor units are reduced in older adults.
C_LIO_LIEstimates of persistent inward currents have a negative relationship with age in the older adults, but not young.
C_LIO_LIThis novel mechanism may play a role in alteration motor firing rates that occurs with aging, which may have consequences for motor control.
C_LI

category: physiology


title: Concealed identity information detection with pupillometry in rapid serial visual presentation

abstract: The concealed information test (CIT) relies on bodily reactions to stimuli that are hidden in mind. However, people can use countermeasures, such as purposely focusing on irrelevant things, to confound the CIT. A new method designed to prevent countermeasures uses rapid serial visual presentation (RSVP) to present stimuli on the fringe of awareness. Previous studies that used RSVP in combination with electroencephalography (EEG) showed that participants exhibit a clear reaction to their real first name, even when they try to prevent such a reaction (i.e. when their name is concealed information). Since EEG is not easily applicable outside the laboratory, we investigated here whether pupil size, which is easier to measure, can also be used to detect concealed identity information. In our first study, participants adopted a fake name, and searched for this name in an RSVP task, while their pupil sizes were recorded. Apart from this fake name, their real name and a control name also appeared in the task. We found pupil dilation in response to the task-irrelevant real name, as compared to control names. However, while most participants showed this effect qualitatively, it was not statistically significant for most participants individually. In a second study, we preregistered the proof-of-concept methodology and replicated the original findings. Taken together, our results show that the current RSVP task with pupillometry can detect concealed identity information at a group level. Further development of the method is needed to create a valid and reliable concealed identity information detector at the individual level.


title: Reconstruction of genome-scale metabolic model for Hansenula polymorpha using RAVEN toolbox

abstract: Genome scale metabolic models (GEMs) provide a useful framework for modeling the metabolism of microorganisms. While the applications of GEMs are wide and far reaching, the reconstruction and continuous curation of such models can be perceived as a tedious and time consuming task. Using the RAVEN toolbox, this protocol practically demonstrates how researchers can create their own GEMs using a homology based approach. To provide a complete example, we reconstruct a draft GEM for the industrially relevant yeast Hansenula polymorpha.

category: systems biology


title: The functional specialization of visual cortex emerges from training parallel pathways with self-supervised predictive learning

abstract: The visual system of mammals is comprised of parallel, hierarchical specialized pathways. Different pathways are specialized in so far as they use representations that are more suitable for supporting specific downstream behaviours. In particular, the clearest example is the specialization of the ventral ("what") and dorsal ("where") pathways of the visual cortex. These two pathways support behaviours related to visual recognition and movement, respectively. To-date, deep neural networks have mostly been used as models of the ventral, recognition pathway. However, it is unknown whether both pathways can be modelled with a single deep ANN. Here, we ask whether a single model with a single loss function can capture the properties of both the ventral and the dorsal pathways. We explore this question using data from mice, who like other mammals, have specialized pathways that appear to support recognition and movement behaviours. We show that when we train a deep neural network architecture with two parallel pathways using a self-supervised predictive loss function, we can outperform other models in fitting mouse visual cortex. Moreover, we can model both the dorsal and ventral pathways. These results demonstrate that a self-supervised predictive learning approach applied to parallel pathway architectures can account for some of the functional specialization seen in mammalian visual systems.


title: Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease

abstract: The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimers disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of A{beta} peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 71 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variations (R332W, S577P, and R654W) and two maturation-competent variations (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of A{beta} secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variations on SorLA protein indicated that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.


title: CCN2 activates ERK-signaling via integrin αv and enhances the interaction of ERK and DUSP6 in lymphatic endothelial cells

abstract: Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated CCN2 signaling in LECs and its effects on lymphangiogenesis. In primary cultured LECs, gene expressions of lymphatic endothelial markers lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), Podoplanin and prospero homeobox 1 (Prox1) and lymphangiogenic factors vascular endothelial cell growth factor c (Vegfc), vascular endothelial cell growth factor d (Vegfd) and fms-related tyrosine kinase 4 (Flt4, also known as Vegfr3) were upregulated by CCN2. Subsequently, we found that CCN2 induced phospho-ERK and that was decreased by suppression of integrin v. CCN2 slightly decreased the growth of LECs due to enhancement of the interaction of ERK and dual specific protein phosphatase 6 (DUSP6), and knockdown of DUSP6 increased CCN2-induced phospho-ERK levels. In in vivo Matrigel plug assays, the number of Podoplanin-positive vessels was increased by exogenous CCN2, and phospho-ERK-positive LEC and DUSP6-positive LEC were detected in CCN2 plugs. These results suggest that CCN2-related lymphangiogenesis is regulated by DUSP6, which enables negative modulation of ERK-signaling.


title: BioCPPNet: Automatic Bioacoustic Source Separation with Deep Neural Networks

abstract: We introduce the Bioacoustic Cocktail Party Problem Network (BioCPPNet), a lightweight, modular, and robust UNet-based machine learning architecture optimized for bioacoustic source separation across diverse biological taxa. Employing learnable or handcrafted encoders, BioCPPNet operates directly on the raw acoustic mixture waveform containing overlapping vocalizations and separates the input waveform into estimates corresponding to the sources in the mixture. Predictions are compared to the reference ground truth waveforms by searching over the space of (output, target) source order permutations, and we train using an objective function motivated by perceptual audio quality. We apply BioCPPNet to several species with unique vocal behavior, including macaques, bottlenose dolphins, and Egyptian fruit bats, and we evaluate reconstruction quality of separated waveforms using the scale-invariant signal-to-distortion ratio (SI-SDR) and downstream identity classification accuracy. We consider mixtures with two or three concurrent conspecific vocalizers, and we examine separation performance in open and closed speaker scenarios. To our knowledge, this paper redefines the state-of-the-art in end-to-end single-channel bioacoustic source separation in a permutation-invariant regime across a heterogeneous set of non-human species. This study serves as a major step toward the deployment of bioacoustic source separation systems for processing substantial volumes of previously unusable data containing overlapping bioacoustic signals.


title: Haplotype analyses reveal novel insights into tomato history and domestication to show long-distance migrations and latitudinal adaptations

abstract: A novel haplotype-based approach that uses Procrustes analysis and automatic classification was used to provide further insights into tomato history and domestication. Agrarian societies domesticated species of interest by introducing complex genetic modifications. For tomatoes, two species, one of which had two botanical varieties, are thought to be involved in its domestication: the fully wild Solanum pimpinellifolium (SP), the wild and semi-domesticated S. lycopersicum var. cerasiforme (SLC) and the cultivated S. l. var. lycopersicum (SLL). The Procrustes approach showed that SP evolved into SLC during a gradual migration from the Peruvian deserts to the Mexican rainforests and that Peruvian and Ecuadorian SLC populations were the result of more recent hybridizations. Our model was supported by independent evidence, including ecological data from the accession collection site and morphological data. Furthermore, we showed that photosynthesis-, and flowering time-related genes were selected during the latitudinal migrations.

category: evolutionary biology


title: Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

abstract: The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

category: genetics


title: A computational screen for alternative genetic codes in over 250,000 genomes

abstract: The genetic code has been proposed to be a "frozen accident", but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force which likely helped drive these codons to low frequency and enable their reassignment.


title: A mutational gradient drives somatic mutation accumulation in mitochondrial DNA and influences germline polymorphisms and genome composition

abstract:
BackgroundMutations in the mitochondrial genome (mtDNA) can cause devastating maternally inherited diseases, while the accumulation of somatic mtDNA mutations is linked to common diseases of aging. Although mtDNA mutations impact human health, the process(es) that give rise to these mutations are unclear and are under considerable debate. We analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing to provide clues to the mechanism by which de novo mutations arise as well as how the genome is replicated.

ResultsWe observe two distinct mutational gradients in G[->]A and T[->]C transitions, but not their complements, that are delimited by the light-strand origin and the control region (CR). The gradients increase with age and are lost in the absence of DNA polymerase {gamma} proofreading activity. A nearly identical pattern is present in human mtDNA somatic mutations. The distribution of mtDNA single nucleotide polymorphisms (SNPs) in the human population and genome base composition across >3,000 vertebrate species mirror this gradient pattern, pointing to evolutionary conservation of this phenomenon. Lastly, high-resolution analysis of the mtDNA control region highlights mutational  hotspots and  cold-spots that strongly align with important regulatory regions.

ConclusionsCollectively, these patterns support an asymmetric strand-displacement mechanism with key regulatory structures in the CR and argue against alternative replication models. The mutational gradient is a fundamental consequence of mtDNA replication that drives somatic mutation accumulation and influences inherited polymorphisms and, over evolutionary timescales, genome composition.


title: A Robust High-throughput Fluorescent Polarization Assay for the Evaluation and Screening of SARS-CoV-2 Fusion Inhibitors

abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious threat to global health. One attractive antiviral target is the membrane fusion mechanism employed by the virus to gain access to the host cell. Here we report a robust protein-based fluorescent polarization assay, that mimicking the formation of the six-helix bundle (6-HB) process during the membrane fusion, for the evaluation and screening of SARS-CoV-2 fusion Inhibitors. The IC50 of known inhibitors, HR2P, EK1, and Salvianolic acid C (Sal C) were measured to be 6 nM, 2.5 nM, and 8.9 {micro}M respectively. In addition, we found Sal A has a slightly lower IC50 (3.9 {micro}M) than Sal C. Interesting, simple caffeic acid can also disrupt the formation of 6-HB with sub-mM concentration. A pilot high throughput screening (HTS) a small marine natural product library validates the assay with a Z factor close to 0.8. We envision the current assay provides a convenient way to screen SARS-CoV-2 fusion inhibitor and assess their binding affinity.


title: A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a

abstract: CRISPR-Cas12a has been widely used for genome editing and diagnostic applications, yet it is not fully understood how RNA-guided DNA recognition activates the sequential cleavage of the non-target strand (NTS) followed by the target strand (TS). Here we used single-molecule magnetic tweezers microscopy, ensemble gel-based assays and nanopore sequencing to explore the coupling of DNA unwinding and cleavage. In addition to dynamic R-loop formation, we also directly observed transient dsDNA unwinding downstream of the 20 bp DNA:RNA hybrid and, following NTS cleavage and prior to TS cleavage, formation of a hyperstable "clamped" Cas12a-DNA intermediate resistant to DNA twisting. Alanine substitution of a conserved aromatic amino acid "gate" in the REC2 domain that normally caps the heteroduplex produced more frequent and extended downstream DNA breathing, a longer-lived twist-resistant state, and a 16-fold faster rate of TS cleavage. We suggest that both breathing and clamping events, regulated by the gate and by NTS cleavage, deliver the unwound TS to the RuvC nuclease and result from previously described REC2 and NUC domain motions.


title: Nanodiscs-based proteomics identify Caj1 as an Hsp40 with affinity for phosphatidic acid lipids

abstract: Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids poor solubility in aqueous solution. Here, we employ nanodiscs - small, water-soluble patches of lipid bilayer encircled with amphipathic scaffold proteins - along with quantitative proteomics to identify lipid-binding proteins in S. cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids, and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of the nanodisc system to identify and characterize protein-lipid interactions that may not be evident using other methods.


title: Multiplexed bioluminescence microscopy via phasor analysis

abstract: Microscopic bioluminescence imaging has been historically challenging due to a lack of detection methods and easily resolved probes. Here we combine bioluminescence with phasor analysis, an optical method commonly used to distinguish spectrally similar fluorophores. Bioluminescent phasor enabled rapid differentiation of multiple luciferase reporters and resonance energy transfer processes. The merger of bioluminescence and phasor analysis provides a platform for routine, time-lapse imaging of collections of cellular features.


title: Vapor mediation as a tool to control micro-nano scale dendritic crystallization and preferential bacterial distribution in drying respiratory droplets

abstract: Deposits of biofluid droplets on surfaces (such as respiratory droplets formed during an expiratory event fallen on surfaces) are composed of the water-based salt-protein solution that may also contain an infection (bacterial/viral).The final patterns of the deposit formed are dictated by the composition of the fluid and flow dynamics within the droplet. This work reports the spatio-temporal, topological regulation of deposits of respiratory fluid droplets and control of motility of bacteria by tweaking flow inside droplets using non-contact vapor-mediated interactions. When evaporated on a glass surface, respiratory droplets form haphazard multiscale dendritic, cruciform-shaped precipitates--using vapor mediation as a tool to control these deposits at the level of nano-micro-millimeter scales. Wemorphologically control dendrite orientation, size and subsequently suppress cruciform-shaped crystals. The nucleation sites are controlled via preferential transfer of solutes in the droplets; thus, achieving control over crystal occurrence and growth dynamics. The active living matter like bacteria is also preferentially segregated with controlled motility without attenuation of its viability and pathogenesis. For the first time, we have experimentally presented a proof-of-concept to control the motion of live active matter like bacteria in a near non-intrusive manner. The methodology can have ramifications in biomedical applications like disease detection, controlling bacterial motility, and bacterial segregation.


title: BioProfiling.jl: Profiling biological perturbations with high-content imaging in single cells and heterogeneous populations

abstract:
MotivationHigh-content imaging screens provide a cost-effective and scalable way to assess cell states across diverse experimental conditions. The analysis of the acquired microscopy images involves assembling and curating morphological measurements of individual cells into morphological profiles suitable for testing biological hypotheses. Despite being a critical step, there is currently no standard approach to morphological profiling and no solution is available for the high-performance Julia programming language.

ResultsHere, we introduce BioProfiling.jl, an efficient end-to-end solution for compiling and filtering informative morphological profiles in Julia. The package contains all the necessary data structures to curate morphological measurements and helper functions to transform, normalize and visualize profiles. Robust statistical distances and permutation tests enable quantification of the significance of the observed changes despite the high fraction of outliers inherent to high-content screens. This package also simplifies visual artifact diagnostics, thus streamlining a bottleneck of morphological analyses. We showcase the features of the package by analyzing a chemical imaging screen, in which the morphological profiles prove to be informative about the compounds mechanisms of action and can be conveniently integrated with the network localization of molecular targets.

AvailabilityThe Julia package is available on GitHub: https://github.com/menchelab/BioProfiling.jl We also provide Jupyter notebooks reproducing our analyses: https://github.com/menchelab/BioProfilingNotebooks

Contactjoerg.menche@univie.ac.at

category: bioinformatics


title: Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome

abstract: The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and reveal that ALC1 preferentially slides nucleosomes away from DNA breaks.


title: Switching an active site helix in dihydrofolate reductase reveals limits to sub-domain modularity

abstract:
To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study sub-domain modularity by creating 20 chimeras of an enzyme, E. coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue -helical sequence is replaced by -helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this -helix are under selection pressure to maintain catalytic activity in DHFR. We performed molecular dynamics simulations using replica exchange with solute-tempering. Chimeras with low catalytic activity exhibit non-helical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although -helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.

Statement of Significance-helices are ubiquitous components of protein structure that exhibit a degree of independent folding behavior, making them plausible structural modules within proteins. Here, we assess the effects of switching the sequence of an -helix in an essential enzyme for -helical sequences from evolutionarily unrelated proteins. The resultant chimeric proteins can still fold but enzymatic activity, stability, and cellular growth rates are negatively affected. Computational investigations reveal how residues in an -helix have been shaped by selection pressure to maintain catalytic activity and a specific, helical conformation of the protein. More broadly, we illustrate how molecular and fitness effects of switching protein segments depend on the protein and cellular context.


title: Notch1 mutation drives clonal expansion in normal esophageal epithelium but impairs tumor growth

abstract: NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age, but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations may promote clonal expansion but impede carcinogenesis1-3. Here we test this hypothesis. Visualizing and sequencing NOTCH1 mutant clones in aging normal human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild type cells, an effect enhanced by loss of the second allele. Notch1 loss alters transcription but has minimal effects on epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. We conclude that Notch1 mutations in normal epithelium are beneficial as wild type Notch1 promotes tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.


title: Identification of SGLT2 inhibitor Ertugliflozin as a treatment for COVID-19 using computational and experimental paradigm

abstract: Drug repurposing can expedite the process of drug development by identifying known drugs which are effective against SARS-CoV-2. The RBD domain of SARS-CoV-2 Spike protein is a promising drug target due to its pivotal role in viral-host attachment. These specific structural domains can be targeted with small molecules or drug to disrupt the viral attachment to the host proteins. In this study, FDA approved Drugbank database were screened using a virtual screening approach and computational chemistry methods. Five drugs were short listed for further profiling based on docking score and binding energies. Further these selected drugs were tested for their in vitro biological activity. There was significant correlation between the prediction from computational studies and the actual RBD-ACE2 binding inhibition by the drugs. Then, we performed a series of studies that mimic some of the biological events seen in COVID-19 patients such as secretion of IL1{beta}, presentation of a more thrombogenic endothelium by production of thrombomodulin and accumulation of inflammatory cells such as monocytes in the lungs. Of all the drugs, most promising drug was Ertugliflozin which is used for type-2 diabetes. This drug possesses several desired properties and may be a good candidate for immediate repurposing for treatment of COVID-19.


title: Neural Regression, Representational Similarity, Model Zoology & Neural Taskonomy at Scale in Rodent Visual Cortex

abstract: How well do deep neural networks fare as models of mouse visual cortex? A majority of research to date suggests results far more mixed than those produced in the modeling of primate visual cortex. Here, we perform a large-scale benchmarking of dozens of deep neural network models in mouse visual cortex with both representational similarity analysis and neural regression. Using the Allen Brain Observatorys 2-photon calcium-imaging dataset of activity in over 6,000 reliable rodent visual cortical neurons recorded in response to natural scenes, we replicate previous findings and resolve previous discrepancies, ultimately demonstrating that modern neural networks can in fact be used to explain activity in the mouse visual cortex to a more reasonable degree than previously suggested. Using our benchmark as an atlas, we offer preliminary answers to overarching questions about levels of analysis (e.g. do models that better predict the representations of individual neurons also predict representational similarity across neural populations?); questions about the properties of models that best predict the visual system overall (e.g. is convolution or category-supervision necessary to better predict neural activity?); and questions about the mapping between biological and artificial representations (e.g. does the information processing hierarchy in deep nets match the anatomical hierarchy of mouse visual cortex?). Along the way, we catalogue a number of models (including vision transformers, MLP-Mixers, normalization free networks, Taskonomy encoders and self-supervised models) outside the traditional circuit of convolutional object recognition. Taken together, our results provide a reference point for future ventures in the deep neural network modeling of mouse visual cortex, hinting at novel combinations of mapping method, architecture, and task to more fully characterize the computational motifs of visual representation in a species so central to neuroscience, but with a perceptual physiology and ecology markedly different from the ones we study in primates.


title: After traumatic brain injury oligodendrocytes regain a plastic phenotype and can become astrocytes

abstract: After acute brain injuries various response cascades are evoked that direct the formation of the glial scar. Here, we report that acute lesions associated with a disruption of the blood-brain barrier trigger a re-programming within the oligodendrocyte lineage. In PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice with cortical injuries, we transiently found PLP transgene-labelled cells with activated GFAP promoter activity adjacent to the lesion site. We termed them AO cells, based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and NG2-CreERT2 mice we observed that major portions of AO cells surprisingly differentiated into astrocytes. Using repeated long-term in vivo two-photon laser-scanning microscopy (2P-LSM) we followed oligodendrocytes after injury. We observed their conversion into astrocytes via the AO cell stage with silencing of the PLP promoter and simultaneous activation of the GFAP promoter. In addition, we provide evidence that this oligodendrocyte-to-astrocyte conversion depends on local cues. At the lesion site higher expression levels of various glial differentiation factors were detected. And indeed, local injection of IL-6 promoted the formation of AO cells. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma. An altered environmental milieu affects gene expression programs of mature oligodendrocytes and induces a plastic differentiation stage with astrogliogenic potential via transitional AO cells.


title: Disturbed retinoid metabolism upon loss of rlbp1a impairs cone function and leads to subretinal lipid deposits and photoreceptor degeneration in the zebrafish retina

abstract:
The RLBP1 gene encodes the 36 kDa cellular retinaldehyde binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod-cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Muller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.

Graphical Abstract

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title: Stromal cell sialylation suppresses T cells in inflammatory tumour microenvironments: A new tumour stromal cell immune checkpoint?

abstract:
Immunosuppressive tumour microenvironments (TME) reduce the effectiveness of immune responses in cancer. Non-haematopoietic mesenchymal stromal cells (MSC), the precursor to cancer associated fibroblasts (CAFs), dictate tumour progression by enhancing immune cell suppression. Hyper-sialylation of glycans promotes immune evasion in cancer, but the role of sialyation in stromal cell-mediated immunosuppression is unknown.

Here we study changes in sialyltransferase (ST) enzymes and associated surface expressed sialic acid in stromal cells following inflammatory and tumour secretome conditioning. We show that tumour conditioned stromal cells have increased levels of sialyltransferases, 2,3/6 linked sialic acid and siglec ligands. In tumour models of solid (colorectal cancer) and haematological (multiple myeloma) stromal rich tumours, stromal cell sialylation is associated with enhanced immunosuppression. Using datasets and patient samples, we confirm that targeting sialylation in tumour stromal cells reverses immune cell exhaustion. Targeting stromal cell sialylation may represent a novel immune checkpoint to reactivate anti-tumour immunity.

category: immunology


title: Probing Affinity, Avidity, Anti-Cooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

abstract: Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behaviour of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behaviour arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e. Fabs). Surprisingly, these sub-stoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.


title: Binding of phosphatidylserine-positive microparticles by PBMCs classifies disease severity in COVID-19 patients

abstract: Infection with SARS-CoV-2 is associated with thromboinflammation, involving thrombotic and inflammatory responses, in many COVID-19 patients. In addition, immune dysfunction occurs in patients characterized by T cell exhaustion and severe lymphopenia. We investigated the distribution of phosphatidylserine (PS), a marker of dying cells, activated platelets, and platelet-derived microparticles (PMP), during the clinical course of COVID-19. We found an unexpectedly high amount of blood cells loaded with PS+ PMPs for weeks after the initial COVID-19 diagnosis. Elevated frequencies of PS+PMP+ PBMCs correlated strongly with increasing disease severity. As a marker, PS outperformed established laboratory markers for inflammation, leucocyte composition, and coagulation, currently used for COVID-19 clinical outcome prognosis. PS+ PMPs preferentially bound to CD8+ T cells with gene expression signatures of proliferating effector rather than memory T cells. As PS+ PMPs carried programmed death-ligand 1 (PD-L1), they may affect T cell expansion or function. Our data provide a novel marker for disease severity and show that PS, which can trigger the blood coagulation cascade, the complement system, and inflammation, resides on activated immune cells. Therefore, PS may serve as a beacon to attract thromboinflammatory processes toward lymphocytes and cause immune dysfunction in COVID-19.


title: A novel bivalent DNA vaccine encoding both spike protein receptor-binding domain and nucleocapsid protein of SARS-CoV-2 to elicit T cell and neutralising antibody responses that cross react with variants

abstract: The efficacy of vaccines targeting SARS-CoV-2 is becoming apparent now that the mRNA and adenovirus vector vaccines that have been approved for emergency use are showing promise. However, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. To improve longevity and future protection against variants, we have designed a DNA vaccine encoding both the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) and its nucleocapsid (N) protein, the latter of which is highly conserved amongst beta coronaviruses. The vaccine elicits strong pro-inflammatory CD4 Th1 and CD8 T-cell responses to both proteins, with these responses being significantly enhanced by fusing the nucleocapsid sequence to a modified Fc domain. We have shown that the vaccine also stimulates high titre antibody responses to RBD which efficiently neutralise in both a pseudotype and live virus neutralisation assay and show cross reactivity with S proteins from the emerging variants Alpha (B.1.1.7) and Beta (B.1.351). This DNA platform can be easily adapted to target variant RBD and N proteins and we show that a vaccine variant encoding the B.1.351 RBD sequence stimulates cross-reactive humoral and T-cell immunity. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for targeting emerging SARS-CoV-2 variants.


title: No evidence of SARS-CoV-2 infection in Neotropical Primates sampled during COVID-19 pandemic in Minas Gerais and Rio Grande do Sul, Brazil.

abstract: In 2019, a new coronavirus disease (COVID-19) was detected in China. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was capable to infect domestic and captive mammals like cats, tigers and minks. Due to genetic similarities, concern about the infection of Non-Human Primates (NHPs) and the establishment of a sylvatic cycle has grown in the Americas. In this study, neotropical primates (NP) were sampled in different areas from Brazil to investigate whether they were infected by SARS-CoV-2. A total of 89 samples from 51 NP of four species were examined. No positive samples were detected via RT-qPCR, regardless of the NHP species, tissue or habitat tested. This work provides the first report on the lack of evidence of circulation of SARS-CoV-2 in NP. The expand of wild animals sampling is necessary to understand their role in the epidemiology of SARS-CoV-2.


title: Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides

abstract:
Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance and epiphytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these pathways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we analysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT-PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature and surface association strongly affect the expression of different polysaccharides under the control of the signalling proteins ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides and WapQ-mediated lipopolysaccharide production are important for leaf adhesion, while -glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection.

HighlightPseudomonas syringae uses the coordinated deployment of polysaccharides to address environmental challenges during plant colonization. Functional redundancy renders individual polysaccharides dispensable during plant infection, but their combined loss impedes pathogenicity.


title: Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids

abstract: The events of the last year have highlighted the complexity of implementing large-scale molecular diagnostic testing for novel pathogens. The purpose of this study was to determine the chemical influences of sample collection media and storage on the stability and detection of viral nucleic acids by qRT-PCR. We studied the mechanism(s) through which viral transport media (VTM) and number of freeze-thaw cycles influenced the analytical sensitivity of qRT-PCR detection of SARS-CoV-2. Our goal is to reinforce testing capabilities and identify weaknesses that could arise in resource-limited environments that do not have well-controlled cold chains. The sensitivity of qRT-PCR analysis was studied in four VTM for synthetic single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) simulants of the SARS-CoV-2 genome. The sensitivity and reproducibility of qRT-PCR for the synthetic ssRNA and dsDNA were found to be highly sensitive to VTM with the best results observed for ssRNA in HBSS and PBS-G. Surprisingly, the presence of epithelial cellular material with the ssRNA increased the sensitivity of the qRT-PCR assay. Repeated freeze-thaw cycling decreased the sensitivity of the qRT-PCR with two noted exceptions. The choice of VTM is critically important to defining the sensitivity of COVID-19 molecular diagnostics assays and this study suggests they can impact upon the stability of the SARS-CoV-2 viral genome. This becomes increasingly important if the virus structure is destabilised before analysis, which can occur due to poor storage conditions. This study suggests that COVID-19 testing performed with glycerol-containing PBS will produce a high level of stability and sensitivity. These results are in agreement with clinical studies reported for patient-derived samples.

category: molecular biology