title: The regeneration factors ERF114 and ERF115 act as transducers of mechanical cues to developmental pathways

abstract:
Plants show an unparalleled regenerative capacity, allowing them to survive severe stress conditions, such as injury, herbivory attack and harsh weather conditions. This potential not only replenishes tissues and restores damaged organs, but can also give rise to whole plant bodies, highlighting the intertwined nature of development and regeneration. It suggests that regeneration and developmental processes respond to the same upstream signals, but how a cell knows which of the two processes to engage is currently unknown. Here, we demonstrate that next to being regulators of regeneration, ETHYENE RESPONSE FACTOR 114 (ERF114) and ERF115 govern developmental growth in the absence of wounding or injury. Increased ERF114 and ERF115 activity is correlated with enhanced xylem maturation and lateral root formation, whereas their knockout results in a decrease in lateral roots and xylem connectivity following grafting. Moreover, we provide evidence that mechanical cues contribute to ERF114 and ERF115 expression in correlation with BZR1 mediated brassinosteroid signaling under both regenerative and developmental conditions. Antagonistically, negative regulation of cell wall extensibility via cell wall-associated mechanosensory FERONIA signaling suppresses their expression under both conditions. Our data suggest a molecular framework in which mechanical perturbations too great to be compensated by adaptive cell wall remodeling results in strong ERF114 and ERF115 expression, switching their role from developmental to regenerative regulators.

One-Sentence SummaryMechanical cues drive ERF114/ERF115 expression during lateral root development as well as regeneration.


title: Phytochrome B regulates reactive oxygen signaling during abiotic and biotic stress in plants

abstract:
Plants are essential for life on Earth converting light into chemical energy in the form of sugars. To adjust for changes in light intensity and quality, and to become as efficient as possible in harnessing light, plants utilize multiple light receptors, signaling, and acclimation mechanisms. In addition to altering plant metabolism, development and growth, light cues sensed by some photoreceptors, such as phytochromes, impact on many plant responses to biotic and abiotic stresses. Central for plant responses to different stresses are reactive oxygen species (ROS) that function as key signaling molecules. Recent studies demonstrated that respiratory burst oxidase homolog (RBOH) proteins that reside at the plasma membrane and produce ROS at the apoplast play a key role in plant responses to different biotic and abiotic stresses. Here we reveal that phytochrome B (phyB) and RBOHs function as part of a key regulatory module that controls ROS production, transcript expression, and plant acclimation to excess light stress. We further show that phyB can regulate ROS production during stress even if it is restricted to the cytosol, and that phyB, RBOHD and RBOHF co-regulate thousands of transcripts in response to light stress. Surprisingly, we found that phyB is also required for ROS accumulation in response to heat, wounding, cold, and bacterial infection. Taken together, our findings reveal that phyB plays a canonical role in plant responses to biotic and abiotic stresses, regulating ROS production, and that phyB and RBOHs function in the same pathway.

Significant StatementAbiotic and biotic stresses cause extensive losses to agricultural production and threaten global food security. Augmenting plant resilience to stressful conditions requires understanding of how plants sense stress. Here we report that the sensing of different abiotic and biotic stresses that result in the production of the key stress-response signaling molecules, reactive oxygen species, requires the plant photoreceptor protein phytochrome B. We further show that in contrast to its many nuclear functions, phytochrome B regulates reactive oxygen production by plasma membrane-localized respiratory burst oxidase homologs while localized to the cytosol. Our findings reveal the existence of a rapid stress response regulatory mechanism requiring phytochrome B and reactive oxygen species, essential for plant acclimation to stress.

category: plant biology


title: Oxidative Modification of LHC II Associated with Photosystem II and PS I-LHC I-LHC II Membranes

abstract: Under aerobic conditions the production of Reactive Oxygen Species (ROS) by electron transport chains is unavoidable, and occurs in both autotrophic and heterotrophic organisms. In photosynthetic organisms both Photosystem II (PS II) and Photosystem I (PS I), in addition to the cytochrome b6/f complex, are demonstrated sources of ROS. All of these membrane protein complexes exhibit oxidative damage when isolated from field-grown plant material. An additional possible source of ROS in PS I and PS II is the distal, chlorophyll-containing light-harvesting array LHC II, which is present in both photosystems. These serve as possible sources of 1O2 produced by the interaction of 3O2 with 3chl* produced by intersystem crossing. We have hypothesized that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in a subset of the spinach LHC II proteins (Lhcb1 and Lhcb2) that were associated with either PS II membranes (i.e. BBYs) or PS I-LHC I-LHC II membranes, both of which were isolated from field-grown spinach. We identified oxidatively modified residues by high-resolution tandem mass spectrometry. Interestingly, two different patterns of oxidative modification were evident for the Lhcb1 and Lhcb2 proteins from these different sources. In the LHC II associated with PS II membranes, oxidized residues were identified to be located on the stromal surface of Lhcb1 and, to a much lesser extent, Lhcb2. Relatively few oxidized residues were identified as buried in the hydrophobic core of these proteins. The LHC II associated with PS I-LHC I-LHC II membranes, however, exhibited fewer surface-oxidized residues but, rather a large number of oxidative modifications buried in the hydrophobic core regions of both Lhcb1 and Lhcb2, adjacent to the chlorophyll prosthetic groups. These results appear to indicate that ROS, specifically 1O2, can modify the Lhcb proteins associated with both photosystems and that the LHC II associated with PS II membranes represent a different population from the LHC II associated with PS I-LHC I-LHC II membranes.


title: Differential regulation of flower transpiration during abiotic stress in plants

abstract:
Heat waves, occurring during droughts, can have a devastating impact on yield, especially if they happen during the flowering and seed set stages of the crop cycle. Global warming and climate change are driving an alarming increase in the frequency and intensity of combined drought and heat stress episodes, critically threatening global food security. Previous studies revealed that during a combination of drought and heat stress stomata on leaves of many plants are closed, preventing cooling by transpiration. Because high temperature is detrimental to reproductive processes, essential for plant yield, we measured the inner temperature, transpiration, and sepal stomatal aperture of closed soybean flowers, developing on plants subjected to a combination of drought and heat stress. Here, we report that during a combination of drought and heat stress soybean plants prioritize transpiration through flowers over transpiration through leaves by opening their flower stomata, while keeping their leaf stomata closed. This acclimation strategy, termed  differential transpiration, lowers flower inner temperature by about 2-3{degrees}C, protecting reproductive processes at the expense of vegetative tissues. Manipulating stomatal regulation, stomatal size and/or stomatal density of flowers could therefore serve as a viable strategy to enhance the yield of different crops and mitigate some of the current and future impacts of global warming and climate change on agriculture.

One sentence summaryDuring stress conditions that result in higher flower inner temperature plants use a differential transpiration strategy to protect reproductive processes at the expense of vegetative tissues.


title: Differential EDS1 requirement for cell death activities of plant TIR-domain proteins

abstract:
Toll/interleukin-1 Receptor (TIR) domains are integral to immune systems across all domains of life. TIRs exist as single-domain and as larger receptor or adaptor proteins. In plants, TIRs constitute N-terminal domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors. Although TIR-NLR and TIR signaling requires the Enhanced disease susceptibility 1 (EDS1) protein family, TIR domains persist in species that have incomplete or no EDS1 members. To assess whether particular TIR groups appear with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algae and land plant species, we identify four conserved TIR groups, two of which are TIR-NLRs present in eudicots and two are more widespread. Presence of one TIR-only protein group is highly correlated with EDS1 and members of this group elicit EDS1-dependent cell death. By contrast, a more widely represented TIR group of TIR-NB-WD40/TPR (TNP) proteins (formerly called XTNX) has at least one member which can induce EDS1-independent cell death. Our data provide a new phylogeny-based plant TIR classification and identify TIR groups that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity.

One sentence summaryLand plants have evolved four conserved TIR groups


title: Pontin/Reptin-associated complexes differentially impact plant development and viral pathology

abstract: Pontin and Reptin are essential eukaryotic AAA+ ATPases that work together in several multiprotein complexes, contributing to chromatin remodeling and TARGET OF RAPAMCYIN (TOR) kinase complex assembly, among other functions. Null alleles of pontin or reptin are gametophyte lethal in plants, which has hindered studies of their crucial roles in plant biology. Here, we used virus-induced gene silencing (VIGS) to interrogate the functions of Pontin and Reptin in plant growth and physiology, focusing on Nicotiana benthamiana, a model species for the agriculturally significant Solanaceae family. Silencing either Pontin or Reptin caused pleiotropic developmental and physiological reprogramming, including aberrant leaf shape, reduced apical growth, delayed flowering, increased branching, chlorosis, and decreased spread of the RNA viruses Tobacco mosaic virus (TMV) and Potato virus X (PVX). To dissect these pleiotropic phenotypes, we took a comparative approach and silenced expression of key genes that encode subunits of each of the major Pontin/Reptin-associated chromatin remodeling or TOR complexes (INO80, SWR-C/PIE1, TIP60, TOR, and TELO2). We found that many of the pontin/reptin phenotypes could be attributed specifically to disruption of one of these complexes, with tip60 and tor knockdown plants each phenocopying a large subset of pontin/reptin phenotypes. We conclude that Pontin/Reptin complexes are crucial for proper plant development, physiology, and stress responses, highlighting the multifaceted roles these conserved enzymes have evolved in eukaryotic cells.


title: Mutation-related apparent myelin, not axon density, drives white matter differences in premanifest Huntington's disease: Evidence from in vivo ultra-strong gradient MRI

abstract: White matter (WM) alterations have been observed early in Huntingtons disease (HD) progression but their role in the disease-pathophysiology remains unknown. We exploited ultra-strong-gradient MRI to tease apart contributions of myelin (with the magnetization transfer ratio), and axon density (with the restricted volume fraction from the Composite Hindered and Restricted Model of Diffusion) to WM differences between premanifest HD patients and age- and sex-matched controls. Diffusion tensor MRI (DT-MRI) measures were also assessed. We used tractometry to investigate region-specific changes across callosal segments with well-characterized early- and late-myelinating axonal populations, while brain-wise alterations were explored with tract-based cluster analysis (TBCA). Behavioural measures were included to explore disease-associated brain-function relationships. We detected lower myelin in the rostrum of patients (tractometry: p = 0.0343; TBCA: p = 0.030), but higher myelin in their splenium (p = 0.016). Importantly, patients myelin and mutation size were positively associated (all p-values < 0.01), indicating that increased myelination might be a direct result of the mutation. Finally, myelin was higher than controls in younger patients but lower in older patients (p = 0.003), suggesting detrimental effects of increased myelination later in the course of the disease. Higher FR in patients left cortico-spinal tract (CST) (p = 0.03) was detected, and was found to be positively associated with MTR in the posterior callosum (p = 0.033), possibly suggesting compensation to myelin alterations. This comprehensive, ultra-strong gradient MRI investigation provides novel evidence of CAG-driven myelin alterations in premanifest HD which may reflect neurodevelopmental, rather than neurodegenerative disease-associated changes.

category: neuroscience


title: Carbon-based Fluorescent Antibody Nanoprobes as Brain Tumour Glioblastoma Diagnostics.

abstract:
The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters.

Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems.

category: immunology


title: Carbon Dot-based Fluorescent Antibody Nanoprobes as Brain Tumour Glioblastoma Diagnostics.


title: Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermis in vivo using two-photon excited FLIM

abstract: Macrophages (M{Phi}s) are important immune effector cells that promote (M1 M{Phi}s) or inhibit (M2 M{Phi}s) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, is not fully understood because of the lack of non-invasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free non-invasive method, can visualize M{Phi}s in human dermis in vivo. We demonstrate in vitro that human dermal M{Phi}s exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized M{Phi}s, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 M{Phi}s with a sensitivity of 0.88{+/-}0.04 and 0.82{+/-}0.03 and a specificity of 0.89{+/-}0.03 and 0.90{+/-}0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of M{Phi}s in health and disease.

category: biophysics


title: The coagulation factor IX (F9) loss of function prevents the cell cycle arrest induced by CDK4/6 inhibitors treatment

abstract: During this last decade the development of pro-senescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and have showed efficacy in reducing tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their anti-tumour activity. Here, using a functional genome wide CRISPR/Cas9 genetic screen we found several genes that synergistically participate in the proliferation arrest induced by the CDK4/6 inhibitor, Palbociclib. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout reduces senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitors response that will be useful to design new therapeutic strategies in personalized medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

category: cancer biology


title: Cross-modal Semantic Relationships Guide Spontaneous Orienting in Real-life Scenes

abstract: In real-world scenes, the different objects and events available to our senses are interconnected within a rich web of semantic associations. These semantic links help parse information and make sense of the environment. For example, during goal-directed attention, characteristic everyday life object sounds help speed up visual search for these objects in natural and dynamic environments. However, it is not known whether semantic correspondences also play a role under spontaneous observation. Here, we investigated this question addressing whether crossmodal semantic congruence can drive spontaneous, overt visual attention in free-viewing conditions. We used eye-tracking whilst participants (N=45) viewed video clips of realistic complex scenes presented alongside sounds of varying semantic congruency with objects within the videos. We found that characteristic sounds increased the probability of looking, the number of fixations, and the total dwell time on the semantically corresponding visual objects, in comparison to when the same scenes were presented with semantically neutral sounds or just with background noise only. Our results suggest that crossmodal semantic congruence has an impact on spontaneous gaze and eye movements, and therefore on how attention samples information in a free viewing paradigm. Our findings extend beyond known effects of object-based crossmodal interactions with simple stimuli and shed new light upon how audio-visual semantically congruent relationships play out in everyday life scenarios.


title: Imaging microvasculature network evolution and neurodegeneration with precise photothrombosis approach

abstract: In the cerebral cortex, the vasculature plays important homeostatic functions, especially at the highly connected complex capillary networks. The association of focal capillary ischemia with the neurodegenerative disease as well as the laminar vascular dynamics have prompted studies of vascular micro-occlusion via photothrombosis. However, technical challenges of this approach remain, including increased temporal precision of occlusion, increasing the depth of vascular occlusion, understanding how such micro-occlusion impacts local blood flow, and ultimately the neuronal effects of such changes. Here, we have developed a novel approach that employs ultra-fast multiphoton light to induce focal Rose Bengal-induced photothrombosis. We demonstrated induction of highly precise and fast occlusion of microvessels at various types and depths. The change of the microvascular architecture and hemodynamics after occlusion revealed the autoregulation and significant difference between upstream vs downstream in layer 2/3. Further, we found that micro-occlusion at two different layers within the same vascular arbor results in distinct effects on the acute flow redistribution mechanism. To examine neuronal effects of such micro-occlusion, we produced infarct of capillaries surrounding a labeled target neuron and found this induces dramatic and rapid lamina-specific degeneration in neuronal dendritic architecture. In sum, our technique enhanced the precision and power of the photothrombotic study of microvascular function. The current results pointed to the importance of laminar scale regulation within the microvascular network, a finding which may be relevant for models of neurovascular disease.


title: Validating a computational framework for ionicelectrodiffusion with cortical spreading depression as a case study

abstract:
Cortical spreading depression (CSD) is a wave of pronounced depolarization of brain tissue accompanied by substantial shifts in ionic concentrations and cellular swelling. Here, we validate a computational framework for modelling electrical potentials, ionic movement, and cellular swelling in brain tissue during CSD. We consider different model variations representing wild type or knock-out/knock-down mice and systematically compare the numerical results with reports from a selection of experimental studies. We find that the data for several CSD hallmarks obtained computationally, including wave propagation speed, direct current shift duration, peak in extracellular K+ concentration as well as a pronounced shrinkage of extracellular space, are well in line with what has previously been observed experimentally. Further, we assess how key model parameters including cellular diffusivity, structural ratios, membrane water and/or K+ permeabilities affect the set of CSD characteristics.

Significance StatementMovement of ions and molecules in and between cellular compartments is fundamental for brain function. Cortical spreading depression (CSD) is associated with dramatic failure of brain ion homeostasis. Better understanding the sequence of events in CSD could thus provide new insight into physiological processes in the brain. Despite extensive experimental research over the last decades, even basic questions related to mechanisms underlying CSD remain unanswered. Computational modelling can play an important role going forward, since simulation studies can address hypotheses that are difficult to target experimentally. Here, we assess the physiological validity of a novel mathematical framework for detailed modelling of brain electrodiffusion and osmosis - and provide a platform for in silico studies of CSD and other cerebral electro-mechanical phenomena.


title: Public T-Cell Receptors (TCRs) Revisited by Analysis of the Magnitude of Identical and Highly-Similar TCRs in Virus-Specific T-Cell Repertoires of Healthy Individuals

abstract:
Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 antigens of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation.

Significance statementPublic T-cell responses, in which T cells expressing the same T-cell receptor (TCR) are found in different individuals, have been described. However, the magnitude of the contribution of these TCRs to immune responses, defined as prevalence within the population and frequencies within individuals, is not known. In this study we characterized and quantified public T-cell responses within virus-specific memory T cells of healthy individuals by determining identical and highly-similar TCRs recognizing the same antigen and sharing conserved CDR3 motifs. The magnitude of public T-cell responses was surprisingly high and we argue that these dominant TCRs with shared core-sequences could be utilized for diagnostic purposes and may provide attractive TCRs to be used for immunotherapy in immunocompromised patients.


title: Increased expression of viral sensor MDA5 in pancreatic islets and in hormone-negative endocrine cells in recent onset type 1 diabetic donors

abstract:
The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic {beta}-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as Melanoma Differentiation Associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of non-diabetic and T1D donors is still missing. In the present study we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 non-diabetic autoantibody-negative, 2 autoantibody-positive, 8 recent-onset and 2 long-standing T1D).

In non-diabetic control donors, MDA5 was expressed both in - and in {beta}-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in -cells.

In T1D donors, we observed an increased colocalization rate MDA5-glucagon respect to MDA5-insulin in comparison to non-diabetic controls; such increase was more pronounced in recent onset respect to long standing T1D donors. Of note, an increased colocalization rate MDA5-glucagon was found in insulin-deficient-islets (IDI) respect to insulin containing islets (ICI).

Strikingly, in T1D donors we detected the presence of MDA5-positive/hormones-negative endocrine islet-like clusters, putatively deriving from dedifferentiation or neogenesis phoenomena. These clusters were exclusively identified in recent onset donors and not detected in autoantibody-positive non-diabetic or T1D long-standing ones.

In conclusion, we showed that MDA5 is preferentially expressed in -cells and its expression is increased in recent onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.


title: Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution

abstract: NAT10 is an essential enzyme that catalyzes the formation of N4-acetylcytidine (ac4C) in eukaryotic transfer RNA (tRNA) and 18S ribosomal RNA (rRNA). Recent studies in human cells suggested that rRNA acetylation is dependent on SNORD13, a non-canonical box C/D small nucleolar RNA (SNORD) predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10s essential function in pre-rRNA processing remain to be defined. Here, we used CRISPR-Cas9 genome editing to formally demonstrate that SNORD13 is required for acetylation of a single cytidine residue of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for yeast or human cell growth, ribosome biogenesis, translation, and the development of multicellular metazoan model organisms. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation  machinery that led to the characterization of many novel SNORD13 genes in phylogenetically-distant metazoans and more deeply rooted photosynthetic organisms. This includes an atypical SNORD13-like RNA in D. melanogaster which appears to guide ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that C. elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Altogether, our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across the eukaryotic tree of life and raise new questions regarding the biological function and evolutionary persistence of this highly conserved rRNA base modification.

category: molecular biology


title: Consistent predictors of microbial community composition across scales in grasslands reveal low context-dependency

abstract: Environmental circumstances shaping soil microbial communities have been studied extensively, but due to disparate study designs it has been difficult to resolve whether a globally consistent set of predictors exists, or context-dependency prevails. Here, we used a network of 18 grassland sites (11 sampled across regional plant productivity gradients) to examine i) if the same abiotic or biotic factors predict both large- and regional-scale patterns in bacterial and fungal community composition, and ii) if microbial community composition differs consistently with regional plant productivity (low vs high) across different sites. We found that there is high congruence between predictors of microbial community composition across spatial scales; bacteria were predominantly associated with soil properties and fungi with plant community composition. Moreover, there was a microbial community signal that clearly distinguished high and low productivity soils that was shared across worldwide distributed grasslands suggesting that microbial assemblages vary predictably depending on grassland productivity.

category: ecology


title: Mapping trait versus species turnover reveals spatiotemporal variation in functional redundancy in a plant-pollinator network

abstract: Functional overlap between species (redundancy) shapes competitive and mutualistic interactions, determining community responses to perturbations. Most studies view functional redundancy as static, even though individuals within species vary in traits over seasonal or spatial gradients. Consequently, we lack knowledge on trait turnover within species, how functional redundancy spatiotemporally varies, and when and where interaction networks are vulnerable to functional loss. Studying an Arctic bumblebee community, we investigated how body-size turnover over elevation and season shapes their host-plant interactions, and test how sensitive networks are to sequentially losing body-size groups. With trait turnover being larger than species, we found: i) late-season networks were less specialised when nodes comprised functionally similar bumblebees; ii) removal of bumblebee-body-size groups over species accelerated coextinction of host plants, with the magnitude varying in space and time. We demonstrate functional redundancy can vary spatiotemporally, and functional loss impacts interaction partners more than expected from species loss alone.


title: Regional opportunities for Siberian tundra conservation in the next 1000 years

abstract: The biodiversity of tundra areas in northern high latitudes is threatened by invasion of forests under global warming. However, poorly understood nonlinear responses of the treeline ecotone mean the timing and extent of tundra losses are unclear but policymakers need such information to optimize conservation efforts. Our individual based model LAVESI, developed for the Siberian tundra-taiga ecotone, can help improve our understanding. Consequently, we simulated treeline migration trajectories until the end of the millennium, causing a loss of tundra area when advancing north. Our simulations reveal that the treeline follows climate warming with a severe, century-long time lag, which is overcompensated by infilling of stands in the long run even when temperatures cool again. Our simulations reveal that only under ambitious mitigation strategies (RCP 2.6) will ~30% of original tundra areas remain in the north but separated into two disjunct refugia.


title: A subcellular cookie cutter for spatial genomics in human tissue

abstract: Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state of art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of (>20m) for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.

category: genomics


title: DeepSNEM: Deep Signaling Network Embeddings for compound mechanism of action identification

abstract:
MotivationThe analysis and comparison of compounds transcriptomic signatures can help elucidate a compounds Mechanism of Action (MoA) in a biological system. In order to take into account the complexity of the biological system, several computational methods have been developed that utilize prior knowledge of molecular interactions to create a signaling network representation that best explains the compounds effect. However, due to their complex structure, large scale datasets of compound-induced signaling networks and methods specifically tailored to their analysis and comparison are very limited. Our goal is to develop graph deep learning models that are optimized to transform compound-induced signaling networks into high-dimensional representations and investigate their relationship with their respective MoAs.

ResultsWe created a new dataset of compound-induced signaling networks by applying the CARNIVAL network creation pipeline on the gene expression profiles of the CMap dataset. Furthermore, we developed a novel unsupervised graph deep learning pipeline, called deepSNEM, to encode the information in the compound-induced signaling networks in fixed-length high-dimensional representations. The core of deepSNEM is a graph transformer network, trained to maximize the mutual information between whole-graph and sub-graph representations that belong to similar perturbations. By clustering the deepSNEM embeddings, using the k-means algorithm, we were able to identify distinct clusters that are significantly enriched for mTOR, topoisomerase, HDAC and protein synthesis inhibitors respectively. Additionally, we developed a subgraph importance pipeline and identified important nodes and subgraphs that were found to be directly related to the most prevalent MoA of the assigned cluster. As a use case, deepSNEM was applied on compounds gene expression profiles from various experimental platforms (MicroArrays and RNA sequencing) and the results indicate that correct hypotheses can be generated regarding their MoA.

Availability and ImplementationThe source code and pre-trained deepSNEM models are available at https://github.com/BioSysLab/deepSNEM.

ContactEmail for correspondence: leo@mail.ntua.gr.

Supplementary informationAccompanying supplementary material are available online.

category: bioinformatics


title: Kv3 channels contribute to the excitability of sub-populations of spinal cord neurons in lamina VII

abstract:
Autonomic parasympathetic preganglionic neurons (PGN) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be due to recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within lamina VII of the lumbosacral spinal cord, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphological analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.

Significance statementNeural circuits in the spinal cord and pons mediate the micturition reflex. The spinal cord drives bladder contraction during micturition through the activation of parasympathetic preganglionic neurons in lamina VII of the lumbosacral spinal cord. Despite the significant contribution of these neurons to a crucial physiological reflex, neurons in this region have been under-characterised. This study therefore elucidated and thoroughly characterised distinct neuronal populations in this lamina; we propose that these populations included a fast firing interneuron and subtypes of parasympathetic preganglionic neurons. Further investigation revealed the critical importance of Kv3 channels in the fast firing ability of the interneurons, as well as in synaptic release onto parasympathetic preganglionic neurons.


title: Induction of mitochondrial heat shock proteins and mitochondrial biogenesis in endothelial cells upon acute methylglyoxal stress: Evidence for hormetic autofeedback

abstract: Increased metabolic flux produces potentially harmful side-products, such as reactive dicarbonyl and oxygen species. The reactive dicarbonly methylglyoxal (MG) can impair oxidative capacity, which is downregulated in type 2 diabetes. Heat shock proteins (HSPs) of subfamily A (Hsp70s) promote ATP-dependent processing of damaged proteins during MG exposure which also involve mitochondrial proteins. Since the protection of mitochondrial proteins could promote higher production of reactive metabolites due to increased substrate flux, tight regulation of HspA-mediated protein handling is important. We hypothesized that stress-inducible HspAs (HspA1A/HspA1B) are pivotal for maintaining mitochondrial biogenesis during acute MG-stress. To analyze the role of stress-inducible HspA1A/HspA1B for maintenance of mitochondrial homeostasis during acute MG exposure, we knocked out HSPA1A/HSPA1B in mouse endothelial cells. HSPA1A/HSPA1B KO cells showed upregulation of the mitochondrial chaperones HspA9 (mitochondrial Hsp70/mortalin) and HspD1 (Hsp60) as well as induction of mitochondrial biogenesis upon MG exposure. Increased mitochondrial biogenesis was reflected by elevated mitochondrial branching, total count and area as well as by upregulation of mitochondrial proteins and corresponding transcription factors. Our findings suggest that mitochondrial HspA9 and HspD1 promote mitochondrial biogenesis during acute MG stress, which is counterregulated by HspA1A/HspA1B to prevent mitochondrial overstimulation and to maintain balanced oxidative capacity under metabolic stress conditions. These data support an important role of HSPs in MG-induced hormesis.

category: cell biology


title: A framework for multiplex imaging optimization and reproducible analysis

abstract: Multiplex imaging technologies are increasingly used for single-cell phenotyping and spatial characterization of tissues; however, transparent methods are needed for comparing the performance of platforms, protocols and analytical pipelines. We developed a python software, mplexable, for reproducible image processing and utilize Jupyter notebooks to share our optimization of signal removal, antibody specificity, background correction and batch normalization of the multiplex imaging with a focus on cyclic immunofluorescence (CyCIF). Our work both improves the CyCIF methodology and provides a framework for multiplexed image analytics that can be easily shared and reproduced.


title: Interactions of host defense and hyper-keratinization in psoriasis

abstract:
ObjectivesTo understand the crosstalk between the host and microbiota in psoriatic skin, using a systems biology approach based on transcriptomics and microbiome profiling.

MethodsWe collected the skin tissue biopsies and swabs in both lesion and non-lesion skin of 13 patients with psoriasis (PsO), 15 patients with psoriatic arthritis (PsA), and healthy skin from 12 patients with ankylosing spondylitis (AS). We performed transcriptome sequencing and metagenomics profiling on the local skin sites to study the similarities and differences in the molecular profiles between the three conditions, and the associations between the host defense and microbiota dynamic.

ResultsWe found that lesion and non-lesional samples were remarkably different in terms of their transcriptome profiles. Functional annotation of differentially expressed genes (DEGs) showed a major enrichment in neutrophil activation. By using coexpression gene networks, we identified a gene module that was associated with local psoriasis severity at the site of biopsy. From this module, we extracted a "core" set of genes that were functionally involved in neutrophil activation, epidermal cell differentiation and response to bacteria. Skin microbiome analysis revealed that the abundance of Enhydrobacter, Micrococcus and Leptotrichia were significantly correlated with the "core network" of genes.

ConclusionsWe identified a core network that regulates inflammation and hyper-keratinization in psoriatic skin, and is associated with local disease severity and microbiome composition.

category: systems biology


title: Diversity of Lepidoptera in urban parks at Pasir Gudang, Johor, Malaysia

abstract: Urban parks are a valuable habitat for Lepidoptera, which are often neglected despite their potential role as pollinators and important food source for small animals. Lepidoptera in urban parks in Pasir Gudang are still under-studied. Yet, there is no publication on Lepidoptera in these urban parks. The objectives of this study were to determine and to compare the diversity of Lepidoptera in three urban parks (Laman Rekreasi Sungai Buluh, Tasik Dahlia and Taman Bandar) at Pasir Gudang, Johor. Visual Encounter Survey (VES) method was used to capture the Lepidoptera from February 2021 until April 2021. Four sampling sessions were conducted for each study site. A total of nine species from 117 individuals comprised of family Pieridae (43%), Nymphalidae (29%) and Lycaenidae (28%) were recorded from this study. The most abundant species were Zizula hylax and Elymnias hypermnestra, while Leptosia nina was the least abundant. The results from this study showed that the diversity of Lepidoptera have established in these three urban parks. Diversity of Lepidoptera in Tasik Dahlia was significantly lower compared to the other two study sites. The findings from this study could be useful as a baseline data for future research and conservation management of the order Lepidoptera around the urban parks and indirectly can support the sustainable development of the urban parks.

category: zoology


title: Representation of Drosophila larval behaviors by muscle activity patterns

abstract: How muscle actions are coordinated to realize animal movement is a fundamental question in behavioral study. To obtain the overall muscular activity patterns accompanying behaviors at high spatiotemporal resolution is technically difficult. In this work, we used light sheet microscopy to simultaneously image and analyze the activity, length and orientation of Drosophila larval muscles across body segments at single muscle resolution in nearly free behaviors. For typical behavioral modes such as peristalsis, head cast and turning, larval muscles showed behavioral mode specific activity patterns. Unexpectedly, reorientation of larval head involves muscle tone in the apparently motionless posterior segments. With a STGCN(spatial temporal graph convolution neural network)-Generator model, sequence of larval behavioral poses outlined by morphological patterns of muscles could be accurately predicted based on the time series of ventral but not dorsal muscle activities, and vice versa. Laser ablation of ventral but not dorsal muscles interrupted peristaltic wave and undermined head cast in both frequency and amplitude. Our results provide a simplified muscle activity representation of soft body motion that can be used for probing the key components of animal motor control.


title: Mammalian octopus cells are direction selective to frequency sweeps by synaptic sequence detection

abstract: Octopus cells are remarkable projection neurons of the mammalian cochlear nucleus, with extremely fast membranes and wide frequency tuning. They are considered prime examples of coincidence detectors but are poorly characterized in vivo. We discover that octopus cells are selective to frequency sweep direction, a feature that is absent in their auditory nerve inputs. In vivo intracellular recordings reveal that direction selectivity does not derive from cross-channel coincidence detection but hinges on the amplitudes and activation sequence of auditory nerve inputs tuned to clusters of "hotspot" frequencies. A simple biophysical model of octopus cell excited with real nerve spike trains recreates direction selectivity through interaction of intrinsic membrane conductances with activation sequence of clustered inputs. We conclude that octopus cells are sequence detectors, sensitive to temporal patterns across cochlear frequency channels. The detection of sequences rather than coincidences is a much simpler but powerful operation to extract temporal information.


title: KymoMerge: a new tool for analysis of multichannel kymographs

abstract: Kymograph analysis is commonly used by researchers to study dynamic processes in cells. Current tools widely available only allow for analysis of one channel kymographs. Here we provide a python-coded, open source program to use as a plug-in for ImageJ which creates one kymograph from the kymographs of two separate channels of one time-lapse movie. This add-in, which we call KymoMerge, therefore allows for analysis of only the co-located tracks in multi-channel movies.


title: Distractibility and impulsivity neural states are distinct from attention states and modulate the implementation of attention

abstract: In the context of visual attention, it has been classically assumed that missing the response to a target or erroneously selecting a distractor occurs as a consequence of the (miss)allocation of attention in space. In the present paper, we challenge this view and provide evidence that, in addition to encoding spatial attention, prefrontal neurons also encode a distractibility-to-impulsivity state. Using supervised dimensionality reduction techniques, we identify two partially overlapped neuronal subpopulations associated either with attention or overt behaviour. The degree of overlap accounts for the behavioural gain associated with the good allocation of attention. We further describe the neural variability accounting for distractibility-to-impulsivity behaviour by a two dimensional state associated with optimality in task and responsiveness. Overall, we thus show that behavioural performance arises from the integration of task-specific neuronal processes and pre-existing neuronal states describing task-independent behavioural states, shedding new light on attention disorders such as ADHD.


title: Structural and functional organization of visual responses in the inferior olive of larval zebrafish

abstract: The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. According to a classical theory of cerebellar cortex, the inferior olive (IO) provides Purkinje cells with error information which drives motor learning in the cerebellum. Here we investigate the sensory representations in the IO of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using 2-photon imaging. We found that most IO neurons showed direction selective and binocular responses to visual stimuli and that functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between soma location, axonal projections and functional properties of IO neurons. Taken together, our results suggest that anatomically-defined classes of inferior olive neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.


title: Comparison of TIMS-PASEF quantitative proteomics data-analysis workflows using FragPipe, DIA-NN, and Spectronaut from a user's perspective

abstract: Data-independent acquisition (DIA) methods have gained great popularity in bottom-up quantitative proteomics, as they overcome the irreproducibility and under-sampling limitations of data-dependent acquisition (DDA). diaPASEF, recently developed for the timsTOF Pro mass spectrometers, has brought improvements to DIA, providing additional ion separation (in the ion mobility dimension) and increasing sensitivity. Several studies have benchmarked different workflows for DIA quantitative proteomics, but mostly using instruments from Sciex and Thermo, and therefore, the results are not extrapolable to diaPASEF data. In this work, using a real-life sample set like the one that can be found in any proteomics experiment, we compared the results of analyzing PASEF data with different combinations of library-based and library-free analysis, combining the tools of the FragPipe suite, DIA-NN and including MS1-level LFQ with DDA-PASEF data, and also comparing with the workflows possible in Spectronaut. We verified that library-independent workflows, not so efficient not so long ago, have greatly improved in the recent versions of the software tools, and now perform as well or even better than librarybased ones. We report here information so that the user who is going to conduct a relative quantitative proteomics study using a timsTOF Pro mass spectrometer can make an informed decision on how to acquire (diaPASEF for DIA analysis, or DDA-PASEF for MS1-level LFQ) the samples, and what can be expected depending on the data analysis tool used, among the different alternatives offered by the recently optimized tools for TIMS-PASEF data analysis.


title: Comparison of the somatic TADs and lampbrush chromomere-loop complexes in transcriptionally active prophase I oocytes

abstract: In diplotene oocyte nuclei of all vertebrate species, except mammals, chromosomes lack interchromosomal contacts and chromatin is linearly compartmentalized into distinct chromomere-loop complexes forming lampbrush chromosomes. However, the mechanisms underlying the formation of chromomere-loop complexes remain unexplored. Here we aimed to juxtapose somatic topologically associating domains (TADs), recently identified in chicken embryonic fibroblasts, with chromomere-loop complexes in lampbrush meiotic chromosomes. By measuring 3D-distances and colocalization between linear equidistantly located genomic loci, positioned within one TAD or separated by a TAD border, we confirmed the presence of predicted TADs in chicken embryonic fibroblast nuclei. Using three-colored FISH with BAC probes we mapped equidistant genomic regions included in several sequential somatic TADs on isolated chicken lampbrush chromosomes. Eight genomic regions, each comprising two or three somatic TADs, were mapped to non-overlapping neighboring lampbrush chromatin domains - lateral loops, chromomeres or chromomere-loop complexes. Genomic loci from the neighboring somatic TADs could localize in one lampbrush chromomere-loop complex, while genomic loci belonging to the same somatic TAD could be localized in neighboring lampbrush chromomere- loop domains. In addition, FISH-mapping of BAC probes to the nascent transcripts on the lateral loops indicates transcription of at least 17 protein-coding genes and 2 non-coding RNA genes during the lampbrush stage of chicken oogenesis, including genes involved in oocyte maturation and early embryo development.


title: The ulcerative colitis associated gene FUT8 regulates the quantity and quality of secreted mucins

abstract:
Fucosylation of mucins, the main macrocomponents of the mucus layer that protects the digestive tract from pathogens, increases their viscoelasticity and shear stress resistance. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here we show that the levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients compared to normal individuals. Overexpressing FUT8, as observed in UC, in mucin-producing HT29-18N2 colonic cell line increases trafficking of MUC1 to plasma membrane and secretion of MUC2/MUC5AC. FUT8 depletion (FUT8 KD), instead, causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to shear stress compared to mucins secreted by FUT8 KD cells. These data fit well with the Fut8-/- mice phenotype, which are protected against UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins compared to Fut8+/+ mice. Together, these data reveal that FUT8 optimizes the viscoelastic properties of the extracellular mucous by controlling the quantities of mucins secreted.

SIGNIFICANCE STATEMENTMucins, the major components of the mucous barrier that protects our body from pathogens, are heavily glycosylated proteins. Changes their amounts and properties will alter the viscosity of mucous. Here we show that FUT8, a glycosylation enzyme of the Golgi apparatus, can control the viscosity of secreted mucins. Mucin secreting cells of the distal colon express FUT8, but their levels are altered in Ulcerative colitis patients. As a result, mucous produced by these cells is easily washed away, which exposes them to pathogens. We suggest that a defective mucous production is the main cause of initial inflammation observed in disease. Our findings help in understanding how cells control the quality of mucins and provide a means to prevent Ulcerative colitis.


title: Hepatocytes with a phenotype of substantial CYP3A4 induction generated from drug-associated fulminant hepatitis-derived induced pluripotent stem cells

abstract: Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4). Hepatocytes are often employed to evaluate drug-mediated CYP3A4 induction, but the variation between different cell lots is an issue that needs to be solved. Only a limited number of immortalized hepatocyte cell lines have been reported to date. In this study, we describe the successful generation of hepatocytes from disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with fulminant hepatitis (FH-iPSCs). To examine the CYP3A4 induction potential, FH-iPSCs were induced into hepatocytes. Drug-mediated induction testing revealed that HepaKI exhibited a 57.2-fold increase in CYP3A4 after exposure to rifampicin, relative to control cells. These results suggest that FH-iPSCs are a preferred cell source for in vitro CYP3A4 induction assays.


title: The Astrin/SKAP Complex Reduces Friction at the Kinetochore-Microtubule Interface

abstract: The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation, but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens movement and decreases coordination of metaphase sister kinetochores, and increases tension between them. Using laser ablation to isolate kinetochores bound to polymerizing vs depolymerizing microtubules, we show that without SKAP kinetochores move slower on both polymerizing and depolymerizing microtubules, and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to previously described kinetochore proteins that increase grip on microtubules under force, Astrin-SKAP reduces grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.


title: Hydrothermal Synthesis of Clove Buds-Derived Multifunctional Carbon Dots Passivated with PVP- Antioxidants, Catalysis, and Bioimaging Applications

abstract: The present study reports a one-step synthesis of PVP-passivated clove buds derived carbon dots (PPCCDs) using clove buds and polyvinylpyrrolidone (PVP) as the starting precursors via the hydrothermal route. The adopted technique is facile and environmentally friendly for the production of carbon dots (CDs) with in situ PVP passivation. The study evidenced the significant modulation in optical properties of passivated CDs as compared to non-passivated ones. Structural and morphological studies evidenced the spherical PPCCDs with a diameter of [~] 2 nm and crystalline in nature with an interlayer spacing of 0.33 nm. The PPCCDs showed excellent antioxidant activity against DPPH and superoxide anion radicals and also showed good catalytic activity for the degradation of Rhodamine-B (Rh-B) dye under the studied conditions. Their bioimaging potential was evidenced through live-cell fluorescent imaging with 3T3 and L929 cell lines.


title: Hydrothermal Synthesis of Clove Bud-Derived Multifunctional Carbon Dots Passivated with PVP- Antioxidant, Catalysis, and Bioimaging Applications


title: MiR-513a-5p Aggravates Atherosclerosis Phenotype by Downregulating TFPI2 in Vitro

abstract: This manuscript has been withdrawn by the authors as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.


title: TRPA1: A Potential Prognostic Indicator for Oral Squamous Cell Carcinoma

abstract:
IntroductionTransient receptors are related to oral cancer pain. Previously capsaicin (TRPV1 agonist) was shown to induce cell death in oral cancer cells. We hypothesised that these receptors are present in oral cancer.

MethodWe examined the presence of cannabinoid receptors (CB1 and CB2) and targets (TRPV1, TRPA1, CaV 3.1, CaV 3.2, CaV 3.3) via quantitative polymerase chain reaction (qPCR) in oral cancer cells SCC4, SCC9, SCC25, Cal27, and normal oral cell line OKF6.

ResultCannabinoid receptors are absent in all the cell lines, while TRPA1 is only present in normal cells, but absent in all the oral cancer cell lines. Voltage-gated calcium channels are present in all the cell lines.

Conclusion and Future AspectsTRPA1 could be the possible future prognostic indicator of oral squamous cell carcinoma. Future functionality assays could use precancerous cell lines to follow the loss of TRPA1.


title: OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

abstract: Patients with primary mitochondrial diseases present with fatigue and multi-system disease, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. Integrating data from 17 cohorts of patients with mitochondrial diseases (n=690), we find that clinical mitochondrial disorders increase resting energy expenditure, a state termed hypermetabolism. In a longitudinal cellular model of primary patient-derived fibroblasts from multiple donors, we show that genetic and pharmacological disruptions of oxidative phosphorylation (OxPhos) similarly trigger increased energy consumption in a cell-autonomous manner, despite near-normal OxPhos coupling efficiency. Hypermetabolism is associated with mtDNA instability, activation of the integrated stress response, increased extracellular secretion of age-related cytokines and metabokines including GDF15, as well as an accelerated rate of telomere erosion and epigenetic aging, and a reduced Hayflick limit. Finally, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations to OxPhos dysfunction. Hypermetabolism, or the increased energetic cost of living in mitochondrial diseases, has important biological and clinical implications.


title: Transposon Extermination Reveals Their Adaptive Fitness Contribution

abstract: Transposable Elements are molecular parasites that persist in their host genome by generating new copies to outpace natural selection. Here we measure the parameters governing the copy number dynamics of the fission yeast Tf2 retrotransposons, using experimental and natural populations and a strain where all Tf2 copies are removed. Natural population genomes display active and persistent Tf2 colonies, but in the absence of selection mitotic recombination deletes Tf2 elements at rates that far exceed transposition. We show that Tf2 elements provide a fitness contribution to their host by dynamically rewiring the transcriptional response to metabolic stress. Therefore, Tf2 elements exhibit a mutualistic rather than parasitic behavior toward their host.


title: Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in a Hydrogel Skin Graft Model

abstract:
Regenerating functional skin without the formation of scar tissue remains an important goal for Tissue Engineering. Current hydrogel-based grafts minimize contraction of full-thickness skin wounds and support skin regeneration using adult or neonatal foreskin dermal fibroblasts, which are often expanded in vitro and used after multiple passages. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may also influence the functionality of 3D skin constructs by affecting fibroblast-mediated graft contraction. To validate these predicted differences in fibroblast phenotype and resulting 3D graft model contraction, we isolated porcine dermal fibroblasts of varying donor age for use in a 2D proliferation assay and a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1. However, after 5 passages in culture, there was no difference in contractility between groups. These results show how cellular responses differ based on donor age and time in culture, which could account for important differences in biomanufacturing of 3D hydrogel-based skin grafts. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding.

IMPACT STATEMENTLittle is known about the impact of donor age and time in culture on the contraction of the 3D hydrogel-based graft. These results show how cellular phenotypes differ based on donor age and time in culture, which could account for important inconsistencies in biomanufacturing of skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. Future in vivo studies could help determine the best donor age and time in culture for improved wound healing outcomes or more reproducible in vitro testing constructs.

category: bioengineering


title: The HUSH complex controls brain architecture and protocadherin fidelity

abstract: AbstractFine-tuning of neural connectivity is important for cerebral functions and brain evolution. Protocadherins provide barcodes for neuronal identity as well as synapse formation and expansion of protocadherin cluster genes has been linked to advanced cognitive functions. The tightly controlled stochastic and combinatorial expression of the different protocadherin isoforms in individual neurons provides the molecular basis for neuronal diversity, neuronal network complexity and function of the vertebrate brain. How protocadherins are epigenetically controlled has not yet been fully elucidated. Here we show that the HUSH (human silencing hub) complex containing H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and Microrchidia CW-type zinc finger protein 2 (MORC2), critically controls the fidelity of protocadherin expression. MPP8 and MORC2A are highly expressed in the murine brain and exclusively found in neurons. Genetic inactivation of Mphosph8 (coding for MPP8) or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, MPP8 and MORC2A precisely and selectively suppress the repetitive-like protocadherin gene cluster on mouse chromosome 18 in a H3K9me3-dependent manner, thereby affecting synapse formation. Moreover, we demonstrate that individual MPHOSPH8- or MORC2-deficient neurons in human cerebral organoids express increased numbers of clustered protocadherin isoforms. Our data identify the HUSH complex, previously linked to silencing of repetitive transposable elements, as a key epigenetic regulator of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.


title: Localizing spontaneous memory reprocessing during human sleep

abstract: Reactivation of newly acquired memories during sleep across hippocampal and neocortical systems is proposed to underlie systems memory consolidation. Here, we investigate spontaneous memory reprocessing during sleep by applying machine learning to source space-transformed magnetoencephalographic data in a two-step exploratory and confirmatory study design. We decode memory-related activity from slow oscillations in hippocampus, frontal cortex and precuneus, indicating parallel memory processing during sleep. Moreover, we show complementary roles of hippocampus and neocortex: while gamma activity indicated memory reprocessing in hippocampus, delta and theta frequencies allowed decoding of memory in neocortex. Neocortex and hippocampus were linked through coherent activity and modulation of high-frequency gamma oscillations by theta, a dynamic similar to memory processing during wakefulness. Overall, we noninvasively demonstrate localized, coordinated memory reprocessing in human sleep.


title: COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis

abstract:
BackgroundIn the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options.

MethodUsing an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR.

FindingsCOVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric) and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2.

InterpretationLike IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung disease.

FundingThis work was supported by the National Institutes for Health grants R01-GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780).

One Sentence SummarySevere COVID-19 triggers cellular processes seen in fibrosing Interstitial Lung Disease


title: A recombinant SARS-CoV-2 RBD antigen expressed in insect cells elicits immunogenicity and confirms safety in animal models

abstract:
COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus requires new immunization strategies in addition to the current vaccines approved for human administration. In the present report, the immunological and safety evaluation in mice and hamsters of a subunit vaccine based on the RBD sub-domain with two adjuvants of oil origin is described.

The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions.

In immunization assays in mice and hamsters, the purified RBD formulated with adjuvants based on oil-water emulsifications and squalene was able to stimulate specific neutralizing antibodies and confirm the secretion of IFN-{gamma} after stimulating spleen cells with the purified RBD. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results demonstrate the potential of the purified RBD administered with adjuvants through an intramuscular route, to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection against infection.


title: Myeloid cell-driven nonregenerative pulmonary scarring is conserved in multiple nonhuman primate species regardless of SARS-CoV-2 infection modality

abstract: The novel coronavirus SARS-CoV-2 has caused a worldwide pandemic resulting in widespread efforts in development of animal models that recapitulate human disease for evaluation of medical countermeasures, and to dissect COVID-19 immunopathogenesis. We tested whether route of experimental infection substantially changes COVID-19 disease characteristics in two species (Macaca mulatta; rhesus macaques; RM, Chlorocebus atheiops; African green monkeys; AGM) of nonhuman primates. Species-specific cohorts of RM and AGM Rhesus macaques (Macaca mulatta, RMs) and African green monkeys (Chlorocebus aethiops, AGMs) were experimentally infected with homologous SARS-CoV-2 by either direct mucosal instillation or small particle aerosol in route-discrete subcohorts. Both species demonstrated equivalent infection initially by either exposure route although the magnitude and duration of viral loading was greater in AGMs than that of the RM. Clinical onset was nearly immediate (+1dpi) in mucosally-exposed cohorts whereas aerosol-infected animals began to show signs +7dpi. Myeloid cell responses indicative of the development of pulmonary scarring and extended lack of regenerative capacity in the pulmonary compartment was a conserved pathologic response in both species by either exposure modality. This pathological commonality may be useful in future anti-fibrosis therapeutic evaluations and expands our understanding of how SARS-CoV-2 infection leads to ARDS and functional lung damage.

category: microbiology


title: Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines

abstract: IntroductoryThe evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of many new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccines. To ascertain and rank the risk of VOCs and VOIs, we analyzed their ability to escape from vaccine-induced antibodies. The variants showed differential reductions in neutralization and replication titers by post-vaccination sera. Although the Omicron variant showed the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retained moderate neutralizing activity against that variant. Therefore, vaccination remains the most effective strategy to combat the COVID-19 pandemic.


title: Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

abstract: Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in-silico drug screening studies has been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with 9 drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.


title: Updated range metrics and a global population estimate for the Critically Endangered Philippine Eagle using a spatial ensemble habitat model

abstract: Many range-restricted taxa are currently experiencing population declines yet lack fundamental information regarding distribution and population size. Establishing baseline estimates for both these key biological parameters is however critical for directing conservation planning for at-risk range-restricted species. The International Union for the Conservation of Nature (IUCN) Red List uses three range metrics that define species distributions and inform extinction risk assessments: extent of occurrence (EOO), area of occupancy (AOO) and area of habitat (AOH). However, calculating all three metrics using standard IUCN approaches relies on a geographically representative sample of locations, which for rare species is often spatially biased. Here, we apply model-based interpolation using Species Distribution Models (SDMs), correlating occurrences with remote-sensing covariates, to calculate IUCN range metrics, protected area coverage and a global population estimate for the Critically Endangered Philippine Eagle (Pithecophaga jefferyi). Our final range wide continuous SDM had high predictive accuracy (Continuous Boyce Index = 0.927) and when converted to a binary model estimated an AOH = 23,185 km2, a maximum EOO = 605,759 km2, a minimum EOO = 272,272 km2, with an AOO = 53,867 km2. Based on inferred habitat from the AOH metric, we estimate a global population of 318 breeding pairs (range: 258-362 pairs), or 636 mature individuals, across the Philippine Eagle global range. Protected areas covered 34 % of AOH, 15 % less than the target representation, with the continuous model identifying key habitat as priority conservation areas. We demonstrate that even when occurrences are geographically biased, robust habitat models can be built that enable quantification of baseline IUCN range metrics, protected area coverage, and a population size estimate. In the absence of adequate location data for many rare and threatened taxa, our method is a promising spatial modelling tool with widespread applications, in particular for island endemics facing high extinction risk.


title: Priority conservation areas and a global population estimate for the Critically Endangered Philippine Eagle derived from modelled range metrics using remote sensing habitat characteristics


title: Main protease mutants of SARS-CoV-2 variants remain susceptible to PF-07321332

abstract:
The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics showed that these Mpro variants are similarly catalytically competent as the wildtype. We show that nirmatrelvir has similar potency against the variants as against the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants.

Graphical abstract

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category: biochemistry


title: Main protease mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir (PF-07321332)


title: sgDI-tector: defective interfering viral genome bioinformatics for detection of coronavirus subgenomic RNAs

abstract: Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study the growing number of next-generation sequencing (NGS) data of SARS-CoV-2. We introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. sgDI-tector allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. We produced NGS data and successfully detected the nested set of sgRNAs with the ranking M>ORF3a>N>ORF6>ORF7a>ORF8>S>E>ORF7b. We also compared the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.


title: Hypothermia Suppresses Uncoupling of Oxidative-Phosphorylation after Neonatal Cerebral Hypoxia-Ischemia

abstract: Hypothermia is the best available therapy for neonatal hypoxia-ischemia (HI) brain injury, but its primary mechanisms remain uncertain1. We hypothesize that HI induces, whereas hypothermia represses, uncoupling of oxidative-phosphorylation (OXPHOS)--an increase of the cerebral metabolic rate of oxygen (CMRO2) despite reduction of the mitochondrial energy output2,3. We used a multi-parametric photoacoustic microscopy (PAM) system to compare the effects of HI and post-HI hypothermic treatment on CMRO2 in awake 10-day-old (P10) mice4-9. Here we show that hypoxia (10% O2) elevated CMRO2, but the addition of unilateral carotid artery ligation suppressed CMRO2 and sparked a rapid overshoot of post-HI CMRO2 in the ipsilateral cerebral cortex for at least 2 hours. The post-HI surge of CMRO2 was linked to an increase of mitochondrial oxygen consumption and superoxide outburst, despite reduction of the mitochondrial membrane potential. Notably, post-HI hypothermia blocked the surge of superoxide and CMRO2--primarily by limiting oxygen extraction fraction (OEF)--leading to better preservation of adenosine triphosphate (ATP), creatine (Cr) and N-acetylaspartate (NAA) after HI. Mice that did not receive hypothermia exhibited ~80% reduction of CMRO2 at 24 h post-HI, coupled to a large cortical infarction. These results suggest that mitigation of post-HI uncoupling of OXPHOS is an early and/or pivotal effect of hypothermia. Further, optical measurement of CMRO2 may be a sensitive and non-invasive method to monitor brain damage in hypoxic-ischemic encephalopathy (HIE)10-15.


title: Olfactory sensory preconditioning in Drosophila: role of memory forgetting in gating S1/S2 associations

abstract: Learning and memory storage is a complex process that has proven challenging to tackle. It is likely that, in real nature, the instructive value of reinforcing experiences is acquired rather than innate. The association between seemingly neutral stimuli increases the gamut of possibilities to create meaningful associations and increases the predictive power of moment-by-moment experiences. Here we report physiological and behavioral evidence of olfactory unimodal sensory preconditioning in fruit flies. We show that the presentation of a pair of odors (S1 and S2) before one of them (S1) is associated with electric shocks elicits a conditional response not only to the trained odor (S1) but to the odor previously paired with it (S2). This occurs even if the S2 odor was never presented in contiguity with the aversive stimulus. In addition, we show that inhibition of the small G protein and known forgetting regulator Rac1 facilitates the association between S1/S2 odors. These results indicate that flies can infer value to non-paired odor based on the previous associative structure between odors, and inhibition of Rac1 lengthens the time of olfactory "sensory buffer," allowing linking of neutral odors presented in sequence.


title: TractoInferno: A large-scale, open-source, multi-site database for machine learning dMRI tractography

abstract: TractoInferno is the worlds largest open-source multi-site tractography database, including both research- and clinical-like human acquisitions, aimed specifically at machine learning tractography approaches and related ML algorithms. It provides 284 datasets acquired from 3T scanners across 6 different sites. Available data includes T1-weighted images, single-shell diffusion MRI (dMRI) acquisitions, spherical harmonics fitted to the dMRI signal, fiber ODFs, and reference streamlines for 30 delineated bundles generated using 4 tractography algorithms, as well as masks needed to run tractography algorithms. Manual quality control was additionally performed at multiple steps of the pipeline. We showcase TractoInferno by benchmarking the learn2track algorithm and 5 variations of the same recurrent neural network architecture. Creating the TractoInferno database required approximately 20,000 CPU-hours of processing power, 200 man-hours of manual QC, 3,000 GPU-hours of training baseline models, and 4 Tb of storage, to produce a final database of 350 Gb. By providing a standardized training dataset and evaluation protocol, TractoInferno is an excellent tool to address common issues in machine learning tractography.


title: A large-scale systematic survey of SARS-CoV-2 antibodies reveals recurring molecular features

abstract: In the past two years, the global research in combating COVID-19 pandemic has led to isolation and characterization of numerous human antibodies to the SARS-CoV-2 spike. This enormous collection of antibodies provides an unprecedented opportunity to study the antibody response to a single antigen. From mining information derived from 88 research publications and 13 patents, we have assembled a dataset of [~]8,000 human antibodies to the SARS-CoV-2 spike from >200 donors. Analysis of antibody targeting of different domains of the spike protein reveals a number of common (public) responses to SARS-CoV-2, exemplified via recurring IGHV/IGK(L)V pairs, CDR H3 sequences, IGHD usage, and somatic hypermutation. We further present a proof-of-concept for prediction of antigen specificity using deep learning to differentiate sequences of antibodies to SARS-CoV-2 spike and to influenza hemagglutinin. Overall, this study not only provides an informative resource for antibody and vaccine research, but fundamentally advances our molecular understanding of public antibody responses to a viral pathogen.


title: The RNA helicase DDX6 controls early mouse embryogenesis by repressing aberrant inhibition of BMP signaling through miRNA-mediated gene silencing

abstract:
The evolutionarily conserved RNA helicase DDX6 is a central player of post-transcriptional regulation, but its role during embryogenesis remains elusive. We here demonstrated that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 KO mouse embryos and in vitro epiblast-like cell (EpiLC) induction system. Our study unveiled a great impact of DDX6-mediated RNA regulation on signaling pathways. Deletion of Ddx6 caused the aberrant transcriptional upregulation of the negative regulators of BMP signaling, which accompanied with enhanced Nodal signaling. Ddx6{bigtriangleup}/{bigtriangleup} pluripotent cells acquired higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal expression in the primitive streak likely promoted endoderm cell fate specification while inhibiting mesoderm development. We further clarified the mechanism how DDX6 regulates cell fate determination of pluripotent cells by genetically dissecting major DDX6 pathways: processing body (P-body) formation, translational repression, mRNA decay, and miRNA-mediated silencing. P-body-related functions were dispensable, but the miRNA pathway was essential for the DDX6 function. DDX6 may prevent aberrant transcriptional upregulation of the negative regulators of BMP signaling by repressing translation of certain transcription factors through the interaction with miRNA-induced silencing complexes (miRISCs). Overall, this delineates how DDX6 affects development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function.

Author summaryGene expression occurs through the two steps: transcription (DNA to RNA) and translation (RNA to protein). Cells have very sophisticated regulatory processes working on various levels for the accurate gene expression. Post-transcriptional regulation, which includes all RNA-related controls, is crucial because it enables fine-tuning and rapid alteration of gene expression. RNA- binding proteins and non-coding RNAs are the two main players of post-transcriptional regulation. DDX6, the subject of our study, is an RNA-binding protein, more specifically an RNA helicase, which can unwind or rearrange RNA secondary structures. Its diverse molecular and cellular functions have been reported, but its embryogenic role is unknown. Here, we describe DDX6 function during early mouse embryogenesis and the underlying mechanism using genetic methodology. DDX6 enables proper cell lineage specification of pluripotent stem cells by mainly regulating BMP signaling through miRNA-mediated gene silencing. As DDX6- mediated RNA regulation affected signaling pathways, the loss of Ddx6 had a wide impact on developmental processes from pluripotency to embryo patterning. In addition, we identified which DDX6 molecular function is essential during early embryogenesis by genetically dissecting its main pathways.

category: genetics


title: Benchmarking metagenomics classifiers on ancient viral DNA: a simulation study

abstract:
Owing to technological advances in ancient DNA, it is now possible to sequence viruses from the past to track down their origin and evolution. However, ancient DNA data is considerably more degraded and contaminated than modern data making the identification of ancient viral genomes particularly challenging. Several methods to characterise the modern microbiome (and, within this, the virome) have been developed; in particular, tools that assign sequenced reads to specific taxa in order to characterise the organisms present in a sample of interest. While these existing tools are routinely used in modern data, their performance when applied to ancient microbiome data to screen for ancient viruses remains unknown.

In this work, we conducted an extensive simulation study using public viral sequences to establish which tool is the most suitable to screen ancient samples for human DNA viruses. We compared the performance of four widely used classifiers, namely Centrifuge, Kraken2, DIAMOND and MetaPhlAn2, in correctly assigning sequencing reads to the corresponding viruses. To do so, we simulated reads by adding noise typical of ancient DNA to a set of publicly available human DNA viral sequences and to the human genome. We fragmented the DNA into different lengths, added sequencing error and C to T and G to A deamination substitutions at the read termini. Then we measured the resulting sensitivity and precision for all classifiers.

Across most simulations, more than 228 out of the 233 simulated viruses are recovered by Centrifuge, Kraken2 and DIAMOND, in contrast to MetaPhlAn2 which recovers only around one third. Overall, Centrifuge and Kraken2 have the best performance with the highest values of sensitivity and precision. We found that deamination damage has little impact on the performance of the classifiers, less than the sequencing error and the length of the reads. Since Centrifuge can handle short reads (in contrast to DIAMOND and Kraken2 with default settings) and since it achieves the highest sensitivity and precision at the species level across all the simulations performed, it is our recommended tool. Regardless of the tool used, our simulations indicate that, for ancient human studies, users should use strict filters to remove all reads of potential human origin. Finally, we recommend to verify which species are present in the database used, as it might happen that default databases lack sequences for viruses of interest.


title: Increased Brain Volumetric Measurement Precision from Multi-Site 3D T1-weighted 3T Magnetic Resonance Imaging by Correcting Geometric Distortions

abstract:
Magnetic resonance imaging (MRI) scanner-specific geometric distortions may contribute to scanner induced variability and decrease volumetric measurement precision for multi-site studies. The purpose of this study was to determine whether geometric distortion correction increases the precision of brain volumetric measurements in a multi-site multi-scanner study. Geometric distortion variation was quantified over a one-year period at 10 sites using the distortion fields estimated from monthly 3D T1-weighted MRI geometrical phantom scans. The variability of volume and distance measurements were quantified using synthetic volumes and a standard quantitative MRI (qMRI) phantom. The effects of geometric distortion corrections on MRI derived volumetric measurements of the human brain were assessed in two subjects scanned on each of the 10 MRI scanners and in 150 subjects with cerebrovascaular disease (CVD) acquired across imaging sites.

Geometric distortions were found to vary substantially between different MRI scanners but were relatively stable on each scanner over a one-year interval. Geometric distortions varied spatially, increasing in severity with distance from the magnet isocenter. In measurements made with the qMRI phantom, the geometric distortion correction decreased the standard deviation of volumetric assessments by 35% and distance measurements by 42%. The average coefficient of variance decreased by 16% in gray matter and white matter volume estimates in the two subjects scanned on the 10 MRI scanners. Geometric distortion correction using an up-to-date correction field is recommended to increase precision in volumetric measurements made from MRI images.


title: Positive feedback promotes terrestrial emergence behaviour in an amphibious fish

abstract: Major ecological transitions such as the invasion of land by aquatic vertebrates have been hypothesised to be facilitated by positive feedback between habitat choice and phenotypic plasticity. We tested whether aquatic hypoxia, emergence behaviour, and plastic changes in gill surface area could create such a positive feedback loop and drive an amphibious fish to spend increasing amounts of time out of water. We found that terrestrially acclimated amphibious mangrove rivulus Kryptolebias marmoratus were more sensitive to, and less tolerant of, aquatic hypoxia relative to water-acclimated fish, which are necessary trade-offs for positive feedback to occur. Next, we acclimated fish to normoxic or hypoxic water with the opportunity to emerge for 7d to test the predictions that fish in hypoxic conditions should regularly leave water, reduce gill surface area, and become less hypoxia tolerant. Consistent with these predictions, fish in severe hypoxia spent almost 50% of the time out of water and coverage of the gill lamellae by an inter-lamellar cell mass almost doubled. Hypoxia acclimated fish were also more sensitive to acute aquatic hypoxia (emergence at higher oxygen levels), and lost equilibrium faster in hypoxic water compared to control fish. Thus, we show that a positive feedback loop develops in amphibious fish where emergence behaviour begets further emergence behaviour, driven by gill remodelling which reduces aquatic respiratory function. Such a scenario may explain how amphibious behaviour has repeatedly evolved in fishes that occupy hypoxic aquatic habitats despite the associated challenges of life on land.

category: physiology