title: Bayesian modeling and simulation to inform rare disease drug development early decision-making: application to Duchenne muscular dystrophy

abstract: Rare disease clinical trials are constrained to small sample sizes and may lack placebo-control, leading to challenges in drug development. This paper proposes a Bayesian model-based framework for early go/no-go decision making in rare disease drug development, using Duchenne muscular dystrophy (DMD) as an example. Early go/no-go decisions were based on projections of long-term functional outcomes from a Bayesian model-based analysis of short-term trial data informed by prior knowledge based on 6MWT natural history literature data in DMD patients. Frequentist hypothesis tests were also applied as a reference analysis method. A number of combinations of hypothetical trial designs, drug effects and cohort comparison methods were assessed.The proposed Bayesian model-based framework was superior to the frequentist method for making go/no-go decisions across all trial designs and cohort comparison methods in DMD. The average decision accuracy rates across all trial designs for the Bayesian and frequentist analysis methods were 45.8 and 8.98%, respectively. A decision accuracy rate of at least 50% was achieved for 42 and 7% of the trial designs under the Bayesian and frequentist analysis methods, respectively. The frequentist method was limited to the short-term trial data only, while the Bayesian methods were informed with both the short-term data and prior information. The specific results of the DMD case study were limited due to incomplete specification of individual-specific covariates in the natural history literature data and should be reevaluated using a full natural history dataset. These limitations aside, the framework presented provides a proof of concept for the utility of Bayesian model-based methods for decision making in rare disease trials.


title: The epoxyeicosatrienoic pathway is intact in endothelial and smooth muscle cells exposed to aldosterone excess

abstract:
ObjectivesEndothelial dysfunction (ED) is considered to be a major driver of the increased incidence of cardiovascular disease in primary aldosteronism (PA). Whether the epoxyeicosatrienoic acid (EET) pathway, involving the release of beneficial endothelium-derived lipid mediators, contributes to ED in PA is unknown. Preclinical evidence suggests this pathway to be relevant in the pathogenesis in various models of experimental hypertension. In addition, an orally available soluble epoxide hydrolase inhibitor, which halts the breakdown of EETs, has already passed a phase 1 clinical trial.

We, therefore, exposed primary human coronary artery endothelial cells to 1 nM aldosterone.

MethodsWe used qPCR to investigate changes in the expression levels of essential genes for the synthesis and degradation of EETs as well as mass spectrometry to determine endothelial synthetic capacity to release EETs upon stimulation. We also assessed primary human coronary artery smooth muscle cells for expression of putative EET receptor ion channels or downstream mediators as well as for the calcium response to EETs using calcium imaging.

ResultsNo major aldosterone-related expression changes were detected on the endothelial as well as the smooth muscle side. Stimulated release of endothelial EETs was unaffected. Likewise, the smooth muscle calcium response was unchanged after aldosterone excess.

ConclusionsThe EET pathway is not negatively affected by increased aldosterone concentrations as seen in PA. Modulating the EET pathway with therapeutic intent in patients with PA might therefore be assessed in future preclinical and clinical trials to address ED.

category: physiology


title: Small in size, big on taste: metabolomics analysis of flavor compounds from Philippine garlic

abstract: Philippine garlic (Allium sativum L.) is arguably known to pack flavor and aroma in smaller bulbs compared to imported varieties saturating the local market. In this study, ethanolic extracts of Philippine garlic cultivars were profiled using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF). {gamma}-Glu dipeptides, oligosaccharides and lipids were determined in Philippine garlic cultivars through bioinformatics analysis in GNPS Molecular Networking Platform and fragmentation analysis. Multivariate statistical analysis using XCMS Online showed the abundance of {gamma}-Glu allyl cysteine in Batanes-sourced garlic while {gamma}-Glu propenyl cysteine, {gamma}-Glu methyl cysteine, and alliin are enriched in the Ilocos cultivar. Principal component analysis showed that the {gamma}-Glu dipeptides found in local garlic influenced their distinct separation across PC1 from imported varieties. This presence of high levels of {gamma}-Glu dipeptides and probiotic oligosaccharides may potentially contribute to the superior flavor and nutritional benefits of Philippine garlic.

category: systems biology


title: What The Brain Does As We Speak

abstract: AO_SCPLOWBSTRACTC_SCPLOWLanguage is a defining human behavior and is dependent on networks interactions amongst broadly distributed neuronal substrates. Transient dynamics between language regions that underpin speech production have long been postulated, yet have proven challenging to evaluate empirically. We used direct intracranial recordings during single word production to create a finely resolved spatiotemporal atlas (134 patients, 25810 electrodes, 40278 words) of the entire language-dominant cortex and used this to derive single-trial state-space sequences of network motifs. We derived 5 discrete neural states during the production of each word, distinguished by unique patterns of distributed cortical interaction. This interactive model was significantly better than a model of the same design but lacking interactions between regions in explaining observed activity. Our results eschew strict functional attribution to localized cortical populations, supporting instead the idea that cognitive processes are better explained by distributed metastable network states.

category: neuroscience


title: A nested cortical hierarchy of neural states underlies event segmentation in the human brain

abstract: A fundamental aspect of human experience is that it is segmented into discrete events. This may be underpinned by transitions between distinct neural states. Using an innovative data-driven state segmentation method, we investigate how neural states are organized across the cortical hierarchy and where in the cortex neural state boundaries and perceived event boundaries overlap. Our results show that neural state boundaries are organized in a temporal cortical hierarchy, with short states in primary sensory regions, and long states in lateral and medial prefrontal cortex. State boundaries are shared within and between groups of brain regions that resemble well-known functional networks. Perceived event boundaries overlap with neural state boundaries across large parts of the cortical hierarchy, particularly when those state boundaries demarcate a strong transition or are shared between brain regions. Taken together, these findings suggest that a partially nested cortical hierarchy of neural states forms the basis of event segmentation.


title: A partially nested cortical hierarchy of neural states underlies event segmentation in the human brain


title: Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-inflammatory Cytokines

abstract:
BackgroundMicroglia safeguard the CNS against injuries and pathogens by inducing an inflammatory response. When exposed to anti-inflammatory cytokines, these cells possess the ability to switch from an inflammatory to an immunosuppressive phenotype. Cancer cells exploit this property to evade the immune system, and elicit an anti-inflammatory microenvironment that facilitates tumor attachment and growth.

ObjectiveThe tumor-supportive biological processes that are activated in microglia cells in response to anti-inflammatory cytokines released from cancer cells were explored with mass spectrometry and proteomic technologies.

MethodsSerum-depleted and non-depleted human microglia cells (HMC3) were treated with a cocktail of IL-4, IL-13, IL-10, TGF{beta}, and CCL2. The cellular protein extracts were analyzed by LC-MS/MS. Using functional annotation clustering tools, statistically significant proteins that displayed a change in abundance between cytokine-treated and non-treated cells were mapped to their biological networks and pathways.

ResultsThe proteomic analysis of HMC3 cells enabled the identification of [~]10,000 proteins. Stimulation with anti-inflammatory cytokines resulted in the activation of distinct, yet integrated clusters of proteins that trigger downstream a number of tumor-promoting biological processes. The observed changes could be classified into four major categories, i.e., mitochondrial gene expression, ECM remodeling, immune response, and impaired cell cycle progression. Intracellular immune activation was mediated mainly by the transducers of MAPK, STAT, TGF{beta}, NFKB, and integrin signaling pathways. Abundant collagen formation along with the expression of additional receptors, matrix components, growth factors, proteases and protease inhibitors, enabled ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that correlated ECM reorganization with cytoskeletal re-arrangements supportive of cell migration. Antigen processing/presentation was represented by HLA class I histocompatibility antigens, and correlated with upregulated proteasomal subunits, and vesicular/viral transport and secretory processes. Immunosuppressive and proangiogenic chemokines were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors and ECM proteins indicated the presence of such capabilities.

ConclusionsComprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a microglia phenotype that provides novel insights into the tumor microenvironment-driven mechanisms that fuel cancer development in the brain.

category: immunology


title: Morphogenetic coupling leads to pattern emergence in the pre somitic mesoderm

abstract: As tissues elongate, cell rearrangement alters positional information in manner that must be accounted for to generate gene expression pattern. How this is achieved during paraxial mesoderm elongation is unknown. By reverse-engineering gene regulatory networks that predict single cell expression trajectories across the tissue, we find a network capable of recapitulating the full range of dynamic differentiation profiles observed both in vivo and in vitro. Simulating gene expression profiles on in toto cell tracking data sets reveal that temporal exposure to Wnt and FGF is generated by cell movement. The absence of reversal in gene expression towards a more premature gene expression state predicts the generation of aberrant tbx6 expression in the posterior progenitor zone that we then confirm by quantitative single cell imaging. Taken together, these results demonstrate cell rearrangement tunes the dynamics of mesoderm progenitor differentiation to generate pattern emergence as a function of temporal Wnt and FGF exposure.

category: developmental biology


title: Cell Rearrangement Generates Pattern Emergence as a Function of Temporal Morphogen Exposure.


title: The impacts of hydropower on freshwater macroinvertebrate richness: A global meta-analysis

abstract: Hydroelectric dams and their reservoirs have been suggested to affect freshwater biodiversity. However, studies investigating the consequences of hydroelectric dams and reservoirs on macroinvertebrate richness have reached opposite conclusions. We carried out a meta-analysis devised to elucidate the effects of hydropower dams and their reservoirs on macroinvertebrates richness while accounting for the potential role played by moderators such as biomes, impact types, study designs, sampling seasons and gears. We used a random and mixed effect model, combined with robust variance estimation, to conduct the meta-analysis on 72 pairs of observations (i.e., impacted versus reference) extracted from 17 studies (more than one observation per study). We observed a large range of effect sizes, from very negative to very positive impacts of hydropower. However, according to this meta-analysis, hydropower dams and their reservoirs did not have an overall clear, directional and statistically significant effect on macroinvertebrate richness. We tried to account for the large variability in effect sizes using moderators, but none of the moderators included in the meta-analysis had statistically significant effect. This suggests that some other moderators, which were unavailable for the 17 studies included in this meta-analysis, might be important (e.g., temperature, granulometry, wave disturbance and macrophytes) and that macroinvertebrate richness may be driven by local, smaller scale processes. As new studies become available, it would be interesting to keep enriching this meta-analysis, as well as collecting local habitat variables, to see if we could finally draw statistically significant conclusions about the impacts of hydropower on macroinvertebrate richness.

category: ecology


title: Evolution and connectivity influence the persistence and recovery of coral reefs under climate change in the Caribbean, Southwest Pacific, and Coral Triangle

abstract: Corals are experiencing unprecedented decline from climate change-induced mass bleaching events. Dispersal not only contributes to coral reef persistence through demographic rescue but can also hinder or facilitate evolutionary adaptation. Locations of reefs that are likely to survive future warming therefore remain largely unknown, particularly within the context of both ecological and evolutionary processes across complex seascapes that differ in temperature range, strength of connectivity, network size, and other characteristics. Here, we used eco-evolutionary simulations to examine coral adaptation to warming across reef networks in the Caribbean, the Southwest Pacific, and the Coral Triangle. We assessed the factors associated with coral persistence in multiple reef systems to understand which results are general and which are sensitive to particular geographic contexts. We found that evolution can be critical in preventing extinction and facilitating the long-term recovery of coral communities in all regions. Furthermore, the strength of immigration to a reef (destination strength) and current sea surface temperature robustly predicted reef persistence across all reef networks and across temperature projections. However, we found higher initial coral cover, slower recovery, and more evolutionary lag in the Coral Triangle, which has a greater number of reefs and more larval settlement than the other regions. We also found the lowest projected future coral cover in the Caribbean. These findings suggest that coral reef persistence depends on ecology, evolution, and habitat network characteristics, and that, under an emissions stabilization scenario (RCP 4.5), recovery may be possible over multiple centuries.


title: TRNP1 sequence, function and regulation co-evolve with cortical folding in mammals

abstract: Genomes can be seen as notebooks of evolution that contain unique information on successful genetic experiments (Wright 2001). This allows to identify conserved genomic sequences (Zoonomia Consortium 2020) and is very useful e.g. for finding disease-associated variants (Kircher et al. 2014). Additional information from genome comparisons across species can be leveraged when considering phenotypic variance across species. Here, we exemplify this principle in a cross-species association study by testing whether structural or regulatory changes in TRNP1 correlate with changes in brain size and cortical folding in mammals. We find that the rate of TRNP1 protein evolution ({omega}) correlates best with the rate of cortical folding and that TRNP1 proteins from species with more cortical folding induce higher proliferation rates in neural stem cells from murine cerebral cortex. Furthermore, we compare the activity of putative cis-regulatory elements of TRNP1 in a massively parallel reporter assay and identify one element that correlates with cortical folding in Old World Monkeys and Apes. Our analyses indicate that coding and regulatory changes in TRNP1 modulated its activity to adjust cortical folding during mammalian evolution and exemplify how evolutionary information can be leveraged by cross-species association studies.

category: evolutionary biology


title: Ancient genomes reveal hybridisation between extinct short-faced bears and the extant spectacled bear (Tremarctos ornatus)

abstract: Two genera and multiple species of short-faced bear from the Americas went extinct during or toward the end of the Pleistocene, and all belonged to the endemic New World subfamily Tremarctinae [1-7]. Two of these species were giants, growing in excess of 1,000 kg [6, 8, 9], but it remains uncertain how these extinct bears were related to the sole surviving short-faced bear: the spectacled bear (Tremarctos ornatus). Ancient mitochondrial DNA has recently suggested phylogenetic relationships among these lineages that conflict with interpretations based on morphology [1, 10-12]. However, widespread hybridisation and incomplete lineage sorting among extant bears mean that the mitochondrial phylogeny frequently does not reflect the true species tree [13, 14]. Here we present ancient nuclear genome sequences from representatives of the two extinct short-faced bear genera, Arctotherium and Arctodus. Our new data support a third hypothesis for the relationships among short-faced bears, which conflicts with existing mitochondrial and morphological data. Based on genome-wide D-statistics, we suggest that the extant spectacled bear derives substantial ancestry from Pleistocene hybridisation with an extinct short-faced bear lineage, resulting in a discordant phylogenetic signal between the mitochondrion and portions of the nuclear genome.


title: Identification of Novel Genetic Susceptibility Loci for Thoracic and Abdominal Aortic Aneurysms Via Genome-Wide Association Study Using the UK Biobank Cohort

abstract:
BackgroundThoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) are known to have a strong genetic component.

Methods and ResultsIn a genome-wide association study (GWAS) using the UK Biobank, we analyzed the genomes of 1,363 individuals with AAA compared to 27,260 age, ancestry, and sex-matched controls (1:20 case:control study design). A similar analysis was repeated for 435 individuals with TAA compared to 8,700 controls. Polymorphism with minor allele frequency (MAF) >0.5% were evaluated.

We identified novel loci near LINC01021, ATOH8 and JAK2 genes that achieved genome-wide significance for AAA (p-value <5x10-8), in addition to three known loci. For TAA, three novel loci in CTNNA3, FRMD6 and MBP achieved genome-wide significance. There was no overlap in the genes associated with AAAs and TAAs. Additionally, we identified a linkage group of high-frequency variants (MAFs [~]10%) encompassing FBN1, the causal gene for Marfan syndrome, which was associated with TAA. In Finngen PheWeb, this FBN1 haplotype was associated with aortic dissection. Finally, we found that baseline bradycardia was associated with TAA, but not AAA.

ConclusionsOur GWAS found that AAA and TAA were associated with distinct sets of genes, suggesting distinct underlying genetic architecture. We also found association between baseline bradycardia and TAA. These findings, including JAK2 association, offer plausible mechanistic and therapeutic insights. We also found a common FBN1 linkage group that is associated with TAA and aortic dissection in patients who do not have Marfan syndrome. These FBN1 variants suggest shared pathophysiology between Marfan disease and sporadic TAA.

Condensed AbstractIn genome-wide association study (GWAS) of thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) using UK Biobank database, we found 3 novel loci associated with TAA, and 3 novel loci associated AAA. We also found significant association between baseline bradycardia and TAA. These findings, including JAK2 association, offer plausible mechanistic and therapeutic insights. Additionally, we identified a common FBN1 linkage group associated with TAA in patients who do not have Marfan syndrome. In the FinnGen cohort, this haplotype is associated with aortic dissection. These results suggest a shared pathophysiology between Marfan disease and sporadic TAA.

Study LimitationsAs with any GWAS study, the discovery of novel loci associated with aortopathies does not prove functional causality, and the findings described herein needs to be validated by analysis of other databases, ideally in a patient population of more diverse genetic origins than the UK Biobank. The use of the ICD10 codes to classify disease carriers and noncarriers in a population cohort may not be the most accurate assessment of prevalence of aortopathies. The association between baseline bradycardia and TAA does not take into account the concurrent use of medications that may impact heart rate.

HighlightsO_LIIdentification of 3 novel AAA-associated loci near LINC01021, ATOH8 and JAK2 genes.
C_LIO_LIIdentification of 3 novel TAA-associated loci near CTNNA3, FRMD6 and MBP genes.
C_LIO_LIIdentification of a linkage group of common FBN1 variants associated with non-syndromic TAA in the UK Biobank and with aortic dissection in the FinnGen cohort, strengthening the evidence for a shared pathophysiology between Marfan disease and nonsyndromic aortopathy.
C_LIO_LIAssociation between baseline bradycardia and TAA but not AAA.
C_LI

category: genomics


title: Insights into Mus musculus subspecies population structure across Eurasia revealed by whole-genome sequence analysis

abstract: For more than 100 years, house mice (Mus musculus) have been used as a key animal model in biomedical research. House mice are genetically diverse, yet their genetic background at the global level has not been fully understood. Previous studies suggested that they originated in South Asia and diverged into three major subspecies almost simultaneously, approximately 350,000-500,000 years ago; however, they have spread across the world with the migration of modern humans in prehistoric and historic times ([~]10,000 years ago to present), and undergone secondary contact, which have complicated the genetic landscape of wild house mice. In this study, we sequenced the whole genomes of 98 wild house mice collected from Eurasia, particularly East Asia, Southeast Asia, and South Asia. We found that although wild house mice consist of three major genetic groups corresponding to the three major subspecies, individuals representing admixture between subspecies are much more ubiquitous than previously recognized. Furthermore, several samples showed an incongruent pattern of genealogies between mitochondrial and autosomal genomes. Using samples likely retaining the original genetic components of subspecies with least admixture, we estimated the pattern and timing of divergence among the subspecies. The results are important for understanding the genetic diversity of wild mice on a global level and the information will be particularly useful in future biomedical and evolutionary studies using laboratory mice established from these wild mice.


title: Alternative architecture of the E. coli chemosensory array

abstract: Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

category: biochemistry


title: 13C tracer analysis identifies extensive recycling of endogenous CO2 in vivo

abstract: 13C tracing analysis is increasingly used to monitor cellular metabolism in vivo and in intact cells, but data interpretation is still the key element to unveil the complexity of metabolic activities. We have performed [U-13C]-glucose and [U-13C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). 13C enrichment of metabolites in cultured cells and tissues was determined by liquid chromatography coupled with high-resolution mass spectrometer (LC-HRMS). As expected, citrate M+2 or M+4 is the dominant mass isotopologue in vitro. However, citrate M+1 was unexpectedly the dominant isotopologue in mice receiving [U-13C]-glucose or [U-13C]-glutamine infusion. One plausible explanation is that 13CO2 produced from the oxidation of 13C tracers in vitro is negligible due to the dilution of HCO3 - supplemented to cell culture when sodium bicarbonante is used and diffusible volume of CO2 in the culture incubator, while endogenous 13CO2 in vivo is substantial and is fixed into the TCA cycle, purine, and serine, resulting in M+1 isotopologues. A time course study shows the generation of high abundance citrate M+1 early in plasma, which may serve as a potent non-invasive biomarker of tissue pyruvate carboxylase activity. Altogether, our results show that recycling of endogenous CO2 is substantial in vivo and provides important insights into the experimental design and data interpretation of 13C tracing assays.


title: Automatic Landmark Detection of Human Back Surface from Depth Images via Deep Learning

abstract:
Studying human postural structure is one of the challenging issues among scholars and physicians. The spine is known as the central axis of the body, and due to various genetic and environmental reasons, it could suffer from deformities that cause physical dysfunction and correspondingly reduce peoples quality of life. Radiography is the most common method for detecting these deformities and requires monitoring and follow-up until full treatment; however, it frequently exposes the patient to X-rays and ionization and as a result, cancer risk is increased in the patient and could be highly dangerous for children or pregnant women. To prevent this, several solutions have been proposed using topographic data analysis of the human back surface. The purpose of this research is to provide an entirely safe and non-invasive method to examine the spiral structure and its deformities. Hence, it is attempted to find the exact location of anatomical landmarks on the human back surface, which provides useful and practical information about the status of the human postural structure to the physician.

In this study, using Microsoft Kinect sensor, the depth images from the human back surface of 105 people were recorded and, our proposed approach - Deep convolution neural network-was used as a model to estimate the location of anatomical landmarks. In network architecture, two learning processes, including landmark position and affinity between the two associated landmarks, are successively performed in two separate branches. This is a bottom-up approach; thus, the runtime complexity is considerably reduced, and then the resulting anatomical points are evaluated concerning manual landmarks marked by the operator as the benchmark. Our results showed that 86.9% of PDJ and 80% of PCK. According to the results, this study was more effective than other methods with more than thousands of training data.

category: bioengineering


title: Cryo-EM Structure of Bacterioferritin Nanocages Provides Insight into the Bio-mineralization of Ferritins

abstract: Iron is an essential element involved in various metabolic processes. The ferritin family of proteins forms nanocage assembly and are involved in iron oxidation, storage and mineralization. Although several structures of human ferritin and bacterioferritin subunits have been resolved, there is still no complete structure that shows both the trapped Fe-biomineral cluster along with the nanocage. Furthermore, whereas the mechanism of iron trafficking has been explained using various approaches, an atomic-level description of the pathway and the biomineralization that occurs inside the cavity are lacking. Here, we report three cryo-EM structures of different states of the Streptomyces coelicolor bacterioferritin nanocage (i.e., apo, holo) at 3.4 [A] to 4.6 [A] resolution and the subunit crystal structure at 2.6 [A] resolution. The holo forms show different stages of Fe-biomineral accumulation inside the nanocage and suggest the possibility of a different Fe biomineral accumulation process. The cryo-EM map shows connections between the Fe-biomineral cluster and residues such as Thr157 and Lys42 from the protein shell, which are involved in iron transport. Mutation and truncation of the bacterioferritin residues involved in these connections can significantly reduce iron binding as compared with wild type bacterioferritin. Moreover, S. coelicolor bacterioferritin binds to various DNA fragments, similar to Dps (DNA-binding protein from starved cells) proteins. Collectively, our results represent a prototype for the ferritin nanocage, revealing insight into its biomineralization and the potential channel for ferritin-associated iron trafficking.

category: biophysics


title: Mechanically Transduced Immunosorbent Assay To Measure Protein-Protein Interactions

abstract: Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that indicates binding. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. Here we use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.


title: SSNA1 stabilizes dynamic microtubules and detects microtubule damage

abstract: Sjogrens Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. We find that SSNA1 modulates all parameters of microtubule dynamic instability - slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue. SSNA1 accumulation on dynamic microtubule ends correlates with the growth rate slow-down. Furthermore, SSNA1 prevents catastrophe when soluble tubulin is removed or sequestered by Op18/Stathmin. Finally, SSNA1 detects spastin-induced damage and inhibits spastins severing activity. Therefore, SSNA1 is both a potent microtubule stabilizing protein and a sensor of microtubule damage; activities that likely underlie SSNA1s cellular functions.


title: Mosquito community composition shapes virus prevalence patterns along anthropogenic disturbance gradients

abstract:
Previously unknown pathogens often emerge from primary ecosystems, but there is little knowledge on the mechanism behind. Most studies analyzing the influence of land-use change on pathogen emergence focus on a single host-pathogen system and often observe contrary effects. We studied virus diversity and prevalence patterns in natural and disturbed ecosystems using a multi-host and multi-taxa approach.

We detected 331 viral sequences pertaining to 49 viruses of ten RNA-virus families. Highest host and virus diversity was observed in pristine and intermediately disturbed habitats. The majority of the viruses was detected with low prevalence. However, nine viruses were found frequently of which five viruses increased in prevalence from pristine to disturbed habitats, in congruence with the dilution effect hypothesis. Interestingly, the observed increased prevalence of these five viruses in disturbed habitats was not caused by higher host infection rates but by increased host abundance, an effect tentatively named abundance effect.

Impact statementOur data show that ecosystem disturbance can lead to a turnover in host community composition and that more individuals of a single species is a key driver of virus emergence.


title: Rampant tooth loss across 200 million years of frog evolution

abstract: Teeth have been broadly maintained across most clades of vertebrates but have been lost completely at least once in actinopterygian fishes and several times in amniotes. Using phenotypic data collected from over 500 genera via micro-computed tomography, we provide the first rigorous assessment of the evolutionary history of dentition across all major lineages of amphibians. We demonstrate that dentition is invariably present in caecilians and salamanders, but teeth have been lost completely more than 20 times in frogs, a much higher occurrence of edentulism than in any other vertebrate group. The repeated loss of teeth in anurans is associated with a specialized diet of small invertebrate prey as well as shortening of the lower jaw, but it is not correlated with a reduction in body size. Frogs provide an unparalleled opportunity for investigating the molecular and developmental mechanisms of convergent tooth loss on a large phylogenetic scale.


title: The Integrity of the Speciation Core Complex is necessary for centromeric binding and reproductive isolation in Drosophila

abstract: Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. This overexpression leads to mitotic defects, a misregulation in the expression of transposable elements and a decreased fertility. In this work, we describe a distinct six subunit Speciation Core Complex (SCC) containing HMR and LHR and analyse the effect of Hmr mutations on complex function and integrity. Our experiments suggest that HMR acts as a bridging factor between centromeric chromatin and pericentromeric heterochromatin, which is required for both its physiological function and its ability to cause hybrid male lethality.


title: Cancer cells adapt FAM134B-BiP complex mediated ER-phagy to survive hypoxic stress

abstract: In a tumor microenvironment cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER) which elicit unfolded protein response (UPR) as an adaptive mechanism. UPR activates autophagy enabling the degradation of misfolded/unfolded proteins. More recently, ER-specific autophagy has been implicated in the removal of damaged ER and restoration of ER-homeostasis. Our investigations reveal that during hypoxia induced ER-stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER-homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER-stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia activated proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. Our studies have further led to the identification of a pharmacological agent vitexin that disrupts FAM134B-BiP complex thereby inhibits ER-phagy and suppresses breast cancer progression in vivo.

category: cancer biology


title: Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

abstract: The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. While numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study informs a working model of inter-TMD rearrangement that drives mGluR function.


title: Effects of LSD are differentially modulated in arrestin knockout mice

abstract: Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, the hallucinogenic side-effects of psychedelics often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT2A serotonin receptor (5-HT2AR). 5-HT2AR activation stimulates Gq- and {beta}-arrestin-({beta}Arr) mediated signaling. To separate effects of these signaling modes, we have used {beta}Arr1 and {beta}Arr2 mice. We find that LSD stimulates motor activities to similar extents in WT and {beta}Arr1-KO mice, with non-significant effects in {beta}Arr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose poking in WT and {beta}Arr1-KO animals. In contrast, LSD only slightly stimulates head twitches in {beta}Arr2-KO mice, without effects on retrograde walking or nose poking. The 5-HT2AR antagonist MDL100907 (MDL) blocks these LSD effects. LSD also disrupts prepulse inhibition (PPI) in WT and {beta}Arr1-KOs; PPI is unaffected in {beta}Arr2-KOs. MDL restores PPI in WT mice, but this antagonist is without effect and haloperidol is required in {beta}Arr1-KOs. LSD produces a biphasic body-temperature response in WT mice, a monophasic response in {beta}Arr1-KOs, and is without effect in {beta}Arr2 mutants. Both MDL and the 5-HT1AR antagonist, WAY 100635 (WAY), block the effects of LSD on body temperatures in WT mice, whereas WAY is effective in {beta}Arr1-KOs. Collectively, these results reveal that LSD produces diverse behavioral effects through {beta}Arr1 and {beta}Arr2, and that LSDs psychedelic drug-like actions appear to require {beta}Arr2.


title: Macroscopic quantities of collective brain activity during wakefulness and anesthesia

abstract: The study of states of arousal is key to understand the principles of consciousness. Yet, how different brain states emerge from the collective activity of brain regions remains unknown. Here, we studied the fMRI brain activity of monkeys during wakefulness and anesthesia-induced loss of consciousness. Using maximum entropy models, we derived collective, macroscopic properties that quantify the systems capabilities to produce work, to contain information and to transmit it, and that indicate a phase transition from critical awake dynamics to supercritical anesthetized states. Moreover, information-theoretic measures identified those parameters that impacted the most the network dynamics. We found that changes in brain state and in state of consciousness primarily depended on changes in network couplings of insular, cingulate, and parietal cortices. Our findings suggest that the brain state transition underlying the loss of consciousness is predominantly driven by the uncoupling of specific brain regions from the rest of the network.


title: LAMP3 is critical for surfactant homeostasis in mice

abstract: Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.


title: waveRAPID(R) - a robust assay for high-throughput kinetic screens with the Creoptix(R) WAVEsystem

abstract:
Surface-based biophysical methods for measuring binding kinetics of molecular interactions, such as Surface Plasmon Resonance (SPR) or Grating-Coupled Interferometry (GCI), are now well established and widely used in drug discovery. Increasing throughput is an often-cited need in the drug discovery process, and this has been achieved with new instrument generations where multiple interactions are measured in parallel, shortening the total measurement times and enabling new application areas within the field. Here, we present the development of a novel technology called waveRAPID for a further - up to ten-fold - increase in throughput, consisting of an injection method using a single sample. Instead of sequentially injecting increasing analyte concentrations for constant durations, the analyte is injected at a single concentration in short pulses of increasing durations. A major advantage of the new method is its ability to determine kinetics from a single well of a micro-titer plate, making it uniquely suitable for kinetic screening. We present the fundamentals of this approach using a small molecule model system for experimental validation and comparing kinetic parameters to traditional methods. By varying experimental conditions, we furthermore assess the robustness of this new technique.

Finally, we discuss its potential for improving hit quality and shortening cycle times in the areas of fragment screening, low molecule weight compound screening, and hit-to-lead optimization.


title: In vivo magnetic resonance spectroscopy of hyperpolarized  pyruvate in a male guinea pig model of life-long Western diet consumption and non-alcoholic fatty liver disease development

abstract:
BACKGROUNDAlterations in glycolysis and oxidative pathways are central to the increasing incidence of non-alcoholic fatty liver disease (NAFLD), highlighting a need for in vivo, non-invasive technologies to understand the development of hepatic metabolic aberrations in lean NAFLD.

PURPOSE/HYPOTHESISTo use hyperpolarized magnetic resonance spectroscopy (MRS) and proton density fat fraction (PDFF) MRI techniques to investigate effects of a chronic, life-long exposure to the Western Diet (WD) in a model of NAFLD and identify cellular metabolite changes and correlations related to enzyme activity. It is hypothesized that exposure to the WD will result in NAFLD in association with altered pyruvate metabolism.

STUDY TYPEProspective POPULATION/SUBJECTS/PHANTOM/SPECIMEN/ANIMAL MODEL: 28 male guinea pigs were weaned onto a control diet or WD.

FIELD STRENGTH/SEQUENCE3T; T1, T2, IDEAL, broadband PRESS MRS.

ASSESSMENTMedian PDFF was calculated in the liver and hind limbs. [1-13C]pyruvate dynamic MRS in the liver was quantified by the time to peak (TTP), calculated as the time from pyruvate peak to metabolite peak. After a recovery period, animals were euthanized, and tissue was analyzed for lipid and cholesterol concentration and enzyme level and activity.

STATISTICAL TESTSUnpaired Students t-tests were used to determine differences in measurements between the two diet groups. The Pearson correlation coefficient was calculated to determine correlations between measurements.

RESULTSLife-long WD consumption resulted in significantly higher liver PDFF correlated with elevated triglyceride content in the liver. The WD group exhibited a decreased TTP for lactate production, and ex vivo analysis highlighted increased liver lactate dehydrogenase (LDH) activity. DATA CONCLUSION: PDFF MRI results suggest differential fat deposition patterns occurring in animals fed a life-long WD, corresponding with increased liver triglyceride levels characteristic of lean NAFLD. The decreased liver lactate TTP and increased ex vivo LDH activity suggest lipid accumulation occurs in association with a shift from oxidative metabolism to anaerobic glycolytic metabolism in WD livers.


title: Active microrheology using pulsed optical tweezers to probe viscoelasticity of Lamin A towards diagnosis of laminopathies

abstract:
Lamins are nucleoskeletal proteins of mammalian cells that stabilize the structure and maintain the rigidity of the nucleus. These type V intermediate filament proteins which are predominantly of A and B types provide necessary tensile strength to the nucleus. Single amino acid missense mutations occurring all over the lamin A protein form a cluster of human diseases termed as laminopathies, a few of which principally affect the muscle and cardiac tissues responsible for load bearing functionalities of the body. One such mutation is lamin A350P which causes dilated cardiomyopathy in patients. It is likely that a change from alanine to proline in the -helical 2B rod domain of the protein might severely disrupt the propensity of the filaments to polymerise into functional higher order structures required to form a fully functional lamina with its characteristic elasticity. In this study, we validate for the very first time, the application of active microrheology employing oscillating optical tweezers to investigate any alterations in the visco-elastic parameters of the mutant protein meshwork in vitro, which might translate into possible changes in nuclear plasticity. We confirm our findings from this robust yet fast method by imaging both the wild type and mutant lamins using a super resolution microscope, and observe changes in the mesh size which explain our measured changes in the viscoelastic parameters of the lamins. This method could naturally be extended to conduct microrheological measurements on any intermediate filament protein or any protein endowed with elastic behavior, with minor schematic modifications, thus bearing significant implications in laminopathies and other diseases which are associated with changes in structural rigidity of any cellular organelle.

SignificanceLamin A mutations produce an array of diseases termed as laminopathies which are primarily characterized by alteration of elastic behavior of the nucleus which in turn leads to defects in mechanotransduction. This is the first report in the lamin arena which shows a fast, accurate and direct quantification of elastic moduli of lamin A using optical tweezers-based microrheology. This has very significant implications and can be registered to be a robust and universal method that could also be suitably used for probing changes in elastic properties of any proteins or surfactants in a disease scenario such as SARS-Cov2 (Covid-19), which is pandemic at this time.


title: A novel class of sulfur-containing aminolipids widespread in marine roseobacters

abstract: Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated SalA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modelling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. SalA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

category: microbiology


title: The glycosphingolipid inhibitor eliglustat inhibits autophagy in osteoclasts to increase bone mass and reduce myeloma bone disease

abstract:
Multiple myeloma (MM) is a fatal hematological malignancy, where the majority of patients are diagnosed with, or develop, destructive and debilitating osteolytic bone lesions. Current treatments for MM bone disease such as the bisphosphonate zoledronic acid can result in deleterious side effects at high doses. In this study, eliglustat, an FDA approved glycosphingolipid inhibitor, was shown to reduce MM bone disease in preclinical models of MM. Mechanistically, eliglustat alters the lipid composition and plasma membrane fluidity and acts as an autophagy flux inhibitor in bone-resorbing osteoclasts (OC). Autophagic degradation of the signaling molecule TRAF3 is key step in OC differentiation; this was prevented by eliglustat in OC precursors. In addition, eliglustat works depend on TRAF3 in vivo. Furthermore, the combination of eliglustat and zoledronic acid was found to have an additive effect to reduce MM bone disease, suggesting the potential for combination therapies that would allow for drug dose reductions. Taken together, this project identifies a novel mechanism in which glycosphingolipid inhibition reduces osteoclastogenesis via autophagy and highlights the translational potential of eliglustat for the treatment of bone loss disorders such as MM.

One Sentence SummaryTranslational use of eliglustat as an autophagy inhibitor to limit bone lesions in multiple myeloma.

category: cell biology


title: The differentiation of pluripotent stem cells towards transplantable endothelial progenitor cells

abstract: Endothelial progenitor cells (EPCs) and endothelial cells (ECs) have been applied in the clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, the lack of sufficient transplantable cells before the deterioration of disease condition is a current limitation to apply cell therapy in patients. It is necessary to differentiate pluripotent stem cells (PSCs) into EPCs and identify their characteristics. Comparing previously reported methods of human PSCs-derived ECs, we optimized a highly efficient differentiation protocol to obtain cells that match the phenotype of isolated EPCs from healthy donors. The protocol is compatible with chemically defined medium (CDM), it could produce a large number of clinically applicable cells with low cost. Moreover, we also found PSCs-derived EPCs express CD133, have some characteristics of mesenchymal stem cells and are capable of homing to repair blood vessels in zebrafish xenograft assays. In addition, we further revealed that IPAH PSCs-derived EPCs have higher expression of proliferation-related genes and lower expression of immune-related genes than normal EPCs and PSCs-derived EPCs through microarray analysis. In conclusion, we optimized a highly efficient differentiation protocol to obtain PSCs-derived EPCs with the phenotypic and molecular characteristics of EPCs from healthy donors which distinguished them from EPCs from PAH.


title: Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern

abstract: The leaf epidermis is a dynamic biomechanical shell that integrates growth across spatial scales to influence organ morphology. Pavement cells, the fundamental unit of this tissue, morph irreversibly into highly lobed cells that drive planar leaf expansion. Here we define how tissue-scale cell wall tensile forces and the microtubule-cellulose synthase systems pattern interdigitated growth in real-time. A morphologically potent subset of cortical microtubules span the periclinal and anticlinal cell faces to pattern cellulose fibers that generate a patch of anisotropic wall. The result is local polarized growth that is mechanically coupled to the adjacent cell via a pectin-rich middle lamella, and this drives lobe formation. Finite element pavement cell models revealed cell wall tensile stress as an upstream patterning element that links cell- and tissue-scale biomechanical parameters to interdigitated growth. Cell lobing in leaves is evolutionarily conserved, occurs in multiple cell types, and is associated with important agronomic traits. Our general mechanistic models of lobe formation provide a foundation to analyze the cellular basis of leaf morphology and function.


title: Genetic Analysis of Right Heart Structure and Function in 40,000 People

abstract: The heart evolved hundreds of millions of years ago. During mammalian evolution, the cardiovascular system developed with complete separation between pulmonary and systemic circulations incorporated into a single pump with chambers dedicated to each circulation. A lower pressure right heart chamber supplies deoxygenated blood to the lungs, while a high pressure left heart chamber supplies oxygenated blood to the rest of the body. Due to the complexity of morphogenic cardiac looping and septation required to form these two chambers, congenital heart diseases often involve maldevelopment of the evolutionarily recent right heart chamber. Additionally, some diseases predominantly affect structures of the right heart, including arrhythmogenic right ventricular cardiomyopathy (ARVC) and pulmonary hypertension. To gain insight into right heart structure and function, we fine-tuned deep learning models to recognize the right atrium, the right ventricle, and the pulmonary artery, and then used those models to measure right heart structures in over 40,000 individuals from the UK Biobank with magnetic resonance imaging. We found associations between these measurements and clinical disease including pulmonary hypertension and dilated cardiomyopathy. We then conducted genome-wide association studies, identifying 104 distinct loci associated with at least one right heart measurement. Several of these loci were found near genes previously linked with congenital heart disease, such as NKX2-5, TBX3, WNT9B, and GATA4. We also observed interesting commonalities and differences in association patterns at genetic loci linked with both right and left ventricular measurements. Finally, we found that a polygenic predictor of right ventricular end systolic volume was associated with incident dilated cardiomyopathy (HR 1.28 per standard deviation; P = 2.4E-10), and remained a significant predictor of disease even after accounting for a left ventricular polygenic score. Harnessing deep learning to perform large-scale cardiac phenotyping, our results yield insights into the genetic and clinical determinants of right heart structure and function.

category: genetics


title: Engineering Threshold-Based Selection Systems

abstract: Using model organisms to identify novel therapeutic targets is frequently constrained by pre-existing genetic toolkits. To expedite positive selection for identification of novel downstream effectors, we engineered conditional expression of activated CED-10/Rac to disrupt C. elegans embryonic morphogenesis, titrated to 100% lethality. The strategy of engineering thresholds for positive selection using experimental animals was validated with pharmacological and genetic suppression and is generalizable to diverse molecular processes and experimental systems.


title: Activity pulses induce spontaneous flow reversals in viscoelastic environments

abstract: Complex interactions between cellular systems and their surrounding extracellular matrices are emerging as important mechanical regulators of cell functions such as proliferation, motility, and cell death, and such cellular systems are often characterized by pulsating acto-myosin activities. Here, using an active gel model, we numerically explore the spontaneous flow generation by activity pulses in the presence of a viscoelastic medium. The results show that cross-talk between the activity-induced deformations of the viscoelastic surroundings with the time-dependent response of the active medium to these deformations can lead to the reversal of spontaneously generated active flows. We explain the mechanism behind this phenomenon based on the interaction between the active flow and the viscoelastic medium. We show the importance of relaxation timescales of both the polymers and the active particles and provide a phase-space over which such spontaneous flow reversals can be observed. Our results suggest new experiments investigating the role of controlled pulses of activity in living systems ensnared in complex mircoenvironments.


title: Expectation-Based Gist Facilitation: Rapid Scene Understanding and the Role of Top-Down Information

abstract: Scene meaning is processed rapidly, with  gist extracted even when presentation duration spans a few dozen milliseconds. This has led some to suggest a primacy of bottom-up information. However, gist research has typically relied on showing successions of unrelated scene images, contrary to our everyday experience in which the world unfolds around us in a predictable manner. Thus, we investigated whether top-down information - in the form of observers predictions of an upcoming scene - facilitates gist processing. Within each trial, participants (N=336) experienced a series of images, organised to represent an approach to a destination (e.g., walking down a sidewalk), followed by a final target scene either congruous or incongruous with the expected destination (e.g., a store interior or a bedroom). Over a series of behavioural experiments, we found that: appropriate expectations facilitated gist processing; inappropriate expectations interfered with gist processing; the effect of congruency was driven by provision of contextual information rather than the thematic coherence of approach images, and; expectation-based facilitation was most apparent when destination duration was most curtailed. We then investigated the neural correlates of predictability on scene processing using ERP (N=26). Congruency-related differences were found in a putative scene-selective ERP component, related to integrating visual properties (P2), and in later components related to contextual integration including semantic and syntactic coherence (N400 and P600, respectively). Taken together, these results suggest that in real-world situations, top-down predictions of an upcoming scene influence even the earliest stages of its processing, affecting both the integration of visual properties and meaning.


title: IL-1-driven stromal-neutrophil interaction in deep ulcers defines a pathotype of therapy non-responsive inflammatory bowel disease

abstract: Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology, and in situ localisation, we describe heterogeneity of the tissular inflammatory response in IBD treatment failure. Among inflammatory pathotypes, we found high neutrophil infiltration, activation of fibroblasts, and vascular remodelling at sites of deep ulceration was a feature of non-response to several anti-inflammatory therapies. Activated fibroblasts in the ulcer bed display neutrophil chemoattractant properties that are IL-1R- but not TNF-dependent. The identification of distinct, localised, tissular pathotypes associated with treatment non-response will aid precision targeting of current therapeutics and provide a biological rationale for IL-1 signalling blockade in ulcerating disease.


title: The cryo-EM structure of vesivirus 2117 highlights functional variations in entry pathways for viruses in different clades of the vesivirus genus.

abstract: Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the Caliciviridae, 2117 is classified within the Vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense RNA containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22 amino-acid insertion in the sequence of the FCV major capsid protein that forms a  cantilevered arm, which plays an important role in both receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the Vesivirus genus.


title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

abstract: Previous studies have shown that G9a, a lysine methyltransferase, inhibits autophagy by repressing the transcription of autophagy genes. Here, we demonstrate a novel mechanism whereby G9a/GLP inhibit autophagy through post-translational modification of ATG12, a protein critical for the initiation of autophagosome formation. Under non-stress conditions, G9a/GLP directly methylate ATG12. The methylated ATG12 undergoes ubiquitin-mediated protein degradation, thereby inhibiting autophagy induction. By contrast, under stress conditions that elevate intracellular Ca2+ levels, the activated calpain system cleaves the G9a/GLP proteins, leading to G9a/GLP protein degradation. The reduced G9a/GLP levels allow ATG12 to accumulate and form the ATG12-ATG5 conjugate, thus expediting autophagy initiation. Collectively, our findings reveal a distinct signaling pathway that links cellular stress responses involving Ca2+/calpain to G9a/GLP-mediated autophagy regulation. Moreover, our model proposes that the methylation status of ATG12 is a molecular rheostat that controls autophagy induction.

category: molecular biology


title: The nucleoid-associated protein Gbn binds to GATC sequences and affects sporulation and antibiotic production in Streptomyces

abstract:
Bacterial chromosome structure is organized by a diverse group of proteins collectively referred to as nucleoid-associated proteins (NAPs). Many NAPs have been well studied in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of Actinobacteria NAPs and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein was designated Gbn for GATC-binding NAP. Deletion of gbn led to alterations in development and antibiotic production in Streptomyces coelicolor. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2800 binding regions, encompassing some 3600 GATCWT motifs, which comprise 55% of all such motifs in the S. coelicolor genome. DNA binding of Gbn in vitro increased DNA stiffness but not compaction, suggesting a role in regulation rather than chromosome organization. Transcriptomics analysis showed that Gbn binding generally leads to reduced gene expression. The DNA binding profiles were nearly identical between vegetative and aerial growth. Exceptions are SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognises the rare leucine codon CUA, respectively, which nearly exclusively bound during vegetative growth. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes.

IMPORTANCEA large part of the chemical space of bioactive natural products is derived from Actinobacteria. Many of the biosynthetic gene clusters for these compounds are cryptic, in others words, they are expressed in nature but not in the laboratory. Understanding the global regulatory networks that control gene expression is key to the development of approaches to activate this biosynthetic potential. Chromosome structure has a major impact on the control of gene expression. In bacteria, the organization of chromosome structure is mediated by a diverse group of proteins referred to collectively as nucleoid-associated proteins (NAPs), which play an important role in the control of gene expression, nucleoid structure and DNA repair. We here present the discovery of a novel and extremely pleiotropic NAP, which we refer to as Gbn. Gbn is a sporulation-specific protein that occurs only in the Actinobacteria and binds to GATC sequences, with a subtle but broad effect on global gene expression. The discovery of Gbn is a new step towards better understanding of how gene expression and chromosome structure is governed in antibiotic-producing streptomycetes.


title: Systems-wide analysis of the GATC-binding nucleoid-associated protein Gbn and its impact on Streptomyces development


title: Repurposing of Synaptonemal Complex Proteins for Kinetochores in Kinetoplastida

abstract: Chromosome segregation in eukaryotes is driven by a macromolecular protein complex called the kinetochore that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. In contrast, unicellular flagellates of the class Kinetoplastida have a unique set of kinetochore components. The evolutionary origin and history of these kinetochores remains unknown. Here, we report evidence of homology between three kinetoplastid kinetochore proteins KKT16-18 and axial element components of the synaptonemal complex, such as the SYCP2:SYCP3 multimers found in vertebrates. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. Using a sensitive homology detection protocol, we identify divergent orthologues of SYCP2:SYCP3 in most eukaryotic supergroups including other experimentally established axial element components, such as Red1 and Rec10 in budding and fission yeast, and the ASY3:ASY4 multimers in land plants. These searches also identify KKT16-18 as part of this rapidly evolving protein family. The widespread presence of the SYCP2-3 gene family in extant eukaryotes suggests that the synaptonemal complex was likely present in the last eukaryotic common ancestor. We found at least twelve independent duplications of the SYCP2-3 gene family throughout the eukaryotic tree of life, providing opportunities for new functional complexes to arise, including KKT16-18 in Trypanosoma brucei. We propose that kinetoplastids evolved their unique kinetochore system by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.


title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

abstract: Mutations in the Cl-/H+ exchanger CLC7 and its subunit OSTM1 result in osteopetrosis, lysosomal disorders, and pigmentation defects in mice and humans. How CLC7/OSTM1 regulates pigmentation in skin and hair melanocytes remains unexplored. In human epidermal melanocytes, we found CLC7/OSTM1 localized to melanosomes, the organelles in which melanin is synthesized, where it negatively regulates melanin production. Using a novel ratiometric melanosomal pH indicator, we showed that CLC7 acidifies melanosomes, opposing the function of the oculocutaneous albinism II (OCA2) Cl- ion channel. The de novo CLC7 variant (CLC7-Y715C) that causes albinism in humans and mice, decreased melanocytes pigmentation, which was restored by coexpression of OCA2. Remarkably, the enlarged hyperacidic vacuoles caused by CLC7-Y715C were also rescued by OCA2 coexpression in both melanocytes and non-melanocytic cells. Our data uncover a novel mechanism by which CLC7 regulates melanocyte pigmentation and identifies OCA2 as a tool to counteract the effects of CLC7 activating mutations.


title: Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors

abstract:
Heterotrimeric G proteins constitute the primary transducers of G protein-coupled receptor (GPCR) signaling. Besides mediating ligand-induced GPCR activation, G proteins transduce basal levels of activity in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Several generations of optical biosensors were developed and optimized to monitor GPCR ligand-induced G protein activation, however, quantitative approaches to detect constitutively active GPCRs are not available. Here, we designed and validated a set of eight bioluminescence-resonance-energy-transfer (BRET)-based G protein sensors, covering all four major families of G proteins, and established a protocol to identify constitutive GPCR/G protein signaling in living cells. These sensors rely on the encoding of all three G protein subunits on a single plasmid, enabling their cellular expression at desired relative levels and resulting in reduced signal variability in mammalian cells. Based on this sensor platform, we further present here an experimental protocol to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will aid in the characterization of constitutively active GPCRs and the exploration of their role in health and disease.

One Sentence SummaryThis Resource article describes the validation of a biophysical approach to directly assess the constitutive signaling activity of G protein-coupled receptors through heterotrimeric G proteins in living cells using optical biosensors.


title: Characterization of a novel Fgf10CreERT2 knock-in mouse line targeting postnatal lung Fgf10 lineages

abstract: Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a Fgf10CreERT2 line (with the CreERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki-v1, to target Fgf10Pos cells. While this line allowed fairly efficient and specific labeling of Fgf10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus on our research. We report here the generation and validation of a new Fgf10CreERT2 (called thereafter Fgf10Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. This new Fgf10Ki-v2 line exhibited comparable Fgf10 expression level to their wild type counterpart. However, a disconnection between the Fgf10 and the Cre expression was observed in Fgf10Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki-v2 mice. Bio-informatics analysis shows that the 3UTR, where the CreERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Significantly, Fgf10Ki-v2 mouse is capable of significantly targeting Fgf10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki-v2 line opens the way for future mechanistic experiments involving the postnatal lung.


title: Strong transgenerational effects but no genetic adaptation in zooplankton 24 years after an abrupt +10°C climate change

abstract:
The climate is currently warming fast, threatening biodiversity all over the globe. Adaptation is often rapid when the environment changes quickly, but for climate warming very little evidence is available. Here, we investigate the pattern of adaptation to an extreme +10{degrees}C climate change in the wild, following the introduction of brine shrimp Artemia franciscana from San Francisco Bay, USA, to Vinh Chau saltern in Vietnam. We use a resurrection ecology approach, hatching diapause eggs from the ancestral population and the introduced population after 13 and 24 years (resp. ~54 and ~100 generations). In a series of coordinated experiments, we determined whether the introduced Artemia show increased tolerance to higher temperatures, and the extent to which genetic adaptation, developmental plasticity, transgenerational effects, and local microbiome differences contributed to this tolerance. We find that introduced brine shrimp do show increased phenotypic tolerance to warming. Yet strikingly, these changes do not have an additive genetic component, are not caused by mitochondrial genetic variation, and are not caused by epigenetic marks set by adult parents exposed to warming. Further, we do not find any developmental plasticity in response to warming, nor any protective effect of heat-tolerant local microbiota. We conclude that the evolution of shrimps extreme thermal tolerance is only due to transgenerational (great)grandparental effects, possibly epigenetic marks set by parents who were exposed to high temperatures as juveniles. This finding challenges standard models of genetic and plastic adaptive responses, and our conception of how species may cope with climate warming.

Significance statementAdaptation is often rapid when environments change quickly, but for climate warming little evidence is available. Many studies report no genetic responses due to pre-existing plasticity, while others point towards epigenetics and microbiota effects. In this study, we take advantage of a natural experiment to study all of these effects. We use a set of coordinated experiments and a  resurrection ecology approach, reviving resting eggs of brine shrimp up to100 generations after their introduction from a temperate to a tropical saltern. We find that heat adaptation occurs, but heritability is fully "missing". Plasticity and microbiota play no role either, indicating that only transgenerational (great)grandmaternal effects are involved. This finding prompts us to reconsider the relative importance of the different possible mechanisms by which phenotypic change can occur, especially in response to temperature variation.


title: Enhanced genetic analysis of type 1 diabetes by selecting variants on both effect size and significance, and by integration with autoimmune thyroid disease

abstract: For polygenic traits, associations with genetic variants can be detected over many chromosome regions, owing to the availability oflarge sample sizes. Most variants, however, have small effects on disease risk and, therefore, unravelling the causal variants, target genes, and biology of these variants is challenging. Here, we define the Bigger or False Discovery Rate (BFDR) as the probability that either a variant is a false-positive or a randomly drawn, true-positive association exceeds it in effect size. Using the BFDR, we identified 302 previously unreported signals with larger effect associations with type 1 diabetes and autoimmune thyroid disease. Out of 239 genome-wide significant signals in both diseases, only 66 (28%) show evidence for having a large effect using the BFDR, further demonstrating how using a combination of effect size and significance, rather than significance alone, is important in identifying SNPs and candidate genes for further investigation.


title: Alternative splice variants and germline polymorphisms in human immunoglobulin light chain genes

abstract: Immunoglobulin loci are rich in germline polymorphisms and identification of novel polymorphic variants can be facilitated by germline inference of B cell receptor repertoires. Germline gene inference is complicated by somatic hypermutations, errors arising from PCR amplification, and DNA sequencing as well as from the varying length of reference alleles. Inference of light chain genes is even more challenging than inference of heavy chain genes due to large gene duplication events on the kappa locus as well as absence of D genes in the rearranged light chain transcripts. Here, we analyzed the light chain cDNA sequences from naive BCR repertoires of a Norwegian cohort of 100 individuals. We optimized light chain allele inference by tweaking parameters within TIgGER functions, extending the germline reference sequences, and establishing mismatch frequency patterns at polymorphic positions to filter out false positive candidates. As a result, we identified 48 previously unreported variants of light chain variable genes. Altogether, we selected 14 candidates for novel light chain polymorphisms for validation and successfully validated 11 by Sanger sequencing. Additional clustering of light chain 5UTR, L-PART1 and L-PART2 revealed partial intron retention in alternative splice variants in 11 kappa and 9 lambda V alleles. The alternatively spliced transcripts were only observed in genes with low expression levels, suggesting a possible role in expression regulation. Our results provide novel insight into germline variation in human light chain immunoglobulin loci.


title: Analysis of mitochondrial genome methylation using Nanopore single-molecule sequencing

abstract: The level and the biological significance of mitochondrial DNA (mtDNA) methylation in human cells is a controversial topic. Using long-read third-generation sequencing technology, mtDNA methylation can be detected directly from the sequencing data, which overcomes previously suggested biases, introduced by bisulfite treatment-dependent methods. We investigated mtDNA from whole blood-derived DNA and established a workflow to detect CpG methylation with Nanopolish. In order to obtain native mtDNA, we adjusted a whole-genome sequencing protocol and performed ligation library preparation and Nanopore sequencing. To validate the workflow, 897bp of methylated and unmethylated synthetic DNA samples at different dilution ratios were sequenced and CpG methylation was detected. Interestingly, we observed that reads with higher methylation in the synthetic DNA did not pass Guppy calling, possibly affecting conclusions about DNA methylation in Nanopore sequencing. We detected in all blood-derived samples overall low-level methylation across the mitochondrial genome, with exceptions at certain CpG sites. Our results suggest that Nanopore sequencing is capable of detecting low-level mtDNA methylation. However, further refinement of the bioinformatical pipelines including Guppy failed reads are recommended.


title: Suppression of miR-199a-5p alleviates ulcerative colitis by upregulating endoplasmic reticulum stress component XBP1

abstract: Aims: This study aimed to explore the biological activities of miR-199a-5p in dextran sulphate sodium (DSS)-induced ulcerative colitis and apoptosis and identify the direct target of miR-199a-5p in this process. Main methods: HT-29 cells and C57BL/6 mice were used to examine the function of miR-199a-5p in vitro and in vivo, respectively. Expression of miRNA and mRNA was measured using quantitative real-time PCR and western blotting was used to measure the change in protein expression. Flow cytometry was subsequently employed to determine cell apoptosis, and a luciferase assay was used to confirm the direct target of miR-199a-5p. Results: Expression of miR-199a-5p was increased by DSS treatment in mice. In parallel, miR-199a-5p is found to be involved in endoplasmic reticulum stress (ERS) and cell apoptosis in HT-29 cells, and its upregulation induced ERS, apoptosis, weight loss, and ulcerative colitis in mice in vivo, which could be prevented by the suppression of miR-199a-5p. Luciferase assay confirmed that the 3' untranslated region (3'-UTR) of XBP1 is the target binding site of miR-199a-5p. Conclusion: miR-199a-5p promotes ulcerative colitis and cell apoptosis by targeting the 3'-UTR of XBP1. Our findings reveal a new regulatory mechanism for ERS signaling and suggest that miR-199a-5p might be a potential target for UC therapy.


title: CD8+ T cell immunogenicity induced by endogenous EVs engineered by antigens fused to a truncated Nefmut EV-anchoring protein

abstract:
Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicits a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective to a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. Furthermore, the EV-anchoring function was preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. By analyzing the immune responses induced after intramuscular injection of DNA vectors expressing fusion products based on the four viral antigens, we found that the Nefmut C-terminal deletion did not impact on the levels of antigen -specific CD8+ T lymphocytes as evaluated by IFN-{gamma} EliSpot analysis and intracellular cytokine staining. In addition, immune responses at distal sites remained unaffected, as indicated by the similar percentages of SARS-CoV-2 S1- and S2-specific CD8+ T cells detected in spleens and lung airways of mice injected with DNA vectors expressing the viral antigens fused with either Nefmut or NefmutPL.

We concluded that the C-terminal Nefmut truncation does not affect stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, NefmutPL represents a safer alternative to full-length Nefmut for the design of CD8+ T cell vaccines for humans.


title: The Case for Retaining Natural Language Descriptions of Phenotypes in Plant Databases and a Web Application as Proof of Concept

abstract:
MotivationFinding similarity across phenotypic descriptions is not straightforward, with previous successes in computation requiring significant expert data curation. Natural language processing of free text phenotype descriptions is often easier to apply than intensive curation. It is therefore critical to understand the extent to which these techniques can be used to organize and analyze biological datasets and enable biological discoveries.

ResultsA wide variety of approaches from the natural language processing domain perform as well as similarity metrics over curated annotations for predicting shared phenotypes. These approaches also show promise both for helping curators organize and work through large datasets as well as for enabling researchers to explore relationships among available phenotype descriptions. Here we generate networks of phenotype similarity and share a web application for querying a dataset of associated plant genes using these text mining approaches. Example situations and species for which application of these techniques is most useful are discussed.

AvailabilityThe dataset used in this work is available at https://git.io/JTutQ. The code for the analysis performed here is available at https://git.io/JTutN and https://git.io/JTuqv. The code for the web application discussed here is available at https://git.io/Jtv9J, and the application itself is available at https://quoats.dill-picl.org/.

category: bioinformatics


title: Improving RNA branching predictions: advances and limitations

abstract: Minimum free energy prediction of RNA secondary structures is based on the Nearest Neighbor Thermodynamics Model. While such predictions are typically good, the accuracy can vary widely even for short sequences, and the branching thermodynamics are an important factor in this variance. Recently, the simplest model for multiloop energetics -- a linear function of the number of branches and unpaired nucleotides -- was found to be the best. Subsequently, a parametric analysis demonstrated that per family accuracy can be improved by changing the weightings in this linear function. However, the extent of improvement was not known due to the ad hoc method used to find the new parameters. Here we develop a branch-and-bound algorithm that finds the set of optimal parameters with the highest average accuracy for a given set of sequences. Our analysis shows that the previous ad hoc parameters are nearly optimal for tRNA and 5S rRNA sequences on both training and testing sets. Moreover, cross-family improvement is possible but more difficult because competing parameter regions favor different families. The results also indicate that restricting the unpaired nucleotide penalty to small values is warranted. This reduction makes analyzing longer sequences using the present techniques more feasible.


title: Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferative disease in mice via altered Runx1 splicing

abstract:
Acute myeloid leukemia (AML) is driven by numerous molecular events that contribute to disease progression. Herein, we identified hnRNP K overexpression as a recurrent abnormality in AML that is associated with inferior patient outcomes. In murine hematopoietic stem and progenitor cells, hnRNP K overexpression altered self-renewal and differentiation potential. Furthermore, murine transplantation models revealed that hnRNP K overexpression resulted in fatal myeloproliferative phenotypes. Using unbiased approaches, we discovered a direct relationship between hnRNP K and RUNX1--a master transcriptional regulator of hematopoiesis often dysregulated in leukemia. Molecular analyses revealed hnRNP K-dependent alternative splicing of RUNX1, resulting in the generation of a functionally distinct isoform. Taken together, we have established hnRNP K as an oncogene in myeloid leukemia that binds RUNX1 RNA, altering its splicing and subsequent transcriptional activity. These findings shed new light on a mechanism of myeloid leukemogenesis, paving the way for new drug discovery efforts.

HighlightsO_LIhnRNP K, an RNA binding protein, is overexpressed in AML and correlates with poor clinical outcomes
C_LIO_LIhnRNP K overexpression in murine HSPCs drives fatal myeloproliferative phenotypes in mice
C_LIO_LIhnRNP Ks oncogenicity can be attributed, at least in part, to its ability to bind and influence the splicing of the RUNX1 transcript
C_LI


title: Long-term antitumor CD8+ T cell immunity induced by endogenously engineered extracellular vesicles

abstract:
We developed a novel approach to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is an innovative vaccination approach employing a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) that has lost the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nefmut traffics large amounts of antigens fused to it into EVs spontaneously released by cells expressing the Nefmut_based DNA vector. We previously provided evidence that the inoculation in mice of a DNA vector expressing the Nefmut/HPV16-E7 fusion protein induced an E7-specific CTL immune response as detected 2 weeks after the second immunization. In an effort to optimize the anti-HPV16 CD8+ T cell immune response, we found that the co-injection of DNA vectors expressing Nefmut fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that we observed in mice injected with the single vectors. When TC-1 cells, i.e., a tumor cell line co-expressing E6 and E7, were implanted before immunization, all mice survived until day 44, whereas no mice injected with either void or Nefmut_expressing vectors survived until day 32 after tumor implantation. A substantial part of mice (7 out of 12) cleared the tumor. When cured mice were re-challenged with a second sub cute implantation of TC-1 cells, and followed for additional 135 days, whereas none of them developed tumors. Both E6- and E7-specific CD8+ T immunity was still detectable at the end of the observation time.

Hence, the immunity elicited by engineered EVs, besides curing already developed tumors, is strong enough to guarantee the resistance to additional tumor attack. This results is of relevance for therapy against both metastatic and relapsing tumors.


title: Larval nutrition impacts survival to adulthood, body size, and the allometric scaling of metabolic rate in adult honeybees

abstract:
Resting metabolic rate (RMR) is a fundamental physiological measure linked to numerous aspects of organismal function, including lifespan. Although dietary restriction in insects during larval growth/development affects adult RMR, the impact of larval diet quality on adult RMR has not been studied. Using in vitro rearing to control larval diet quality, we determined the effect of dietary protein and carbohydrate on honeybee survival-to-adulthood, time-to-eclosion, body mass/size and adult RMR. High carbohydrate larval diets increased survival-to-adulthood and time-to-eclosion compared to both low carbohydrate and high protein diets. Upon emergence, bees reared on the high protein diet were smaller and lighter than those reared on other diets, whilst those raised on the high carbohydrate diet varied more in body mass. Newly emerged adult bees reared on the high carbohydrate diet showed a significantly steeper increase in allometric scaling of RMR compared to those reared on other diets. This suggests that diet quality influences survival-to-adulthood, time-to-eclosion, and the allometric scaling of RMR. Given that agricultural intensification and increasing urbanisation have led to a decrease in both forage availability and dietary diversity for bees, our results are critical to improving understanding of the impacts of poor developmental nutrition on bee growth/development and physiology.

Summary statementWe show, for the first time, that the nutritional quality of insect larval diets affects the scaling of metabolic rate with body mass in newly emerged adult honeybees.

category: physiology


title: A Mathematical Model which Examines Age-Related Stochastic Fluctuations in DNA Maintenance Methylation

abstract: Due to its complexity and its ubiquitous nature the ageing process remains an enduring biological puzzle. Many molecular mechanisms and biochemical process have become synonymous with ageing. However, recent findings have pinpointed epigenetics as having a key role in ageing and healthspan. In particular age related changes to DNA methylation offer the possibility of monitoring the trajectory of biological ageing and could even be used to predict the onset of diseases such as cancer, Alzheimers disease and cardiovascular disease. At the molecular level emerging evidence strongly suggests the regulatory processes which govern DNA methylation are subject to intracellular stochasticity. It is challenging to fully understand the impact of stochasticity on DNA methylation levels at the molecular level experimentally. An ideal solution is to use mathematical models to capture the essence of the stochasticity and its outcomes. In this paper we present a novel stochastic model which accounts for specific methylation levels within a gene promoter. We quantify the uncertainty of the eventual cite-specific methylation levels for different values of methylation age, depending on the initial methylation levels. Our model predicts the observed bistable levels in CpG islands. In addition, simulations with various levels of noise indicate that uncertainty predominantly spreads through the hypermethylated region of stability, especially for large values of input noise. A key outcome of the model is that CpG islands with intermediate methylation levels tend to be more susceptible to dramatic DNA methylation changes towards both hypomethylation and hypermethylation, due to increasing methylation age.

category: systems biology


title: Osteomacs support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model

abstract: Osteal macrophages (osteomacs) support osteoblast function and promote bone anabolism, but their contribution to osteoporosis has not been explored. While mouse ovariectomy models have been repeatedly used, variation in strain, experimental design and assessment modalities, have contributed to no single model being confirmed as comprehensively replicating the full gamut of osteoporosis pathological manifestations. We validated an ovariectomy model in adult C3H/HeJ mice and demonstrated that it presents with human post-menopausal osteoporosis features, including reduced bone volume in axial and appendicular bone and bone loss in both trabecular and cortical bone including increased cortical porosity. Bone loss was associated with increased osteoclasts on trabecular and endocortical bone and decreased osteoblasts on trabecular bone. Importantly, this OVX model was characterised by delayed fracture healing. Using this validated model, we demonstrated that osteomacs are increased post-ovariectomy on both trabecular and endocortical bone. Dual F4/80 (pan-macrophage marker) and TRAP staining revealed osteomacs frequently located near TRAP+ osteoclasts and containing TRAP+ intracellular vesicles. Using an in vivo inducible macrophage depletion model that does not simultaneously deplete osteoclasts, we observed that osteomac loss was associated with elevated extracellular TRAP in bone marrow interstitium and increased serum TRAP. Using in vitro high-resolution confocal imaging of mixed osteoclast-macrophage cultures on bone substrate, we observed macrophages juxtaposed to osteoclast basolateral functional secretory domains scavenging degraded bone by-products. These data demonstrate a role for osteomacs in supporting osteoclastic bone resorption through phagocytosis and sequestration of resorption by-products. Finally, using Siglec1 knockout mice, we demonstrated that loss of the macrophage-restricted molecule Siglec-1/CD169 is sufficient to cause age-associated low bone mass, emphasizing the macrophages, independent of osteoclasts, contribute to optimal skeletal health. Overall, our data expose a novel role for osteomacs in supporting osteoclast function and provide the first evidence of their involvement in osteoporosis pathogenesis.


title: Tn7-CRISPR-Cas12K elements manage pathway choice using truncated repeat-spacer units to target tRNA attachment sites

abstract: CRISPR-Cas systems provide a defense against mobile elements. These defense systems have been naturally coopted multiple times for guide RNA-directed transposition by Tn7-like transposons. Elements associated with a type I-F CRISPR-Cas system categorize guide RNAs, maintaining a standard CRISPR array capable of acquiring new spacers targeting other mobile elements while maintaining a special guide RNA allowing integration into a conserved site in the chromosome called an attachment site. We show here that Tn7-like elements associated with a type V-K (Cas12K-based) system use a similar strategy to target diverse tRNA genes as attachment sites. These guides are encoded as truncated minimal repeat-spacer units and are found in distinct locations. Multiple pieces of information support that V-K guide RNAs are acquired using a type I-D adaptation system, but remain private to the V-K transposition process. This catalog of Cas12K elements and naturally occurring insertions will help future work engineering precision integration systems.