title: Quality-controlled R-loop meta-analysis reveals the characteristics of R-Loop consensus regions

abstract: R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA during transcription. While the pathological consequences of R-loops have been well-studied to date, the locations, classes, and dynamics of physiological R-loops remain poorly understood. R-loop mapping studies provide insight into R-loop dynamics, but their findings are challenging to generalize. This is due to the narrow biological scope of individual studies, the limitations of each mapping modality, and, in some cases, poor data quality. In this study, we reprocessed 693 R-loop mapping datasets from a wide array of biological conditions and mapping modalities. From this data resource, we developed an accurate method for R-loop data quality control, and we reveal the extent of poor-quality data within previously published studies. We then identified a set of high-confidence R-loop mapping samples and used them to define consensus R-loop sites called "R-loop regions" (RL regions). In the process, we revealed the stark divergence between S9.6 and dRNH-based R-loop mapping methods and identified biologically meaningful subtypes of both constitutive and variable R-loops. Taken together, this work provides a much-needed method to assess R-loop data quality and reveals intriguing aspects of R-loop biology.

category: bioinformatics


title: Elexacaftor/Tezacaftor/Ivacaftor alters branching morphogenesis of the mouse embryonic lung

abstract:
IntroductionCFTR modulators triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) has proven to clinically benefit homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of the potential impact of these modulators on the development of the foetus are mandatory.

MaterialsWe used the early mouse embryonic lung organ culture model to analyse ex vivo the lung branching process and the relative expression of Fgf10, Fgfr2IIIb, Shh, and Hhip development regulator genes in different conditions: standard culture medium, treatment with ETI or with Forskolin {+/-} Inh172. Development of lung branching and distal bud caliber were evaluated in lung explants from heterozygous F508del Cftrtm1Eur/+ and control Cftrtm1Eur+/+ (WT) mouse embryos at E12.5 during pseudo-glandular stage.

ResultsExposure to ETI of the Cftrtm1Eur/+ and WT lung explants induced a significant decrease in lung branching after 48h culture and the percentage of terminal bud dilations was significantly increased. These results were recapitulated by cAMP-dependent CFTR continuous activation by Forskolin and reversed by addition of Inh172.

ETI induced a significant decrease in Fgf10, Fgfr2IIIb, Shh and Hhip expression in lung explants of both E12.5 Cftrtm1Eur/+ and WT embryos treated with ETI for 72h.

ConclusionOur results provide evidence that the triple association Elexacaftor/Tezacaftor/Ivacaftor alters lung branching morphogenesis of WT and heterozygous F508del mouse embryos during the pseudo-glandular stage. Those results argue for a close monitoring of pregnancies in patients treated with these drugs.

Plain LanguageO_ST_ABSIntroductionC_ST_ABSThe triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) improves homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of this treatment on the development of the foetus are lacking. We incubated lungs of murine foetus not carrying CFTR mutation or F508del heterozygous. We show that ETI induces significant defect of lung development and the formation of cysts. These results are at least partly due to CFTR activation. Those results argue for a close monitoring of pregnancies in patients treated with these drugs.

category: pharmacology and toxicology


title: Unbiased Proteomics, Histochemistry, and Mitochondrial DNA Copy Number Reveal Better Mitochondrial Health in Muscle of High Functioning Octogenarians

abstract:
BackgroundMaster athletes prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear.

MethodsWe performed muscle biopsies in 15 octogenarian world class track and field masters athletes (MA) and 14 non-athlete age/sex-matched controls (NA) to provide insights into mechanisms for preserving function in advanced age. Muscle samples were assessed for respiratory compromised fibers, mtDNA copy number, and proteomics by liquid-chromatography mass spectrometry.

ResultsMost of the [~]800 differentially represented proteins in MA versus NA pertained to mitochondria structure/function such as electron transport capacity (ETC), cristae formation, mitochondrial biogenesis, and mtDNA-encoded proteins. In contrast, proteins from the spliceosome complex and nuclear pore were downregulated in MA. Consistent with proteomics data, MA had fewer respiratory compromised fibers, higher mtDNA copy number, and an increased protein ratio of the cristae-bound ETC subunits relative to the outer mitochondrial membrane protein voltage dependent anion channel. There was a substantial overlap of proteins overrepresented in MA versus NA with proteins that decline with aging and which are higher in physically active than sedentary individuals. However, we also found 176 proteins related to mitochondria that are uniquely differentially expressed in MA.

DiscussionWe conclude that high function in advanced age is associated with preserving mitochondrial structure/function proteins, with under-representation of proteins involved in the spliceosome and nuclear pore complex. Whereas many of these differences in MA appear related to their physical activity habits, others may reflect unique biological (e.g., gene, environment) mechanisms that preserve muscle integrity and function with aging.

category: physiology


title: LRH-1/NR5A2 regulates the PTGS2-PGE2-PTGER1 signalling axis contributing to islet survival and antidiabetic actions of the agonist BL001

abstract: We have previously described a role of LRH-1/NR5A2 in islet morphogenesis during postnatal development and reported that the treatment with BL001, an agonist of LRH-1/NR5A2, protects islets against-stress induced apoptosis and reverts hyperglycemia in 3 mouse models of Type 1 Diabetes Mellitus (T1DM). Islet transcriptome profiling revealed that most differentially expressed genes after BL001 treatment are involved in immunomodulation, among them, the increase in PTGS2/COX2 expression. Herein, we dissected the cellular and molecular branches of the BL001/LRH-1/NR5A2 signalling axis in order to chart the mode of action confering beta cell protection and hyperglycaemia reversion. We found that constitutive LRH-1/NR5A2 ablation within the insulin expression domain (RIP-Cre mouse model) caused a significant beta cell mass reduction characterized by blunted proliferation correlating with animal growth retardation, weight loss and hypoglycemia, leading to lethality before weaning. Using an inducible approach (pdx1PBCreER mouse model), specific deletion of LRH-1/NR5A2 in adult beta cells abolished the anti diabetic effect of BL001 in streptozotocin treated mice, correlating with complete beta-cell mass destruction. Additionally, BL001 induced Ptgs2 expression, was blunted in islets lacking LRH-1/NR5A2. The combined BL001/cytokine treatment did not further stimulate Ptgs2 expression above levels detected with cytokine alone yet secreted PGE2 levels were increased 5-fold. Inactivation of PTGS2 blunted induction of the target and its product PGE2 in islets treated with cytokines alone or with BL001. Importantly, PTGS2 inactivated islets were refractory to the BL001 protective effect under cytokine attack as evidenced by increased Bax expression levels, cytochrome C release and cleaved PARP. The PTGER1 antagonist ONO-8130, but not the PTGER4 antagonist L-161,982, negated BL001-mediated islet survival. Our results establish that the beneficial properties of BL001 against stress-induced cell death are specifically conveyed by LRH-1/NR5A2 activation in beta cells and downstream stimulation of the PTGS2-PGE2/PTGER1 signalling axis.


title: Isolation of gametes and zygotes from Setaria viridis

abstract: Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C4 crops because it has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C4 crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms.

category: plant biology


title: Learning from ingroup experiences changes intergroup impressions

abstract: Living in multicultural societies, humans form impressions towards individuals of their own social groups (ingroup members) and of different social groups (outgroup members). Some psychological theories predict that intergroup impressions are mainly shaped by experiences with outgroup individuals ("outgroup focused theories"), while other theories predict that ingroup experiences play a dominant role ("ingroup focused theories"). Here we test predictions from these two psychological theories by estimating how intergroup impressions are dynamically shaped when people learn from both ingroup and outgroup experiences. Participants expected to receive painful shocks but were saved from pain by different ingroup or outgroup members in 75% of all trials. We measured neural responses using functional magnetic resonance imaging (fMRI), and participants rated their social closeness as well as impressions towards the ingroup and the outgroup. Behavioral results showed an initial ingroup bias in impression ratings which was significantly reduced over the course of learning. Computational learning models revealed that these changes in intergroup impressions were predicted by the weight given to ingroup prediction errors. The weight of the ingroup prediction error and its effect on intergroup impression change was stronger the more individuals identified with their ingroup. On the neural level, the left inferior parietal lobule (IPL) encoded more negative prediction errors for the ingroup compared to the outgroup. Moreover, stronger weight for ingroup prediction errors was related to increased neural coupling between the left IPL and the anterior insula (AI). This coupling further predicted learning-related changes in intergroup impressions. Together, our work provides computational and neural evidence for "ingroup focused theories", highlighting the importance of ingroup experiences in shaping social impressions in intergroup settings.

category: neuroscience


title: Time-of-day influences on skill acquisition and consolidation after physical and mental practices

abstract: Time-of-day influences both physical and mental performance. Its impact on motor learning is, however, not well established yet. Here, using a finger tapping-task, we investigated the time-of-day effect on skill acquisition (i.e., immediately after a physical or mental practice session) and consolidation (i.e., 24 hours later). Two groups (one physical and one mental) were trained in the morning (10 a.m.) and two others (one physical and one mental) in the afternoon (3 p.m.). We found an enhancement of motor skill following both types of practice, whatever the time of the day, with a better acquisition for the physical than the mental group. Interestingly, there was a better consolidation for both groups when the training session was scheduled in the afternoon. Overall, our results indicate that the time-of-day positively influences motor skill consolidation and thus must be considered to optimize training protocols in sport and clinical domains to potentiate motor (re)learning.


title: ConvNets Develop Organizational Principles of the Visual Cortex when using Ganglion Cell-Based Sampling

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe distribution of retinal ganglion cells in primate visual systems portrays a densely distributed central region, with an incrementally decreasing cell density as the angle of visual eccentricity increases. This results in a non-uniform sampling of the retinal image that resembles a wheelbarrow distortion. We propose that this sampling gives rise to several organizational properties of the primate visual system, including cortical magnification, linear relationship between eccentricity and receptive field sizes, eccentricity-dependent drop-off in spatial-frequency preference, and radial bias. We test this hypothesis by training a convolutional neural network to classify the orientation of sine gratings and Gabor stimuli, resampled according to retinal ganglion cell distributions. Our simulations show that introducing this sampling step gives rise to the aforementioned organizational principles in convolutional layers while only minimally affecting their classification performance. This lends credence to the notion that the retinal ganglion cell distribution is an important factor for the emergence of these organizational principles in visual systems.


title: THE CONSTITUTIVE ACTIVITY OF THE HISTAMINE H1 RECEPTOR INTERACTION WITH THE NMDA RECEPTOR CONSEQUENCES: IN EPILEPSY

abstract:
ObjectivesTo explore the epileptogenic effects of several drugs specific to the antihistaminic H1 receptor on NMDA and GABA responses.

MethodsWith patch-clamp whole-cell recordings of hippocampal neurons of rats, we observe the effects of several histaminergic agonists and antagonists on the NMDA and GABA currents. During the NMDA rundown and GABA rundown, we applied the drugs and observe the effect on the responses, and compare them to control conditions.

ResultsMepyramine with nanomolar concentrations increases significantly the NMDA responses about 35 %, this effect is mimicked by another Anti H1 drug Triprolidine. Histamine alone has no effect on NMDA rundown but 100 M Histamine can reduce partially the effect of Mepyramine. An H1 receptor agonist 2,3 Bromophenyl Histamine also shows the same properties as Histamine. The H1 receptor of Histamine seems to be crucial during these experiments, blockade of its constitutive activity by Mepyramine induces a significant increase of the NMDA responses. On the GABA response, the same nanomolar dose of Mepyramine has no effect on the rundown and we also observed no effect of Histamine on the GABA rundown.

SignificanceAn interaction between Histamine H1 receptor and the NMDA is revelated by using Mepyramine in nanomolar concentration, Histamine has no effect on these responses also in these experiments Mepyramine acts as an inverse agonist blocking the constitutive activity of the H1 receptor. The constitutive activity of the H1 receptor seems to be crucial in the regulation of NMDA receptor activities.

Keys Points The use of antihistaminic H1 drugs can facilitate the apparition of epileptic seizures,
During rundown experiments, Mepyramine, a high potent H1 antagonist increased the NMDA responses, while Histamine as no effect.
On GABA rundown both Mepyramine and Histamine show no effect.
Constitutive activity of the Histamine H1 receptor seems to be crucial for the NMDA receptor activity.


title: Surprise: a unified theory and experimental predictions

abstract:
Surprising events trigger measurable brain activity and influence human behavior by affecting learning, memory, and decision-making. Currently there is, however, no consensus on the definition of surprise. Here we identify 16 mathematical definitions of surprise in a unifying framework, show how these definitions relate to each other, and prove under what conditions they are indistinguishable. We classify these surprise measures into four main categories: (i) change-point detection surprise, (ii) information gain surprise, (iii) prediction surprise, and (iv) confidence-correction surprise. We design experimental paradigms where different categories make different predictions: we show that surprise-modulation of the speed of learning leads to sensible adaptive behavior only for change-point detection surprise whereas surprise-seeking leads to sensible exploration strategies only for information gain surprise. However, since neither change-point detection surprise nor information gain surprise perfectly reflect the definition of  surprise in natural language, a combination of prediction surprise and confidence-correction surprise is needed to capture intuitive aspects of surprise perception. We formalize this combination in a new definition of surprise with testable experimental predictions. We conclude that there cannot be a single surprise measure with all functions and properties previously attributed to surprise. Consequently, we postulate that multiple neural mechanisms exist to detect and signal different aspects of surprise.

Author noteAM is grateful to Vasiliki Liakoni, Martin Barry, and Valentin Schmutz for many useful discussions in the course of the last few years, and to Andrew Barto for insightful discussions through and after EPFL Neuro Symposium 2021 on "Surprise, Curiosity and Reward: from Neuroscience to AI". We thank K. Robbins and collaborators for their publicly available experimental data (Robbins et al., 2018). All code needed to reproduce the results reported here will be made publicly available after publication acceptance. This research was supported by Swiss National Science Foundation (no. 200020_184615). Correspondence concerning this article should be addressed to Alireza Modirshanechi, School of Computer and Communication Sciences and School of Life Sciences, EPFL, Lausanne, Switzerland. E-mail: alireza.modirshanechi@epfl.ch.


title: Simulated attack reveals how lesions affect network properties in post-stroke aphasia

abstract: Aphasia is one of the most prevalent cognitive syndromes caused by stroke. The rarity of premorbid imaging and heterogeneity of lesion size and extent obfuscates the links between the local effects of the lesion, global anatomical network organization, and aphasia symptoms. We applied a simulated attack approach to examine the effects of 39 stroke lesions on network topology by simulating their effects in a control sample of 36 healthy brain networks. We focused on measures of global network organization thought to support overall brain function and resilience in the whole brain and within the left hemisphere. After removing lesion volume from the network topology measures and behavioral scores (the Western Aphasia Battery Aphasia Quotient; WAB-AQ), four behavioral factor scores obtained from a neuropsychological battery, and a factor sum), we compared the behavioral variance accounted for by simulated post-stroke connectomes to that observed in the randomly permuted data. Overall, global measures of network topology in the whole brain and left hemisphere accounted for 10% variance or more of the WAB-AQ and the lexical factor score beyond lesion volume and null permutations. Streamline networks provided more reliable point estimates than FA networks. Edge weights and network efficiency were weighted most highly in predicting the WAB-AQ for FA networks. Overall, our results suggest that global network measures can provide modest statistical value predicting overall aphasia severity, but less value in predicting specific behaviors. Variability in estimates could be induced by premorbid ability, deafferentation and diaschisis, and neuroplasticity following stroke.


title: Caste, sex, and parasitism influence brain plasticity in a social wasp

abstract: Phenotypic plasticity is the capacity of a single genotype to exhibit different phenotypes, and can be an adaptive response to specific environmental and social conditions. Social insects are particularly well-suited to study plasticity, because the division of labor amongst females and the different life histories of males and females are associated with specific sensory needs. Here, we take advantage of the social wasp Polistes dominula to explore if brain plasticity is influenced by caste and sex, and the exploitation by the parasite Xenos vesparum. Within sexes, males had proportionally larger optic lobes, while females, regardless of caste, had larger antennal lobes, which is consistent with sensory needs of sex-specific life histories. Within castes, reproductive females had larger calyces, as predicted by their sensory needs for extensive within-colony interactions and forming winter aggregations, than workers who spend more time foraging for nest material and prey. Surprisingly, parasites had different effects on female and male hosts. Female workers were castrated and behaviorally manipulated by female or male parasites, but only showed moderate differences in relative allocation of different brain tissue compared to non-parasitized workers. In contrast, the testes and behavior of parasitized males were essentially unaffected, but they had smaller brains and greater relative volume of most sensory brain regions than non-parasitized males. Our results are consistent with caste and sex mediating brain plasticity in P. dominula and that the parasites manipulation can also drive differential allocation of brain regions depending on host sex.


title: Proteomic and single-cell transcriptomic dissection of human plasmacytoid dendritic cell response to influenza virus

abstract: Plasmacytoid dendritic cells [pDCs] represent a rare innate immune subset uniquely endowed with the capacity to produce substantial amounts of type-I interferons [IFN-I]. This function of pDCs is critical for effective antiviral defenses and has been implicated in autoimmunity. While IFN-I and select cytokines have been recognized as pDC secreted products, a comprehensive agnostic profiling of the pDC secretome in response to a physiologic stimulus has not been reported. We applied LC-MS/MS to catalogue the repertoire of proteins secreted by pDCs in response to challenge with live influenza H1N1. Additionally, using single-cell RNA-seq [scRNA-seq], we perform multidimensional analyses of pDC transcriptional diversification following stimulation. Our data reveal an abundance of protein species released by pDCs in addition to IFN-I, and evidence highly specialized roles within the pDC population ranging from dedicated cytokine super-producers to cells with APC-like functions. Moreover, dynamic expression of transcription factors and surface markers characterize activated pDC fates.

category: immunology


title: Low selectivity index of ivermectin and macrocyclic lactones on SARS-CoV2 replication in vitro argues against their therapeutic use for COVID-19.

abstract: There are very limited antiviral therapeutic options for coronavirus infections, therefore global drug re-purposing efforts are paramount to identify available compounds that could provide clinical benefits to patients with COVID-19. Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin demonstrated anti-SARS-CoV-2 activity in vitro in human pulmonary cells in comparison to VeroE6 (with EC50 of 1-3 M). Similarly, the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin reduced SARS-CoV-2 replication in vitro (with EC50 of 2-5 M). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index <10 for all compounds. In conclusion, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se, due to low selectivity index. This is discussed in regards to recent clinical COVID studies on ivermectin.

category: microbiology


title: Leishmania amazonensis sabotages host cell SUMOylation for intracellular survival

abstract: Leishmania parasites use elaborate virulence mechanisms to invade and thrive in macrophages. These virulence mechanisms inhibit host cell defense responses and generate a specialized replicative niche, the parasitophorous vacuole. In this work, we performed a genome-wide RNAi screen in Drosophila macrophage-like cells to identify host factors necessary for Leishmania amazonensis infection. This screen identified 52 conserved genes required specifically for parasite entry, including several components of the SUMOylation machinery. Further studies in mammalian macrophages found that L. amazonensis infection inhibited SUMOylation within infected macrophages and this inhibition enhanced parasitophorous vacuole growth and parasite proliferation through modulation of multiple genes especially ATP6V0D2, which in turn effects CD36 expression and cholesterol levels. Together, these data suggest that parasites actively sabotage host SUMOylation and alter host transcription to improve their intracellular niche and enhance their replication.

category: cell biology


title: A phosphoinositide and RAB switch controls early macropinocytosis

abstract:
Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluids into giant vesicles known as macropinosomes. This mechanism is used by immune cells to sample the surroundings for antigens and can be exploited by cancer cells for nutrient uptake. What determines the fate of macropinosomes after they have been internalized is largely unknown. Here we investigate the role of the phosphatidylinositol 3-kinase VPS34/PIK3C3 and its product phosphatidylinositol 3-phosphate (PtdIns3P) in macropinosome fate determination. Inhibition of VPS34 led to a decrease in macropinosome survival and fluid phase uptake as well as preventing recruitment of early endosomal factors, including the small GTPase RAB5 and its effectors, to the forming macropinosomes. Instead, forming macropinosomes under VPS34 inhibition accumulated regulators of endocytic recycling, including RAB8A, RAB10, RAB11A, and PtdIns4P, which led to fusion of macropinosomes with the plasma membrane.

Whereas RAB5 was critical for macropinosome formation, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a PtdIns3P-controlled switch that balances macropinosome fate between the default, endolysosomal maturation and an alternative, secretory route.


title: Demographic history shapes genomic variation in an intracellular parasite with a wide geographic distribution

abstract: Analyzing variation in a species genomic diversity can provide insights into its historical demography, biogeography and population structure, and thus, its ecology and evolution. Although such studies are rarely undertaken for parasites, they can be highly revealing because of the parasites coevolutionary relationships with hosts. Modes of reproduction and transmission are thought to be strong determinants of genomic diversity for parasites and vary widely among microsporidia (fungal-related intracellular parasites), which are known to have high intraspecific genetic diversity and interspecific variation in genome architecture. Here we explore genomic variation in the microsporidium Hamiltosporidium, a parasite of the freshwater crustacean Daphnia magna, looking especially at which factors contribute to nucleotide variation. Genomic samples from 18 Eurasian populations and a new, long-read based reference genome were used to determine the roles that reproduction mode, transmission mode and geography play in determining population structure and demographic history. We demonstrate two main H. tvaerminnensis lineages and a pattern of isolation-by-distance, but note an absence of congruence between these two parasite lineages and the two Eurasian host lineages. We suggest a comparatively recent parasite spread through Northern Eurasian host populations after a change from vertical to mixed-mode transmission and the loss of sexual reproduction. While gaining knowledge about the ecology and evolution of this focal parasite, we also identify common features that shape variation in genomic diversity for many parasites, e.g., distinct modes of reproduction and the intertwining of host-parasite demographies.

category: evolutionary biology


title: Prioritising Autoimmunity Risk Variants for Functional Analyses by Fine-Mapping Mutations Under Natural Selection

abstract: Pathogens imposed selective pressure on humans and shaped genetic variation in immunity genes. This can also be true for a fraction of causal variants implicated in chronic inflammatory disorders. Hence, locating adaptive mutations among candidate variants for these disorders can be a promising way to prioritize and decipher their functional response to microbial stimuli and contribution to pathogenesis. This idea has been discussed for decades, but challenges in locating adaptive SNPs hindered its application in practice. Our study addresses this issue and shows that a fraction of candidate variants for inflammatory conditions evolved under moderate and weak selection regimes (sweeps), and such variants are mappable. Using a novel powerful local-tree-based methodology, we show that 204 out of 593 risk loci for 21 autoimmune disorders contain at least one candidate SNP with strong evidence of selection. More importantly, in 28% of cases, these candidates for causal variants colocalize with SNPs under natural selection that we fine-mapped in this study. Causal SNPs under selection represent promising targets for functional experiments. Such experiments will help decipher molecular events triggered by infectious agents, a likely early event in autoimmunity. Finally, we show that a large fraction (60%) of candidate variants are either hitchhikers or linked with the selected mutation. Our findings, thus, support both hitchhiking and natural selection models, with the latter having important practical implications in medicine.

category: genetics


title: Sex significantly impacts the function of major depression-linked variants in vivo

abstract:
Genome-wide association studies have discovered blocks of common variants--likely transcriptional-regulatory--associated with major depressive disorder (MDD), though the functional subset and their biological impacts remain unknown. Likewise, why depression occurs in females more frequently than males is unclear. We therefore tested the hypothesis that risk-associated functional variants interact with sex and produce greater impact in female brains. We developed methods to directly measure regulatory variant activity and sex interactions using massively parallel reporter assays (MPRAs) in the mouse brain in vivo, in a cell type-specific manner. We measured activity of >1,000 variants from >30 MDD loci, identifying extensive sex-by-allele effects in mature hippocampal neurons and suggesting sex-differentiated impacts of genetic risk may underlie sex bias in disease. Unbiased informatics approaches indicated that functional MDD variants recurrently disrupt sex hormone receptor binding sequences. We confirmed this with MPRAs in neonatal brains, comparing brains undergoing the masculinizing hormone surge to hormonally-quiescent juveniles. Our study provides novel insights into the influence of age, biological sex, and cell type on regulatory-variant function, and provides a framework for in vivo parallel assays to functionally define interactions between organismal variables like sex and regulatory variation.

One-Sentence SummaryMassively parallel assays in vivo identified extensive functional and sex-interacting common variants in depression risk loci.


title: Evaluating the heterogeneous effect of extended incubation to blastocyst transfer on the implantation outcome via causal inference

abstract:
In IVF treatments, extended culture to single blastocyst-transfer is the recommended protocol over cleavage-stage transfer. However, evidence-based criteria for assessing the heterogeneous implications on implantation outcome are lacking. To estimate the causal effect of blastocyst-transfer on implantation outcome, we assembled a multicenter dataset of embryo time-lapse imaging. The data includes a natural source of randomness and has a strong claim for satisfying the assumptions needed for valid causal inference. By fitting a causal forest model, we assessed the  Transfer Lift, which quantifies the probability difference in embryo implantation if transferred as a blastocyst versus cleavage-stage. Blastocyst transfer increased the average implantation rate, however we revealed a subpopulation of negative Transfer Lift embryos whose implantation potential is predicted to increase via cleavage-stage transfer. We provide day-of-transfer decision-support tools that are retrospectively estimated to improve implantation rate by 32%, thus demonstrating the efficacy of embryo-level causal inference in reproductive medicine.

One Sentence SummaryA causal inference model predicts the heterogeneous effect of prolonged incubation to blastocyst transfer on embryo implantation, thus providing means for optimizing pregnancy rates in IVF treatments.

category: bioengineering


title: Coiled Coil Crosslinked Alginate Hydrogels Dampen Macrophage-Driven Inflammation

abstract: Alginate hydrogels are widely used for tissue engineering and regenerative medicine due to their excellent biocompatibility. A facile and commonly used strategy to crosslink alginate is the addition of Ca2+ that leads to hydrogelation. However, extracellular Ca2+ is a secondary messenger in activating inflammasome pathways following physical injury or pathogenic insult leading to persistent inflammation and scaffold rejection. Here we present graft copolymers of charge complementary heterodimeric coiled coil (CC) peptides and alginate that undergo supramolecular self-assembly to form Ca2+ free alginate hydrogels. The formation of heterodimeric CCs was confirmed using circular dichroism spectroscopy and scanning electron microscopy revealed a significant difference in pore size between Ca2+ and CC crosslinked gels. The resulting hydrogels were self-supporting and display shear-thinning and shear-recovery properties. In response to lipopolysaccharide (LPS) stimulation, peritoneal macrophages and bone marrow derived dendritic cells cultured in the CC crosslinked gels exhibited a 10-fold reduction in secretion of the proinflammatory cytokine IL-1{beta} compared to Ca2+ crosslinked gels. A similar respose was also observed in vivo upon peritoneal delivery of Ca2+ or CC crosslinked gels. Analysis of peritoneal lavage showed that macrophages in mice injected with Ca2+ crosslinked gels display a more inflammatory phenotype compared to macrophages from mice injected with CC crosslinked gels. These results suggest that CC peptides by virtue of their tunable sequence-structure-function relationship and mild gelation conditions are promising alternative crosslinkers for alginate and other biopolymer scaffolds used in tissue engineering.


title: Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris

abstract: Harnessing the unique biochemical capabilities of non-model microorganisms would expand the array of biomanufacturing substrates, process conditions, and products. There are non-model microorganisms that fix nitrogen and carbon dioxide, derive energy from light, catabolize methane and lignin-derived aromatics, are tolerant to physiochemical stresses and harsh environmental conditions, store lipids in large quantities, and produce hydrogen. Model microorganisms often only break down simple sugars and require low stress conditions, but they have been engineered for the sustainable manufacture of numerous products, such as fragrances, pharmaceuticals, cosmetics, surfactants, and specialty chemicals, often by using tools from synthetic biology. Transferring complex pathways with all of the needed cofactors, energy sources, and cellular conditions from a non-model microorganism to a common chassis has proven to be exceedingly difficult. Utilization of unique biochemical capabilities could also be achieved by engineering the host; although, synthetic biology tools developed for model microbes often do not perform as designed in other microorganisms. The metabolically versatile Rhodopseudomonas palustris CGA009, a purple non-sulfur bacterium, catabolizes aromatic compounds derived from lignin in both aerobic and anaerobic conditions and can use light, inorganic, and organic compounds for its source of energy. R. palustris utilizes three nitrogenase isozymes to fulfill its nitrogen requirements while also generating hydrogen. Furthermore, the bacterium produces two forms of RuBisCo in response to carbon dioxide/bicarbonate availability. While this potential chassis harbors many beneficial traits, stable heterologous gene expression has been problematic due to its intrinsic resistance to many antibiotics and the lack of synthetic biology parts investigated in this microbe. To address these problems, we have characterized gene expression and plasmid maintenance for different selection markers, started a synthetic biology toolbox specifically for the photosynthetic R. palustris, including origins of replication, fluorescent reporters, terminators, and 5 untranslated regions, and employed the microbes endogenous plasmid for exogenous protein production. This work provides essential synthetic biology tools for engineering R. palustris many unique biochemical processes and has helped define the principles for expressing heterologous genes in this promising microbe through a methodology that could be applied to other non-model microorganisms.


title: Base-resolution prediction of transcription factor binding signals by a deep learning framework

abstract:
Transcription factors (TFs) play an important role in regulating gene expression, thus the identification of the sites bound by them has become a fundamental step for molecular and cellular biology. In this paper, we developed a deep learning framework leveraging existing fully convolutional neural networks (FCN) to predict TF-DNA binding signals at the base-resolution level, called FCNsignal. The proposed FCNsignal can simultaneously achieve the following tasks: (i) modeling the base-resolution signals of binding regions; (ii) discriminating binding or non-binding regions; (iii) locating TF-DNA binding regions; (iv) predicting binding motifs. The experimental results on 53 TF ChIP-seq datasets and 6 chromatin accessibility ATAC-seq datasets show that our proposed framework outperforms some existing state-of-the-art methods. In addition, we explored to use the trained FCNsignal to locate all potential TF-DNA binding regions on a whole chromosome and predict DNA sequences of arbitrary length, and the results show that our framework can find most of the known binding regions and accept sequences of arbitrary length. Furthermore, we demonstrated the potential ability of our framework in discovering causal disease-associated single-nucleotide polymorphisms (SNPs) through a series of experiments.

Author summaryIdentification of transcription factor binding sites (TFBSs) is fundamental to study gene regulatory networks in biological systems, as TFs activate or suppress the transcription of genes by binding to specific TFBSs. With the development of high-throughput sequencing technologies and deep learning (DL), several DL-based approaches have been developed for systematically studying TFBSs, achieving impressive performance. Nevertheless, these methods either excessively focus on discriminating binding or non-binding sequences or individually accomplish multiple TFBSs-associated tasks. In this work, we provide an integrated framework, which utilizes the FCN architecture to predict TF-DNA binding signals at the base-resolution level, to simultaneously study multiple TFBSs-associated tasks. More importantly, we also demonstrate that our proposed framework has the ability to locate all potential TF-DNA binding regions from DNA sequences of arbitrary length. We hope that our framework can provide a new perspective on studying the mechanism of TF-DNA binding and its related tasks.


title: Empirical Bayes functional models for hydrogen deuterium exchange mass spectrometry

abstract: A proteins structure is state-specific and a key determinant of its function. Proteins can undergo subtle structural changes when binding to another protein, small molecule or as a result of environmental perturbations. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a technique to explore differential protein structure by examining the rate of deuterium incorporation for specific peptides. This rate will be altered upon structural perturbation and detecting significant changes to this rate requires a statistical test. To determine rates of incorporation, HDX-MS measurements are frequently made over a time course. However, current statistical testing procedures ignore the correlations in the temporal dimension of the data. Using tools from functional data analysis, we develop a testing procedure that explicitly incorporates a physical model of hydrogen deuterium exchange. To further improve statistical power, we develop an empirical Bayes version of our method, allowing us to borrow information across peptides and stabilise variance estimates for low sample sizes. Our approach has increased power, reduces false positives and improves interpretation over linear model-based approaches. Due to the improved flexibility of our method, we can apply it to a multi-antibody epitope-mapping experiment where current approaches are inapplicable due insufficient flexibility. Hence, our approach allows HDX-MS to be applied in more experimental scenarios and reduces the burden on experimentalists to produce excessive replicates. Our approach is implemented in the R-package "hdxstats": https://github.com/ococrook/hdxstats.


title: Population genomics analysis with RAD, reprised: STACKS 2

abstract: Restriction enzymes have been one of the primary tools in the population genetics toolkit for fifty years, being coupled with each new generation of technology to provide a more detailed view into the genetics of natural populations. Restriction site-Associated DNA protocols, which joined enzymes with short-read sequencing technology have democratized the field of population genomics, providing a means to assay the underlying alleles in scores of populations. More than ten years on, the technique has been widely applied across the tree of life and served as the basis for many different analysis techniques. Here, we provide a detailed protocol to conduct a RAD analysis from experimental design to de novo analysis - including parameter optimization - as well as reference-based analysis, all in SO_SCPLOWTACKSC_SCPLOW version 2, which is designed to work with paired-end reads to assemble RAD loci up to 1000 nucleotides in length. The protocol focuses on major points of friction in the molecular approaches and downstream analysis, with special attention given to validating experimental analyses. Finally, the protocol provides several points of departure for further analysis.


title: Re-expressing coefficients from regression models for inclusion in a meta-analysis

abstract:
BackgroundQuantitative summaries of study results, or meta-analyses, have proven useful in several fields, but pose special challenges when applied to non-randomized studies of exposure in relation to outcome. One such issue arises when summarizing regression results when some studies have log-transformed exposure while others have not. Methods of re-expression have recently been developed, with the goal of generating comparable results across studies regardless of data transformation. We examined the validity of three re-expression methods using simulations and real data examples.

MethodsSimulations were developed to examine the validity of the re-expression methods when the exposure had a skewed distribution. We also identified 15 published studies for which we were able to either access the data and replicate the result, or which had provided regression coefficients ({beta}) from analyses with both log-transformed and untransformed exposure data. For the simulated and real data we re-expressed {beta} as though it had been based on untransformed data, and vice versa. We compared the re-expressed results to fitted regression coefficients. The proportional difference from the fitted {beta} was used to quantitate bias. We also examined the effect of influential observations on the success of the re-expression using the real data examples.

ResultsIn the simulated and real data examples, all three re-expression methods usually gave biased results. The method of Rodriguez-Barranco et al. (2017) tended to give less biased results but with a wider range of bias. For example, in the real data, when re-expressing results as if they had been fit to untransformed exposure, the median estimate was 22% too high, with a range of -40% to 1,904%. In some analyses, the presence of influential observations had a large effect on the validity of the re-expression.

ConclusionsWhen the distribution of exposure is skewed, the re-expression methods examined are likely to give biased results. The bias varied by method, the direction of the re-expression, skewness, influential observations, and in some cases, the median exposure. Meta-analysts using any of these re-expression methods may want to consider the uncertainty, the likely direction and degree of bias, and conduct sensitivity analyses on the re-expressed results.


title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

abstract: Ewing sarcoma is the second most common bone malignancy in children and adolescents. Patients with upfront metastatic or recurrent disease have poor outcomes with 5-year survival rates of <30%. CDC7, also known as DDK (DBF4-dependent kinase), is a serine-threonine kinase that, in coordination with its activation subunit ASK (or DBF4), is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the replication stress response. Due to DDKs diverse roles during replication, coupled with an increased level of genomic instability and R-loop-mediated replication stress within Ewing sarcoma cells, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibitors. Here, we show that treatment with two selective DDK inhibitors, TAK-931 and XL413, results in apoptosis and a significant reduction in cell viability in EWS-FLI1-harboring Ewing sarcoma cell lines. We show that low dose DDK inhibition in Ewing sarcoma cells causes an accumulation of cells in late-S phase with a reduced replication capacity. There is also evidence of premature mitotic entry indicating an inability to properly complete DNA replication in a timely manner upon DDK inhibition. Also, there is a significant increase in the formation of micronuclei and other aberrant mitotic structures upon DDK inhibition in Ewing sarcoma cells indicating a failure to properly progress through S-phase followed by improper mitotic entry/progression, resulting in mitotic catastrophe. Interestingly, we observed minimal signs of mitotic accumulation, despite clear evidence of replication and mitotic stress, suggesting a failure to properly enforce the mitotic checkpoint. Together, these results suggest that Ewing sarcoma cells rely on the activity of DDK to maintain cell viability and suggest that DDK inhibition may prove to be a viable therapeutic strategy for patients with Ewing sarcoma.

category: cancer biology


title: A lineage-specific epigenetic memory of inflammation potentiates Kras-driven pancreatic tumorigenesis

abstract: Inflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner.


title: An epigenetic memory of inflammation controls context-dependent lineage plasticity in the pancreas


title: Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas

abstract: Cancer cells are highly heterogeneous at the transcriptional level and in their epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted Cytometry by Time of Flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of Diffused Intrinsic Pontine Glioma (DIPG). DIPG is a lethal glioma, driven by histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIGP, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem-cell and differentiation markers. Moreover, we demonstrate the use of this high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell-cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.


title: Intratumoral Delivered Novel Circular mRNA Encoding Cytokines for Immune Modulation and Cancer Therapy

abstract: The application of mRNA as a novel kind of vaccine has been proved recently, due to the emergence use authorization (EUA) by FDA for the two COVID-19 mRNA vaccines developed by Moderna and BioNTech. Both of the two vaccines are based on canonical linear mRNA, and encapsulated by lipid nanoparticle (LNP). Circular mRNA, which is found to mediate potent and durable protein expression, is an emerging technology recently. Owing to its simplicity of manufacturing and superior performance of protein expression, circular mRNA is believed to be a disruptor for mRNA area. However, the application of circular mRNA is still at an initiation stage, proof of concept for its usage as future medicine or vaccine is necessary. In the current study, we established a novel kind of circular mRNA, termed C-RNA, based on Echovirus 29 (E29)-derived internal ribosome entry sites (IRES) and newly designed homology arms and RNA spacers. Our results demonstrated that this kind of circular mRNA is able to mediate strong and durable protein expression, compared to typical linear mRNA. Moreover, for the first time, our study demonstrated that direct intratumoral administration of C-RNA encoding a mixture of cytokines achieved successful modulation of intratumoral and systematic anti-tumor immune responses and finally leading to an enhancement of anti-PD-1 antibody-induced tumor repression in syngeneic mouse model. Additionally, after an optimization of the circular mRNA formulation, a significant improvement of C-RNA mediated protein expression was observed. With this optimized formulation, C-RNA induced enhanced anti-tumor effect via intratumoral administration and elicited significant activation of tumor-infiltrated total T cells and CD8+ T cells. Collectively, we established C-RNA, a novel circular mRNA platform, and demonstrated that it can be applied for direct intratumoral administration for cancer therapy.


title: Comprehensive RNA-Seq Reveals Molecular Changes in Kidney Malignancy Among People Living with HIV

abstract:
BackgroundMalignancy of the kidney is a rapidly progressive kidney disease and a major source of morbidity and mortality among people living with HIV (PLWH). Patients with HIV-associated kidey cancer experience higher cancer-specific mortality than the general population, and its mechanism remains poorly understood.

MethodsTo heighten the awareness of kidney malignancy in patients with HIV infection to facilitate the early diagnosis of kidney cancer, we identified 2460 protein-coding transcripts in HIV-associated kidney cancer using comprehensive RNA sequencing (RNA-seq).

ResultsKISS1R, CAIX, and NPTX2 mRNA expression levels were specifically increased in HIV-associated kidney cancer, and UMOD and TMEM213 mRNA were decreased in most cases based on real-time PCR analyses. These findings were similar to those noted for the general population with renal cell carcinoma. Immunohistochemical staining analysis also showed that a total of 16 of 18 kidney malignant cases among PLWH exhibited positive staining for KISS1R and CAIX.

ConclusionPathway analysis of the differentially expressed mRNAs in HIV-associated kidney cancer revealed that several key pathways were involved, including voltage-gated chloride channel activity, distal tubule development, collecting duct development, fructose metabolic processes, and negative regulation of lipase activity. The identified molecular changes in kidney malignancy may offer a helpful explanation for cancer progression and open up new therapeutic avenues that may decrease mortality after a cancer diagnosis among PLWH.


title: Dynamic Flux Balance Analysis Game

abstract: Flux balance analysis (FBA) for microbial communities often assumes a global objective function that all species cooperatively maximize in addition to maximizing their own growth. Combining community FBA with dynamic FBA to understand the time course and steady states of communities typically entails discretizing time and solving a community FBA model at each time point, a time-intensive process. We propose a dynamic community FBA model where species compete for metabolites to grow off of without needing to cooperate to maximize a community-level objective. An efficient method for computing steady state community compositions is provided, as well as methods for determining the stability of a steady state community to perturbations in biomass and invasion by species outside the community. The model and methods are applied to a model of four E. Coli. mutants with elements of competition (for shared metabolites) and cooperation (via mutants being auxotrophic for metabolites exported by other mutants).

category: systems biology


title: Screening for axon regeneration promoting compounds with human iPSC-derived motor neurons

abstract: CNS neurons do not regenerate after injury, leading to permanent functional deficits. Although sensory and motor neuron axons do regrow after peripheral nerve injury, functional outcome is limited due to the incomplete and slow regrowth. The lack of human-relevant assays suitable for large-scale drug screens has limited neuro-repair therapy discovery. To address this we developed a phenotypic screening strategy using human induced pluripotent stem cell-derived motor neurons to identify axon-regeneration promoting compounds and targets. The screens involve both re-plating human motor neurons on chondroitin sulfate proteoglycans and measuring regeneration responses to laser axotomy in spot cultures, and from them we identified multiple hits that promote injured axon regrowth. The top hit blebbistatin, a non-muscle myosin II inhibitor, accelerated axon regeneration and functional recovery after sciatic nerve injury in vivo. Human "injury in a dish" assays are suitable, therefore, to screen for therapeutic interventions that can induce or accelerate axon regeneration.


title: Towards a general framework for modeling large-scale biophysical neuronal networks: a full-scale computational model of the rat dentate gyrus

abstract: Large-scale computational models of the brain are necessary to accurately represent anatomical and functional variability in neuronal biophysics across brain regions and also to capture and study local and global interactions between neuronal populations on a behaviorally-relevant temporal scale. We present the methodology behind and an initial implementation of a novel open-source computational framework for construction, simulation, and analysis of models consisting of millions of neurons on high-performance computing systems, based on the NEURON and CoreNEURON simulators (Carnevale and Hines, 2006, Kumbhar et al., 2019). This framework uses the HDF5 data format and software library (HDF Group, 2021) and includes a data format for storing morphological, synaptic, and connectivity information of large neuronal network models, and an accompanying open-source software library that provides efficient, scalable parallel storage and MPI-based data movement capabilities. We outline our approaches for constructing detailed large-scale biophysical models with topographical connectivity and input stimuli, and present simulation results obtained with a full-scale model of the dentate gyrus constructed with our framework. The model generates sparse and spatially selective population activity that fits well with in-vivo experimental data. Moreover, our approach is fully general and can be applied to modeling other regions of the hippocampal formation in order to rapidly evaluate specific hypotheses about large-scale neural architectural features.


title: In vivo Correlation Tensor MRI reveals microscopic kurtosis in the human brain on a clinical 3T scanner

abstract:
Diffusion MRI (dMRI) has become one of the most important imaging modalities for noninvasively probing tissue microstructure. Diffusion Kurtosis MRI (DKI) quantifies the degree of non-gaussian diffusion, which in turn has been shown to increase sensitivity towards, e.g., disease and orientation mappings in neural tissue. However, the specificity of DKI is limited as different microstructural sources can contribute to the total diffusional kurtosis, including: variance in diffusion tensor magnitudes (Kiso), variance due to intravoxel diffusion anisotropy (Kaniso), and microscopic kurtosis (K) related to restricted diffusion and/or microstructural disorder. The latter in particular is typically ignored in diffusion MRI signal modeling as it is assumed to be negligible. Recently, Correlation Tensor MRI (CTI) based on Double-Diffusion-Encoding (DDE) was introduced for kurtosis source separation and revealed non negligible K in preclinical imaging. Here, we implemented CTI for the first time on a clinical 3T scanner and investigated the kurtosis sources in healthy subjects. A robust framework for kurtosis source separation in humans is introduced, followed by estimation of the relative importance of K in the healthy brain. Using this clinical CTI approach, we find that K significantly contributes to total diffusional kurtosis both in gray and white matter tissue but, as expected, not in the ventricles. The first K maps of the human brain are presented. We find that the spatial distribution of K provides a unique source of contrast, appearing different from isotropic and anisotropic kurtosis counterparts. We further show that ignoring K - as done by many contemporary methods based on multiple gaussian component approximation for kurtosis source estimation - biases the estimation of other kurtosis sources and, perhaps even worse, compromises their interpretation. Finally, a twofold acceleration of CTI is discussed in the context of potential future clinical applications. We conclude that CTI has much potential for future in vivo microstructural characterizations in healthy and pathological tissue.

HighlightsO_LICorrelation Tensor MRI (CTI) was recently proposed to resolve kurtosis sources
C_LIO_LIWe implemented CTI on a 3T scanner to study kurtosis sources in the human brain
C_LIO_LIIsotropic, anisotropic, and microscopic kurtosis sources were successfully resolved
C_LIO_LIMicroscopic kurtosis (K) significantly contributes to overall kurtosis in human brain
C_LIO_LIK provides a novel source of contrast in the human brain in vivo
C_LI


title: Age-dependent Effect Imposed by Rotenone Exposure

abstract:
BackgroundThe increasing awareness that environmental exposure may lead to sporadic neurological disorders has implicated rotenone to the etiology of some neurodegenerative diseases. However, the risk associated with rotenone toxicity remains controversial as a limited amount of research has studied its effects on brain health.

ObjectivesThis work assessed the risk of rotenone exposure to mice of different ages, gender, and duration by examining in vivo effects on brains.

MethodsUsing a mouse model, the impact of rotenone exposure was determined by analyzing the cellular phenotype in the murine brain.

ResultsOur results highlight the neurological susceptibility to long-term rotenone exposure in younger ages. For such, younger mice exhibit seizures and convulsions, resulting in shorter lifespan. At the cellular level, rotenone exposure specifically alters the migrating neuroblast populations in the dentate gyrus and causes disorganized pyramidal neurons in the CA3 within the hippocampus. Our findings, albeit the absence of transgenerational inheritance, demonstrated age-related outcomes from rotenone exposure.

DiscussionWe demonstrated that rotenone exposure specifically influences the population of neuroblasts and pyramidal neurons residing in the hippocampus, a brain region important for learning/memory and associated with convulsive seizure. Our understanding of how exactly rotenone affected region-specific neuronal cells and the molecular mechanism behind exposure risk is still limited. From the perspective of public health, our in vivo study highlights age-related susceptibility to rotenone toxicity. Future investigations in environmental epidemiology should determine whether age and duration of exposure to rotenone in human subjects pertains to the development of seizures or other neurological abnormalities over time.


title: Violet light modulates the central nervous system to regulate memory and mood

abstract: Light stimuli from the external environment serves as a signal. Photoreceptors receive photons at the outer nuclear layer of the retina. Non-visual photoreceptors, such as opsin5 (also known as OPN5 or neuropsin), are expressed in the retinal ganglion cells (RGCs) and hypothalamus to regulate the circadian cycle and body temperature. Here, we show that violet light (VL) stimuli received by OPN5-positive RGCs are transmitted to the habenula brain region. VL improves memory in aged mice and simultaneously increases neural architecture-related genes such as oligodendrocyte-related genes in the hippocampus. In addition, VL improves depressive-like behaviors in the social defeat stress model in an OPN5 dependent manner. Following VL exposure, cFos activation is observed at the nucleus accumbens (NAc) and the paraventricular thalamic nucleus (PVT). Taken together, the results indicate that violet light modulates brain function such as memory and mood by transmitting the signal from RGCs to the habenula region in the brain.


title: Retinal OFF ganglion cells allow detection of quantal shadows at starlight

abstract: Perception of light in darkness requires no more than a handful of photons and this remarkable behavioral performance can be directly linked to a particular retinal circuit - the retinal ON pathway. However, the neural limits to shadow detection in very dim light have remained unresolved. Here, we unravel the neural mechanisms that determine the sensitivity of mice to the dimmest light decrements by measuring signals from the most sensitive ON and OFF retinal ganglion cell types and by correlating their signals with visually guided behavior. We show that mice can detect shadows when only a few photon absorptions are missing among thousands of rods. Behavioral detection of "quantal" shadows relies only on the retinal OFF pathway and is limited by noise and losses of single-photon signals in retinal processing. Thus, in the dim-light regime, light increments and decrements are encoded separately via the ON and OFF retinal pathways, respectively.


title: Directed information sharing in the brain distinguishes perception from its consequences during perceptual bistability

abstract: In the search for the neural basis of conscious experience, perception and its cognitive consequences are typically confounded as neural activity is recorded while participants explicitly report what they experience. Here we present a novel way to disentangle perception from report using eye-movement analysis techniques based on convolutional neural networks and neurodynamical analyses based on information theory. We use a visual bistable stimulus that instantiates two well-known properties of conscious perception: integration and differentiation. At any given moment, observers either experience the stimulus as one integrated unitary percept or as two differentiated objects that are clearly distinct from each other. Using electroencephalography, we show that measures of neural information dynamics of directed information integration and differentiation closely follow participants experience when perceptual transitions were reported. We observed increased directed information from anterior to posterior electrodes (front to back) leading up to the moment the stimulus was reported to switch to the integrated percept and higher information differentiation of anterior signals leading up to reporting the differentiated percept. Crucially, neural integration dynamics were closely linked to perception and even observed in a no-report condition when perceptual transitions were inferred from eye-movements. In contrast, the link between perceptual transitions and neural differentiation was observed solely in the active report condition. Our results therefore suggest a differential role of anterior-posterior network communication vs anterior information differentiation during perception and reporting: while front to back directed information is associated with changes in the content of perception, frontal information differentiation reflects cognitive processes that are consequential of perceptual transitions, not perception per se.


title: Biological Factors and Self-Perception of Stress Predict Human Freeze-Like Responses in the Context of Self-Defence Training and Personal Experience with Violence

abstract: Many animals react to threatening stimuli such as a predator attacks by freezing. However, little experimental research investigated freeze response in humans. Here, we have employed practices commonly used in self-defence training to create two unique scenarios simulating armed physical attacks. Sixty healthy men volunteers divided into three groups of twenty (untrained, trained but unexperienced, trained and experienced) underwent these scenarios accompanied by measurement of biochemical, physiological, and psychological markers of stress. All participants also underwent an fMRI session during which they observed neutral and negative images from the International Affective Picture System (IAPS). Our results show that scenarios simulating physical attacks can induce a freeze-like response in men. In addition, we demonstrate that while electrodermal activity (EDA), subjective stress perception, and brain activity in fMRI predict freeze-like response in men, their effect on freeze-like response is entirely dependent on the level of training and experience of a given individual.


title: Bacterial colonisation dynamics of household plastics in a coastal environment

abstract: Accumulation of plastics in the marine environment has widespread detrimental consequences for ecosystems and wildlife. Marine plastics are rapidly colonised by a wide diversity of bacteria, including human pathogens, posing potential risks to human health. Here, we investigate the effect of polymer type, residence time and estuarine location on bacterial colonisation of common household plastics, including pathogenic bacteria. To do so, we submerged five main household plastic types: low-density PE (LDPE), high-density PE (HDPE), polypropylene (PP), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) at an estuarine site in Cornwall (U.K.) and tracked bacterial colonisation dynamics. Using both culture-dependent and culture-independent approaches, we found that bacteria rapidly colonised plastics irrespective of polymer type. While biofilm community composition changed with colonisation time, no difference was observed between polymer types. Likewise, the presence of pathogenic bacteria, quantified using the insect model Galleria mellonella, increased over a five-week period, with no consistent differences observed between polymer types. Pathogens isolated from plastic biofilms using Galleria enrichment included Serratia and Enterococcus species and harboured a wide range of antimicrobial resistance genes. Our findings show that plastics in coastal waters are rapidly colonised by a wide diversity of bacteria, including known human pathogens, independent of polymer type.

category: ecology


title: Riding into the future on a fly: toward a better understanding of phoresy and avian lice evolution (Phthiraptera) by screening bird carcasses for phoretic lice on hippoboscid flies (Diptera)

abstract: Many phoretic relationships between insects are understudied because of taxonomic impediments. We here illustrate for avian lice riding on hippoboscid flies how new natural history data on phoretic relationships can be acquired quickly with NGS barcoding. Most avian lice are host-specific, but some can arrive on new hosts by riding hippoboscid flies that feed on bird blood. Our summary of the literature yielded 254 published records which we here show to belong to two large and 13 small interaction networks for birds, flies, and lice. In order to generate new records, we then developed a new protocol based on screening bird carcasses sourced by citizen scientists. The inspection of 131 carcasses from Singapore lead to the first record of a Guimaraesiella louse species riding on Ornithoica momiyamai flies collected from a pitta carcass. Phoresy may explain why this louse species is now known from three phylogenetically disparate hosts (Pitta moluccensis, Ficedula zanthopygia; Pardaliparus elegans). A second new case of phoresy enhances a large interaction network dominated by Ornithophila metallica, a cosmopolitan and polyphagous hippoboscid fly species. Overall, we argue that many other two- and three-way phoretic relationships between arthropods (e.g., mites, pseudoscorpiones, beetles, flies) can be resolved using cost-effective large-scale NGS barcoding, which can be used to pre-sort specimens for taxonomic revision and thus partially overcome some taxonomic impediments.


title: Initial approach to the Conservation Genetics of the Guatemalan Beaded Lizard (Heloderma charlesbogerti): A route to genetic rescue

abstract: In this study, samples from 33 Guatemalan Beaded Lizard (Heloderma charlesbogerti) were analyzed for genetic diversity. Twenty-three samples were obtained from wild individuals from two separate population areas, and 10 samples were obtained from captive individuals. Because the seasonally dry tropical forest habitat sampled for this study, is degraded and fragmented, it was hypothesized that beaded lizard populations were small and isolated and would be subject to genetic erosion and an elevated extinction risk. To test this hypothesis, eight microsatellite markers were employed to analyze 22 individual samples from the population of Cabanas, Zacapa, a single individual from the eastern-most population and 10 captive individuals of unknown origin. An average of three alleles per maker was reported for the Cabanas population, evidencing a low genetic diversity. In addition, a recent bottleneck event was detected and an effective population size of 19.6 was estimated. Demographic reconstruction using a Bayesian approach was inconclusive possibly due to a small dataset and shallow coalescence trees obtained with the generated data. No clear structuring pattern was detected for the Cabanas population and most samples from individuals in captivity were found to have similar alleles to the ones from Cabanas. Population designation is challenging without the genotyping of every wild population, but unique alleles were found in captive individuals of unknown origin that could suggest that different genotypes might exist within other, less studied, wild populations. Low genetic diversity, and a small effective population size represent a risk for the Cabanas population facing the threats of isolation, habitat loss and climate change. These findings suggest that genetic management of the Cabanas population might be utilized to avoid high rates of inbreeding and subsequent inbreeding depression.


title: Decision-Making in Rats is Sensitive to Rare and Extreme Events: the Black Swan Avoidance

abstract:
In BriefMost studies assessing decision-making under uncertainty use events with probabilities that are above 10-20 %. Here, to study decision-making in radical uncertainty conditions, Degoulet, Willem, Baunez, Luchini and Pintus provide a novel experimental design that aims at measuring the extent to which rats are sensitive - and how they respond - to extremely rare (below 1% of probability) but extreme events in a four-armed bandit task. Gains (sugar pellets) and losses (time-out punishments) are such that large - but rare - values materialize or not depending on the option chosen. The results show that all rats diversify their choices across options. However, most rats exhibit sensitivity to rare and extreme events despite their sparse occurrence, by combining more often options with extreme gains (Jackpots) and/or avoidance of extreme losses (Black Swans). In general, most rats choices feature one-sided sensitivity in favor of trying more often to avoid extreme losses than to seek extreme gains - that is, they feature Black Swan Avoidance.

Highlights- A novel experiment is proposed to measure whether rats are sensitive - and how they respond - to radical uncertainty materialized by extremely rare and extreme events in a four-armed bandit task;
- Most rats exhibit sensitivity to rare and extreme events;
- Most rats opt more often to avoid rare and extreme losses than to seek rare and extreme gains: they feature Black Swan avoidance.

category: animal behavior and cognition


title: Comparison of Oxidative and Hypoxic Stress Responsive Genes from Meta-Analysis of Public Transcriptomes

abstract: Analysis of RNA-sequencing (RNA-seq) data is an effective means to analyze the gene expression levels under specific conditions and discover new biological knowledge. More than 74000 experimental series with RNA-seq have been stored in public databases as of October 20, 2021. Since this huge amount of expression data accumulated from past studies is a promising source of new biological insights, we focused on a meta-analysis of 1783 runs of RNA-seq data under the conditions of two types of stresses: oxidative stress (OS) and hypoxia. The collected RNA-seq data of OS were organized as the OS dataset to retrieve and analyze differentially expressed genes (DEGs). The OS-induced DEGs were compared with the hypoxia-induced DEGs retrieved from a previous study. The results from the meta-analysis of OS transcriptomes revealed two genes, CRIP1 and CRIP3, which were particularly downregulated, suggesting a relationship between OS and zinc homeostasis. The comparison between meta-analysis of OS and hypoxia showed that several genes were differentially expressed under both stress conditions, and it was inferred that the downregulation of cell cycle-related genes is a mutual biological process in both OS and hypoxia.


title: Validation analysis of EMDB entries

abstract: The Electron Microscopy Data Bank (EMDB) is the central archive of the electron cryo-microscopy (cryo-EM) community for storing and disseminating volume maps and tomograms. With input from the community, EMDB has developed new resources for validation of cryo-EM structures, focussing on the quality of the volume data alone and that of the fit of any models, themselves archived in the Protein Data Bank (PDB), to the volume data. Based on recommendations from community experts, the validation resources are developed in a three-tiered system. Tier 1 covers an extensive and evolving set of validation metrics, including tried and tested as well as more experimental ones, which are calculated for all EMDB entries and presented in the Validation Analysis (VA) web resource. This system is particularly useful for cryo-EM experts, both to validate individual structures and to assess the utility of new validation metrics. Tier 2 comprises a subset of the validation metrics covered by the VA resource that have been subjected to extensive testing and are considered to be useful for specialists as well as non-specialists. These metrics are presented on the entry-specific web pages for the entire archive on the EMDB website. As more experience is gained with the metrics included in the VA resource, it is expected that consensus will emerge in the community regarding a subset that is suitable for inclusion in the tier 2 system. Tier 3, finally, consists of the validation reports and servers that are produced by the Worldwide Protein Data Bank (wwPDB) Consortium. Successful metrics from tier 2 will be proposed for inclusion in the wwPDB validation pipeline and reports. We describe the details of the new resource, with an emphasis on the tier 1 system. The output of all three tiers is publicly available, either through the EMDB website (tiers 1 and 2) or through the wwPDB ftp sites (tier 3), although the content of all three will evolve over time (fastest for tier 1 and slowest for tier 3). It is our hope that these validation resources will help the cryo-EM community to get a better understanding of the quality, and the best ways to assess the quality of cryo-EM structures in EMDB and PDB.


title: Inhibition of BKCa channels protects neonatal hearts against myocardial ischemia and reperfusion injury

abstract: BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channels activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.


title: Influence of Response Criterion on Nociceptive Detection Thresholds and Evoked Potentials

abstract: Pain scientists and clinicians search for objective measures of altered nociceptive processing to study and stratify chronic pain patients. Nociceptive processing can be studied by observing a combination of nociceptive detection thresholds and evoked potentials. However, it is unknown whether the nociceptive detection threshold measured using a Go-/No-Go (GN) procedure can be biased by a response criterion. In this study, we compared nociceptive detection thresholds, psychometric slopes and central evoked potentials obtained during a GN procedure with those obtained during a 2-interval forced choice (2IFC) procedure to determine 1) if the nociceptive detection threshold during a GN procedure is biased by a criterion and 2) to determine if nociceptive evoked potentials observed in response to stimuli around the detection threshold are biased by a criterion. We found that the detection threshold can be higher when assessed using a GN procedure in comparison with the 2IFC procedure. The average P2 component in the central evoked potential showed on-off behavior with respect to stimulus detection and increased proportionally with the detection probability during a GN procedure. These data suggest that nociceptive detection thresholds estimated using a GN procedure are subject to a response criterion.


title: Sex and Circuit Specific Dopamine Transporter Regulation Underlies Unique Behavioral Trajectories of Functional SLC6A3 Coding Variation

abstract: Virtually all neuropsychiatric disorders display sex differences in prevalence, age of onset, and/or clinical symptomology. In sex-biased disorders, one sex is often suggested to harbor protective mechanisms, rendering them resilient to genetic and/or environmental risk factors. Here, we demonstrate sex-biased molecular, pharmacological and behavioral effects induced by the dopamine (DA) transporter (DAT) coding variant Ala559Val, previously identified in subjects diagnosed with the male-biased disorders attention-deficit/hyperactivity disorder and autism spectrum spectrum disorder. In DAT Val559 mice, we identified sex differences in response to psychostimulants, social behavior, and cognitive traits. We reveal a sex by circuit dissociation in D2-type autoreceptor (D2AR) regulation of DAT wherein D2AR-dependent DAT phosphorylation and trafficking, detectable in the male dorsal striatum, does not occur in females but rather is a property of the ventral striatum, predicting sex-specific changes in behavior. Consequently, we found that a subset of altered behaviors can be normalized using the D2R antagonist sulpiride in DAT Val559 mice. Our studies provide a cogent example of how sex shapes the behavioral trajectory of DA signaling perturbations and identify the sex-dependent, locality-selective capacity for D2AR regulation of DAT as an unrecognized determinant of this trajectory. Rather than identifying one sex as resilient, we find that sex can drive alterative behavioral patterns from shared signaling perturbations that may result in females being underreported. Our work underscores the utility of model systems to study the functional intrusions of rare genetic variation to gain insights into pathways underlying normal and perturbed trait domains associated with common neuropsychiatric conditions.


title: Fungal biodiversity in Arctic paleoecosystems assessed by metabarcoding of lake sedimentary ancient DNA

abstract: Fungi are crucial organisms in most ecosystems as they exert ecological key functions and are closely associated with land plants. Fungal community changes may therefore help reveal biodiversity changes in past ecosystems. Lake sediments contain DNA of organisms in the catchment area, which allows reconstructing past biodiversity by using metabarcoding of ancient sedimentary DNA. We developed a novel PCR primer combination for fungal metabarcoding targeting a short amplicon to account for length bias of amplification due to ancient DNA degradation. In-silico PCRs showed higher diversity using this primer combination than using previously established fungal metabarcoding primers. We analyzed existing data from sediment cores from four artic and one boreal lake in Siberia. These cores had been stored for 2-22 years and examined degradation effects of ancient DNA and storage time-related bias in fungal communities. Amplicon size differed between fungal divisions, however, we observed no significant effect of sample age on amplicon length and GC content, suggesting robust results. We also found no indication of post-coring fungal growth during storage distorting ancient fungal communities. Terrestrial soil fungi, including mycorrhizal fungi and saprotrophs, were predominant in all lakes, which supports the use of lake sedimentary ancient DNA for reconstructing terrestrial communities.


title: Multiple hybridization events punctuate the evolutionary trajectory of Malassezia furfur

abstract:
Malassezia species are important fungal skin commensals and are part of the normal microbiota of humans and other animals. However, under certain circumstances these fungi can also display a pathogenic behaviour. For example, Malassezia furfur is a common commensal of human skin, and yet is often responsible for skin disorders but also systemic infections. Comparative genomics analysis of M. furfur revealed that some isolates have a hybrid origin, similar to several other recently described hybrid fungal pathogens. Because hybrid species exhibit genomic plasticity that can impact phenotypes, we sought to elucidate the genomic evolution and phenotypic characteristics of M. furfur hybrids in comparison to their parental lineages. To this end, we performed a comparative genomics analysis between hybrid strains and their presumptive parental lineages, and assessed phenotypic characteristics. Our results provide evidence that at least two distinct hybridization events occurred between the same parental lineages, and that the parental strains may have originally been hybrids themselves. Analysis of the mating-type locus reveals that M. furfur has a pseudobipolar mating system, and provides evidence that after sexual liaisons of mating compatible cells, hybridization involved cell-cell fusion leading to a diploid/aneuploid state. This study provides new insights into the evolutionary trajectory of M. furfur and contributes with valuable genomic resources for future pathogenicity studies.

ImportanceMalassezia furfur is a common commensal member of human/animal microbiota that is also associated with several pathogenic states. Recent studies report involvement of Malassezia species in Crohns disease, a type of inflammatory bowel disease, pancreatic cancer progression, and exacerbation of Cystic fibrosis. A recent genomics analysis of M. furfur revealed the existence of hybrid isolates and identified their putative parental lineages. In this study, we explored the genomic and phenotypic features of these hybrids in comparison to their putative parental lineages. Our results revealed the existence of a pseudobipolar mating system in this species and showed evidence for the occurrence of multiple hybridization events in the evolutionary trajectory of M. furfur. These findings significantly advance our understanding of the evolution of this commensal microbe and are relevant for future studies exploring the role of hybridization in the adaptation to new niches or environments, including the emergence of pathogenicity.


title: Estimated limits of organism-specific training for epitope prediction

abstract:
BackgroundThe identification of linear B-cell epitopes remains an important task in the development of vaccines, therapeutic antibodies and several diagnostic tests. Machine learning predictors are trained to flag potential epitope candidates for experimental validation and currently, most predictors are trained as generalist models using large, heterogeneous data sets. Recently, organism-specific training has been shown to improve prediction performance for data-rich organisms. Unfortunately, for most organisms, large volumes of validated epitope data are not yet available. This article investigates the limits of organism-specific training for epitope prediction. It explores the validity of organism-specific training for data-poor organisms by examining how the size of the training data set affects prediction performance. It also compares the performance of organism-specific training under simulated data-poor conditions to that of models trained using traditional large heterogeneous and hybrid data sets.

ResultsThis work shows how models trained on small organism-specific data sets can outperform similar models trained on (potentially much larger) heterogeneous and mixed data sets. The results reported indicate that as few as 20 labelled peptides from a given pathogen can be sufficient to generate models that outperform widely-used predictors from the literature, which are trained on heterogeneous data. Models trained using more than about 100 to 150 organism-specific peptides perform consistently better than most generalist models across a wide variety of performance measures, and in some cases can even approach the performance of organism-specific models trained on considerably larger data sets.

ConclusionsOrganism-specific training improves linear B-cell epitope prediction performance even in situations when only small training sets are available, which opens new possibilities for the development of bespoke, high-performance predictive models when studying data-poor organisms such as emerging or neglected pathogens.


title: Exploration and analysis of R-loop mapping data with RLBase

abstract: R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA during nascent transcription. In 2012, Ginno et al. introduced the first R-loop mapping method, DNA:RNA immunoprecipitation (DRIP) sequencing. Since that time, dozens of studies have implemented R-loop mapping and new high-resolution techniques have been developed. The resulting datasets have tremendous potential to reveal the causes and consequences of R-loops genome-wide. However, poor quality and variability between mapping approaches pose serious barriers to the meta-analysis of these data. In our recent work, we reprocessed 693 R-loop mapping samples, devising new quality methods, defining a set of high-confidence mapping samples, and then deriving R-loop regions, consensus sites of R-loop formation. This analysis yielded the largest R-loop data resource to date along with novel computational approaches for R-loop mapping analysis. Now, we introduce RLBase, an innovative web server which builds upon those data and software by providing users with the capability to (1) explore hundreds of public R-loop mapping datasets, (2) explore consensus R-loop regions, (3) analyze user-supplied datasets to generate an HTML quality report, and (4) download all the processed data for the 693 samples we previously reprocessed and standardized. In addition to RLBase, we also describe the other software which, along with RLBase, provides a computational framework for R-loop bioinformatics. RLBase, and the rest of these software (termed "RLSuite"), are provided freely under an MIT license and made publicly available: https://gccri.bishop-lab.uthscsa.edu/rlsuite/. RLBase is directly accessible via the following URL: https://gccri.bishop-lab.uthscsa.edu/rlbase/.


title: MS2Rescore: Data-driven rescoring dramatically boosts immunopeptide identification rates

abstract: Immunopeptidomics aims to identify immunopeptides, which are presented on Major Histocompatibility Complexes (MHC) on every cell and can be used to develop vaccines against pathogens and cancer. However, existing immunopeptidomics data analysis pipelines have some major hurdles to overcome, mostly due to the non-tryptic nature of immunopeptides, which complicates their identification. Previously, the machine and deep learning tools MS2PIP and DeepLC have shown to improve tryptic peptide identifications by accurately predicting tandem mass spectrometry (MS2) peak intensities and retention times, respectively, and by using these predictions to rescore peptide-spectrum matches (PSMs) with post-processing tool Percolator. However, MS2PIP was still tailored towards tryptic peptides and fragmentation patterns of immunopeptides are drastically different. To enable MS2PIP-based rescoring of immunopeptide PSMs, we have retrained MS2PIP to include non-tryptic peptides. These newly trained MS2PIP models greatly improve the predictions for immunopeptides and, surprisingly, also for tryptic peptides. Next, the new MS2PIP models, DeepLC, and Percolator were integrated into one software package, called MS2Rescore. Using MS2Rescore, 46% more spectra and 36% more unique peptides were identified at 1% false discovery rate (FDR), with even more extreme differences at 0.1% FDR, in comparison with standard Percolator rescoring. Due to the innovative extraction of MS2PIP-, DeepLC and search engine-based features, MS2Rescore even outperforms current state-of-the-art immunopeptide rescoring efforts. Thus, the integration of the new immunopeptide MS2PIP models, DeepLC, and Percolator into MS2Rescore shows great promise to substantially improve the identification of novel epitopes from immunopeptidomics workflows.


title: Different structures and pathologies of alpha-synuclein fibrils derived from preclinical and postmortem patients of Parkinson's disease

abstract:
-Synuclein (-syn) fibrillar aggregates are the major component of Lewy bodies and Lewy neurites presenting as the pathology hallmark of Parkinsons disease (PD). Studies have shown that -syn is potential to form different conformational fibrils associated with different synucleinopathies, but whether the conformation of -syn fibrils changes in different phases of related diseases is to be explored. Here, we amplified -syn aggregates from the cerebrospinal fluid (CSF) of preclinical (pre-PD) and late-stage postmortem PD (post-PD) patients. Our results show that compared to the CSF of pre-PD, that of post-PD is markedly stronger in seeding in vitro -syn aggregation, and the amplified fibrils are more potent in inducing endogenous -syn aggregation in neurons. Cryo-electron microscopic structures further reveal that the difference between the pre-PD- and post-PD-derived fibrils lies on a minor polymorph which in the pre-PD fibrils is morphologically straight, while in the post-PD fibrils represents a single protofilament assembled by a distinctive conformation of -syn. Our work demonstrates structural and pathological differences between pre-PD and post-PD -syn aggregation and suggests potential alteration of -syn fibrils during the progression of PD clinical phases.

Significance StatementIncreasing evidence support different conformational -syn fibrils in patients with different -synucleinopathies, but whether the conformation of -syn fibrils changes in different phases of related diseases is unknown. Here, we show that -syn fibrils amplified from the cerebrospinal fluid (CSF) of the late-stage postmortem PD (post-PD) patient are more potent in inducing endogenous -syn aggregation in neurons than that amplified from the preclinical (pre-PD) patient. Cryo-EM structures further reveal that the post-PD fibrils contain a novel conformation that is distinct from either the pre-PD fibrils or those previously reported. Our work suggests conformational evolution of -syn fibrils along with PD progression.

category: biophysics


title: Plasticity in Ligand Recognition at Somatostatin Receptors

abstract:
Somatostatin is a signaling peptide that plays a pivotal and wide-ranging role in physiologic processes relating to metabolism and growth through its actions at somatostatin receptors (SSTRs). Members of the somatostatin receptor subfamily, particularly SSTR2, are key drug targets for neuroendocrine neoplasms, with several synthetic peptide agonists currently in clinical use. Here, we show the cryogenic electron microscopy (cryo-EM) structures of active-state SSTR2 in complex with heterotrimeric Gi protein and either the endogenous ligand SST14 or the FDA-approved drug octreotide.

Complemented by biochemical assays and molecular dynamics simulations, these structures reveal key details of ligand recognition, receptor activation, and subtype-selectivity at somatostatin receptors. We find that SSTR ligand recognition is highly diverse, as demonstrated by ligand-induced conformational changes in ECL2, substantial sequence divergence across subtypes in extracellular regions, and loss of ligand binding upon several structurally homologous substitutions between subtypes. Despite this complexity, we rationalize several previously described sources of SSTR subtype selectivity and identify an additional key interaction for SSTR2/3/5 specific binding. These results shed light on the basis of ligand recognition by somatostatin receptors and provide valuable insights for structure-based drug discovery at these targets.