Fixed or random? On the reliability of mixed-effects models for a small number of levels in grouping variables O_LIBiological data are often intrinsically hierarchical. Due to their ability to account for such dependencies, mixed-effects models have become a common analysis technique in ecology and evolution. While many questions around their theoretical foundations and practical applications are solved, one fundamental question is still highly debated: When facing a low number of levels should we model a grouping (blocking, clustering) variable as a random or fixed effect? In such situation, the variance of the random effect is imprecise, but whether this affects the statistical properties of the population effect is unclear.
C_LIO_LIHere, we analyzed the consequences of including a grouping variable as fixed or random effect in the correctly specified and other possible miss-specified models (too simple or too complex models) for data with small number of levels (2 - 8). For all these options, we calculated type I error rates and power. Moreover, we show how these statistical properties change with the study design.
C_LIO_LIWe found that the model choice does not influence the statistical properties of the population effect when the effect is the same at all levels However, if an ecological effect differs among levels, using a random slope and intercept model, and switching to a fixed-effect model only in case of a singular fit, avoids overconfidence in the results. Additionally, power and type I error are strongly influenced by the number of and difference between levels.
C_LIO_LIWe conclude that inferring the correct random effect structure is of high importance to get correct statistical properties. When in doubt, we recommend starting with the simpler model and using model diagnostics to identify missing components. When having identified the correct structure, we encourage to start with a mixed-effects model independent of the number of levels and switch to a fixed-effect model only in case of a singular fit. With these recommendations, we allow for more informative choices about study design and data analysis and thus make ecological inference with mixed-effects models more robust for small number of levels.
C_LI
Scaling effects of temperature on parasitism from individuals to host-parasite systems Parasitism is expected to change in a warmer future, but whether warming leads to substantial increases in parasitism remains unclear. Understanding how warming effects on parasitism in individual hosts (e.g., parasite load) translate to effects on population-level parasitism (e.g., prevalence, R0) remains a major knowledge gap. We analyzed the temperature dependence of parasitism at both host and population levels in thirteen empirical vector-borne host-parasite systems and found a strong, significant positive correlation between the thermal optima of individual- and population-level parasitism. We also found a significant, positive correlation in eleven environmentally-transmitted parasite systems, though several of these systems exhibited thermal optima >5{o}C apart between individual and population levels. Similarly, parasitism thermal optima were close to host performance thermal optima in vector-borne systems but not in environmentally-transmitted systems. We then adapted and simulated simple models for both transmission modes and found a similar pattern to the empirical systems: thermal optima in vector-borne systems were more strongly correlated across scales compared to environmentally-transmitted systems. Generally, our results suggest that information on the temperature-dependence, and specifically the thermal optimum, at either the individual- or population-level should provide a useful--though not quantitatively exact--baseline for predicting temperature dependence at the other level, especially in vector-borne parasite systems. Environmentally-transmitted parasitism may operate by a different set of rules, in which temperature-dependence is decoupled in some systems, requiring the need for trait-based studies of temperature-dependence at individual and population levels.
Integrative small and long RNA-omics analysis of human healing and non-healing wounds discovers cooperating microRNAs as therapeutic targets MicroRNAs (miR) are important posttranscriptional regulators and exhibit a high potential to be utilized in diagnosis and therapy. However, our insufficient knowledge of the miR-mediated gene regulation in human skin wound healing severely hinders the identification of clinically relevant miRs. Here, we performed paired small RNA and long RNA sequencing in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic non-healing venous ulcers (VU). With integrative small and long RNA-omics analysis, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte growth while promoting inflammatory response. Collectively, our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity. Characterizing and explaining impact of disease-associated mutations in proteins without known structures or structural homologues Mutations in human proteins lead to diseases. The structure of these proteins can help understand the mechanism of such diseases and develop therapeutics against them. With improved deep learning techniques such as RoseTTAFold and AlphaFold, we can predict the structure of proteins even in the absence of structural homologues. We modeled and extracted the domains from 553 disease-associated human proteins without known protein structures or close homologues in the Protein Databank (PDB). We noticed that the model quality was higher and the RMSD lower between AlphaFold and RoseTTAFold models for domains that could be assigned to CATH families as compared to those which could only be assigned to Pfam families of unknown structure or could not be assigned to either. We predicted ligand-binding sites, protein-protein interfaces, conserved residues in these predicted structures. We then explored whether the disease-associated missense mutations were in the proximity of these predicted functional sites, if they destabilized the protein structure based on ddG calculations or if they were predicted to be pathogenic. We could explain 80% of these disease-associated mutations based on proximity to functional sites, structural destabilization or pathogenicity. When compared to polymorphisms a larger percentage of disease associated missense mutations were buried, closer to predicted functional sites, predicted as destabilising and/or pathogenic. Usage of models from the two state-of-the-art techniques provide better confidence in our predictions, and we explain 93 additional mutations based on RoseTTAFold models which could not be explained based solely on AlphaFold models. Live imaging YAP signaling in mouse embryo development YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATs kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behavior of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signaling in vivo in many different situations. Informative neural representations of unseen contents during higher-order processing in human brains and deep artificial networks A framework to pinpoint the scope of unconscious processing is critical to improve our models of visual consciousness. Previous research observed brain signatures of unconscious processing in visual cortex but these were not reliably identified. Further, whether unconscious content is represented in high-level stages of the ventral visual stream and linked parieto-frontal areas remains unknown. Using a within-subject, high-precision fMRI approach, we show that unconscious contents can be decoded from multivoxel patterns that are highly distributed alongside the ventral visual pathway and also involving parieto-frontal substrates. Classifiers trained with multivoxel patterns of conscious items generalised to predict the unconscious counterparts, indicating that their neural representations overlap. These findings suggest revisions to models of consciousness such as the neuronal global workspace. We then provide a computational simulation of visual processing/representation without perceptual sensitivity by using deep neural networks performing a similar visual task. The work provides a framework for pinpointing the representation of unconscious knowledge across different task domains. Pulsatile contractions and pattern formation in excitable actomyosin cortex The actin cortex is an active adaptive material, embedded with complex regulatory networks that can sense, generate, and transmit mechanical forces. The cortex exhibits a wide range of dynamic behaviours, from generating pulsatory contractions and travelling waves to forming organised structures. Despite the progress in characterising the biochemical and mechanical components of the actin cortex, the emergent dynamics of this mechanochemical system is poorly understood. Here we develop a reaction-diffusion model for the RhoA signalling network, the upstream regulator for actomyosin assembly and contractility, coupled to an active actomyosin gel, to investigate how the interplay between chemical signalling and mechanical forces regulate stresses and patterns in the cortex. We demonstrate that mechanochemical feedback in the cortex acts to destabilise homogeneous states and robustly generate pulsatile contractions. By tuning active stress in the system, we show that the cortex can generate propagating contraction pulses, form network structures, or exhibit topological turbulence. Super-enhancer signature reveals key mechanisms associated with resistance to non-alcoholicsteatohepatitis in humans with obesity The molecular underpinnings of non-alcoholic steatohepatitis (NASH) development in patients are poorly understood. Active enhancer landscapes are known to determine cell states and behaviors. Super-enhancers, in particular, have helped reveal key disease drivers in several cancer types; however, they remain unexplored in human NASH. To define the enhancer signature of NASH-prone (NP) and NASH-resistant (NR) phenotypes in humans with obesity, we performed chromatin run-on sequencing (ChRO-seq) analysis on liver biopsies of individuals with obesity who were stratified into either NP or NR. We first demonstrated that NP and NR groups exhibit distinct active enhancer signatures. The subsequent identification of NP- and NR-specific super-enhancers revealed the specific genes that are likely the most critical for each of the phenotypes, including HES1 for NP and GATM for NR. Integrative analysis with results from genome-wide association studies of NAFLD and related traits identified disease/trait-loci specific to NP or NR enhancers. Further analysis of the ChRO-seq data pointed to critical roles for serine/glycine metabolism in NASH resistance, which was corroborate by profiling of circulating amino acids in the same patients. Overall, the distinct enhancer signatures of human NP and NR phenotypes revealed key genes, pathways, and transcription factor networks that promote NASH development. Catchet-MS identifies IKZF1-targeting Thalidomide analogues as novel HIV-1 latency reversal agents A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has not been possible. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to describe the protein composition of the latent and activated HIV-1 5LTR. Catchet-MS identified known and novel latent 5LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs that, by targeting IKZF1 for degradation, reverse HIV-1 latency in CD4+T-cells isolated from virally suppressed people living with HIV-1.

One Sentence SummarydCas9 targeted chromatin and histone enrichment for mass spectrometry (Catchet-MS) led to the identification of IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology. Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signaling being particularly affected, especially glutamatergic synapse signaling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to the long-term infection by this parasite. Anchorage by seed mucilage prevents seed dislodgement in high surface flow: a mechanistic investigation O_LIBackground and Aims: Seed mucilage is a common and highly diverse trait shared among thousands of angiosperm species. While long recognized that mucilage allows seeds to anchor to substrates (antitelechory), resisting abiotic and biotic dislodgement, we still lack a mechanistic understanding of this process.
C_LIO_LIMethods: We propose a mechanistic model of how mucilage affects substrate anchorage and fluid resistance, ultimately contributing to dislodgement resistance. To test this model, we subjected mucilaginous seeds of 52 species, varying in eight measured seed traits, to seven days of continuous water flow at a range of dislodgement potentials.
C_LIO_LIKey Results: Supporting our model, mucilage mass increased force necessary to dislodge both dry and wet seeds; our measurement of the dislodgement force of dry mucilage explained time to dislodgement well. The effect size was remarkably large; increasing the standardized mucilage mass by one standard deviation resulted in a 280-fold increase in the time to dislodgement. Fluid resistance was largely dependent on speed of water flow and the seeds modified drag coefficient, but not seed traits. Neither mucilage expansion speed nor mucilage decay rate explained dislodgement potential well.
C_LIO_LIConclusions: Our results suggest that the degree of anchorage to substrate, measured with a simple dislodgement force assay, is highly predictive of mucilaginous seed retention in highly erosive environments. In contrast, we found that other seed and mucilage traits are of lesser importance to anchorage.
C_LI Striatin 3 and MAP4K4 cooperate towards oncogenic growth and tissue invasion in medulloblastoma MAP4K4 has been correlated with increased cell motility and reduced proliferation in mammalian cells. Consequences and regulation of this dichotomous functionality of MAP4K4 in tumor cells remained elusive. We find that MAP4K4 interacts with the STRN3 protein in medulloblastoma and that STRN3 and MAP4K4 exert opposing functions in Hippo tumor suppressor signaling and clonal growth. However, depletion of either STRN3 or MAP4K4 reduces migration and tissue invasion, and concomitant loss of both proteins halts tumor cell growth in the cerebellar tissue. Mechanistically, STRN3 couples MAP4K4 to the protein phosphatase 2A, which inactivates growth repressing activities of MAP4K4. In parallel, STRN3 enables growth factor-induced PKC{theta} activation and the direct phosphorylation of VASPS157 by MAP4K4, which are necessary for efficient migration and tissue invasion. Thus, STRN3 controls MAP4K4 functionality in growth factor-activated tumor cells, and the cooperation of the two proteins promotes invasiveness through nPKCs and VASP and increases proliferation via inactivation of Hippo signaling. Targeting of the STRN3-MAP4K4 cooperation could reactivate Hippo signaling and repress invasiveness in growth factor-driven tumors.

Graphical abstract

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org.highwire.dtl.DTLVardef@102b3c1org.highwire.dtl.DTLVardef@6753ceorg.highwire.dtl.DTLVardef@1af2ff5org.highwire.dtl.DTLVardef@759127_HPS_FORMAT_FIGEXP  M_FIG C_FIG Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Sharpened and mechanically durable carbon fiber electrode arrays for neural interfacing Bioelectric medicine treatments target disorders of the nervous system unresponsive to pharmacological methods. While current stimulation paradigms effectively treat many disorders, the underlying mechanisms are relatively unknown, and current neuroscience recording electrodes are often limited in their specificity to gross averages across many neurons or axons. Here, we develop a novel, durable carbon fiber electrode array adaptable to many neural structures for precise neural recording. Carbon fibers were sharpened using a reproducible blowtorch method that uses the reflection of fibers against the surface of a water bath. The arrays were developed by partially embedding carbon fibers in medical-grade silicone to improve durability. We recorded acute spontaneous electrophysiology from the rat cervical vagus nerve (CVN), feline dorsal root ganglia (DRG), and rat brain. Blowtorching resulted in fibers of 72.3 {+/-} 33.5 degree tip angle with 146.8 {+/-} 17.7 m exposed carbon. Silicone-embedded carbon fiber arrays were durable with 87.5% of fibers remaining after 50,000 passes. Observable neural clusters were recorded using sharpened carbon fiber electrodes from rat CVN (41.8 Vpp), feline DRG (101.1 Vpp), and rat brain (80.7 Vpp). Recordings from the feline DRG included physiologically-relevant signals from increased bladder pressure and cutaneous brushing. These results suggest that this carbon fiber array is a uniquely durable and adaptable neural recording device. In the future, this device may be useful as a bioelectric medicine tool for diagnosis and closed-loop neural control of therapeutic treatments and monitoring systems. Severe COVID-19 shares a common neutrophil activation signature with other acute inflammatory states Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaky disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflamatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention. Royal knifefish generate powerful suction feeding through large neurocranial elevation and high epaxial muscle power Suction feeding in ray-finned fishes involves powerful buccal cavity expansion to accelerate water and food into the mouth. Previous XROMM studies in largemouth bass (Micropterus salmoides), bluegill sunfish (Lepomis macrochirus), and channel catfish (Ictalurus punctatus) have shown that more than 90% of suction power in high performance strikes comes from the axial musculature. Thus, the shape of the axial muscles and skeleton may impact suction feeding mechanics. Royal knifefish (Chitala blanci) have an unusual postcranial morphology, with a ventrally flexed vertebral column and relatively large mass of epaxial muscle. Based on their body shape, we hypothesized that royal knifefish would generate high power strikes by utilizing large neurocranial elevation, vertebral column extension, and epaxial shortening. As predicted, C. blanci generated high suction expansion power compared to the other three species studied to date (up to 160 W), which was achieved by increasing both the rate of volume change and the intraoral subambient pressure. The large epaxial muscle (25% of body mass) shortened at high velocities to produce large neurocranial elevation and vertebral extension (up to 41 deg, combined), as well as high muscle mass-specific power (up to 800 W kg-1). For the highest power strikes, axial muscles generated 95% of the power, and 64% of the axial muscle mass consisted of the epaxial muscles. The epaxial-dominated suction expansion of royal knifefish supports our hypothesis that postcranial morphology may be a strong predictor of suction feeding biomechanics.

SUMMARY STATEMENTRoyal knifefish rely on their distinct postcranial morphology--with a curved vertebral column and large dorsal body muscles--to produce large neurocranial elevation and powerful suction feeding. A multiscale lipid and cellular atlas of the human kidney The human kidney is composed of over 26 cell types that actively coordinate with each other to form higher-order structures, such as the nephron. It is not yet understood how these structures vary throughout a single organ or amongst the same organs within the human population. We have developed an extensive lipid and cellular atlas of the human kidney consisting of over 3 million cells comprising 75,000 functional tissue units (i.e., glomeruli, proximal tubules, distal tubules, and collecting ducts) from 13 human subjects. This atlas was developed using spatially registered and integrated technologies consisting of imaging mass spectrometry, multiplexed immunofluorescence, stained microscopy, and autofluorescence microscopy, to comprehensively probe large (i.e., centimeter-sized) areas of tissue. The cellular organization and lipid profiles of glomeruli, proximal tubules, distal tubules, and collecting ducts were discovered through these multimodal imaging data as well as their intra- and inter-subject variance. Relating the lipid profiles obtained from imaging mass spectrometry to distinct cell types obtained from immunofluorescence allowed us to hypothesize the functional role of specific phospholipids that have not previously been described. These hypotheses include subject characteristics, such as BMI and sex. The integrated data from the aforementioned datasets provide a valuable reference for kidney researchers, are publicly available through the NIH Human Biomolecular Atlas Program (https://portal.hubmapconsortium.org/), and discussed below. SARS-CoV-2 infects lung epithelial cells and induces senescence and an inflammatory response in patients with severe COVID-19 RationaleSARS-CoV-2 infection of the respiratory system can progress to a life threatening multi-systemic disease, mediated via an excess of cytokines ("cytokine storm"), but the molecular mechanisms are poorly understood.

ObjectivesTo investigate whether SARS-CoV-2 may induce cellular senescence in lung epithelial cells, leading to secretion of inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP).

MethodsAutopsy lung tissue samples from eleven COVID-19 patients and sixty age-matched non-infected controls were analysed by immunohistochemistry for SARS-CoV-2 and markers of cellular senescence (SenTraGor, p16INK4A) and key SASP cytokines (interleukin-1{beta}, interleukin-6). We also investigated whether SARS-CoV-2 infection of an epithelial cell line induces senescence and cytokine secretion.

Measurements and Main ResultsSARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in alveolar type-2 (AT2) cells, which also expressed the angiotensin-converting-enzyme 2 (ACE2), a critical entry receptor for this virus. In COVID-19 samples, AT2 cells displayed increased markers of senescence [p16INK4A, SenTraGor staining positivity in 12{+/-}1.2% of cells compared to 1.7{+/-}0.13% in non-infected controls (p<0.001)], with markedly increased expression of interleukin-1{beta} and interleukin-6 (p<0.001). Infection of epithelial cells (Vero E6) with SARS-CoV-2 in-vitro induced senescence and DNA damage (increased SenTraGor and {gamma}-H2AX), and reduced proliferation (Ki67) compared to uninfected control cells (p<0.01).

ConclusionsWe demonstrate that in severe COVID-19 patients, AT2 cells are infected with SARS-CoV-2 and show senescence and expression of proinflammatory cytokines. We also show that SARS-CoV-2 infection of epithelial cells may induce senescence and inflammation, indicating that cellular senescence may be an important molecular mechanism of severe COVID-19. Multivariate genome-wide association study on tissue-sensitive diffusion metrics identifies key molecular pathways for axonal growth, synaptogenesis, and astrocyte-mediated neuroinflammation The molecular determinants of tissue composition of the human brain remain largely unknown. Recent genome-wide association studies (GWAS) on this topic have had limited success due to methodological constraints. Here, we apply advanced whole-brain analyses on multi-shell diffusion imaging data and multivariate GWAS to two large scale imaging genetic datasets (UK Biobank and the Adolescent Brain Cognitive Development study) to identify and validate genetic association signals. We discovered 503 unique genetic loci that have impact on multiple regions of human brain. Among them, more than 79% were validated in either of two large-scale independent imaging datasets. Key molecular pathways involved in axonal growth, astrocyte-mediated neuroinflammation, and synaptogenesis during development were found to significantly impact the measured variations in tissue-specific imaging features. Our results shed new light on the biological determinants of brain tissue composition and their potential overlap with the genetic basis of neuropsychiatric disorders. The important choice of reference environment in microevolutionary climate response predictions It is well documented that individuals of wild populations can adjust to climate change by means of phenotypic plasticity, but few reports on adaptation by means of genetically based microevolution caused by selection. Disentanglement of these separate effects requires that the reference environment (the environmental zero-point) is defined, and this should not be done arbitrarily. The problem is that an error in the reference environment may lead to large errors in predicted microevolution. Together with parameter values and initial mean trait values, the reference environment can be estimated from environmental, phenotypic and fitness data. A prediction error method for this purpose is described, with the feasibility shown by simulations. As shown in a toy example, an estimated reference environment may have large errors, especially for small populations. This may still be a better choice than use of an initial environmental value in a recorded time series, or the mean value, which is often used. Another alternative may be to use the mean value of a past and stationary stochastic environment, which the population is judged to have been fully adapted to, in the sense that the expected geometric mean fitness was at a global maximum. Exceptions are cases with constant phenotypic plasticity, where the microevolutionary changes per generation follow directly from phenotypic and environmental data, independent of the chosen reference environment. Ultrahigh-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Desorption Electrospray Ionization Mass Spectrometry Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultrahigh-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 M 1-hydroxymidazolam and 12 M dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with ~30s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115,200 samples from 300 microtiter plate reads consecutively over 5.54 hours with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high throughput compound screening.

For Table of Contents Only

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org.highwire.dtl.DTLVardef@5f916corg.highwire.dtl.DTLVardef@4473b4org.highwire.dtl.DTLVardef@82e8forg.highwire.dtl.DTLVardef@128b57b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Optimization of avian perching manoeuvres Perching at speed is amongst the most challenging flight behaviours that birds perform1,2, and beyond the capability of current autonomous vehicles. Smaller birds may touchdown by hovering3-8, but larger birds typically swoop upward to perch1,2 - presumably because the adverse scaling of their power margin prohibits slow flapping flight9, and because swooping transfers excess kinetic to potential energy1,2,10. Perching is risky in larger birds6,11, demanding precise control of velocity and pose12-15, but it is unknown how they optimize this challenging manoeuvre. More generally, whereas cruising flight behaviours such as migration and commuting are adapted to minimize cost-of-transport or time-of-flight16, the optimization of unsteady flight manoeuvres remains largely unexplored7,17. Here we show that swooping minimizes neither the time nor energy required to perch safely in Harris hawks Parabuteo unicinctus, but instead minimizes the distance flown under hazardous post-stall conditions. By combining motion capture data from 1,563 flights with flight dynamics modelling, we found that the birds choice of where to transition from powered dive to unpowered climb minimizes the distance from the landing perch over which very high lift coefficients are required. Time and energy are therefore invested to maintain the control authority needed to execute a safe landing, rather than being minimized continuously as in technical applications of autonomous perching under nonlinear feedback control13 and deep reinforcement learning18,19. Naive birds learn this behaviour on-the-fly, so our findings suggest an alternative reward function for reinforcement learning of autonomous perching in air vehicles. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Small extracellular vesicles from young mice prevent frailty, improve healthspan and decrease epigenetic age in old mice. Aging is associated with an increased risk of frailty, disability, comorbidities, institutionalization, falls, fractures, hospitalization, and mortality. Searching for strategies to delay the degenerative changes associated with aging and frailty is interesting. We treated old animals intravenously with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals, and we found an improvement of several functional parameters usually altered with aging, such as motor coordination, grip strength, fatigue resistance, fur regeneration, and renal function. Frailty index analysis showed that 40% of old control mice were frail, whereas none of the old ADSCs-sEVs treated mice were. Molecular and structural benefits in muscle and kidney accompanied this functional improvement. ADSCs-sEVs induced pro-regenerative effects and a decrease in oxidative stress, inflammation, and senescence markers. Moreover, predicted epigenetic age was lower in tissues of old mice treated with ADSCs-sEVs and their metabolome changed to a youth-like pattern. Finally, we gained some insight into the miRNAs contained in sEVs that might be, at least in part, responsible for the effects observed. We propose that young sEVs treatment can be beneficial against frailty and therefore can promote healthy aging. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system.