Feed efficiency of lactating Holstein cows was not as repeatable across diets as within diet over subsequent lactation stages BackgroundImproving feed efficiency has become a common target for dairy farmers to meet the requirement of producing more milk with fewer resources. To improve feed efficiency, a prerequisite is to ensure that the cows identified as most or least efficient will remain as such, independently of diet composition. Therefore, the current research analysed the ability of lactating dairy cows to maintain their feed efficiency while changing the energy density of the diet by changing its concentration in starch and fibre. A total of 60 lactating Holstein cows, including 33 primiparous cows, were first fed a high starch diet-low fibre (diet S+F-), then switched over to a low starch diet-high fibre (diet S-F+). To know if diet affect feed efficiency, we compared the ability of feed efficiency to be maintained within a diet over subsequent lactation stages, known as repeatability of feed efficiency, with its ability to be maintained across diets, known as reproducibility of feed efficiency. To do so we used two indicators: the estimation of the error of repeatability/reproducibility, which is commonly used in metrology, and the coefficient of correlation of concordance (CCC), which is used in biology. The effect of diet change could also lead to a change in cows sorting behaviour which could potentially affect feed efficiency if for example the most efficient cows select more concentrate than the least efficient. We therefore analysed the relationship between the differences in individual feed refusals composition and the differences in feed efficiency. To do so, the composition of each feed refusal was described with its near infra-red (NIR) spectroscopy and was performed on each individual feed ingredient, diet and refusals and used as composition variable. The variability of the NIR spectra of the refusals was described with its principal components thanks to a principal component analysis (PCA). The Pearson correlation was estimated to check the relationship between feed efficiency and refusals composition, i.e. sorting behaviour.

ResultsThe error of reproducibility of feed efficiency across diets was 2.95 MJ/d. This error was significantly larger than the errors of repeatability estimated within diet, which were 2.01 MJ/d within diet S-F+ and 2.40 MJ/d within diet S+F-. The CCC was 0.64 between feed efficiency estimated within diet S+F- and feed efficiency estimated within diet S-F+. This CCC was smaller than the one observed for feed efficiency estimated within diet between two subsequent lactation stages (CCC = 0.72 within diet S+F- and 0.85 within diet S-F+). Feed efficiency was poorly correlated to the first two principal components, which explained 90% of the total variability of the NIR spectra of the individual refusals. This suggests that feed sorting behaviour did not explain differences in feed efficiency.

ConclusionsFeed efficiency was significantly less reproducible across diets than repeatable within the same diet over subsequent lactation stages, but cows ranking for feed efficiency was not significantly affected by diet change. This loss in repeatability across diets could be due to a more pronounced feed sorting subsequent to the change in diet composition. However, the differences in sorting behaviour between cows were not associated to feed efficiency differences in this trial neither with the S+F- diet nor with the S-F+ diet. Those results have to be confirmed on diets having different forage to concentrate ratios to ensure that the least and most efficient cows will not change.
Population differences in the length and early-life dynamics of telomeres among European pied flycatchers Telomere length and shortening rate are increasingly used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between- population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the non-breeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north- south gradient (Finland, Estonia, England, and Spain) predicting negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from mid-latitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level.
Are narrow-ranging species doomed to extinction? Projected dramatic decline in future climate suitability of two highly threatened species Narrow-ranging species are usually omitted from Species distribution models (SDMs) due to statistical constraints, while they are predicted to be particularly vulnerable to climate change. The recently available high-resolution environmental predictors, along with recently developed methods enable to increase the eligibility of narrow-ranging species for SDMs, provided their distribution is well known. We fill a gap of knowledge on the effect of predicted climate change on narrow-ranging species. We modelled the distribution of the golden mantella frog Mantella aurantiaca and the Manapany day gecko Phelsuma inexpectata, for which the distribution of their occurrence records is well documented. Our modelling scheme included a range of processes susceptible to address statistical issues related to narrow-ranging species. We predict an alarming decline in climate suitability in the whole current distribution area of both species by 2070, potentially leading to a complete extinction in most scenarios. We identified the areas with the best climate suitability in the future, but these remain largely suboptimal regarding species climatic niche. The high level of habitat fragmentation suggests that both species likely need to be at least partly translocated. Climate change may not only drive range contractions or distribution shifts in narrow-ranging species, but may lead to the complete extirpation of suitable environments across their entire region. This study suggests that the level of threats of narrow-ranging species already identified as threatened may be underestimated, especially in heterogeneous tropical areas. We stress the need to develop sampling campaigns and implement proactive actions for narrow-ranging species in the tropics. Game of theories: an agent-based model of how variation in contest cost-benefits determines the evolution of contest resolution rules Game-theory based models are used to understand rules that animals use to settle contests over indivisible resources. However, the empirical literature of contests indicates controversial support to models, with some species supporting different models and other species showing no support to any model. Since strategies used to resolve contests may have different associated costs, it is possible that different conditions have determined the evolution of distinct assessment strategies used by animals. We used an agent-based model to explore the importance of the following conditions: resource availability, probability of reproduction with resource, and damage costs on evolution of assessment strategies. We used self- and mutual-assessment models as a heurist framework to build agents with different assessment strategies. In our model, agents competed for resources in scenarios with different combinations of resource availability, probability of reproduction with resource, and damage costs. We found that agents following the self-assessment with damage strategy were prevalent in scenarios with no probability of reproduction without the resource, independently of other variables. We also found that agents following the non-aggressive strategy occurred in all scenarios. However, agents using the non-aggressive strategy were prevalent only in scenarios with probability of reproduction with the resource. Finally, we observed that agents using mutual-assessment occurred only in a scenario with high risk of damage, low availability of resources, and with probability of reproduction without the resource. These results indicate that agents following the self-assessment with damage and non-aggressive strategies may be able to stay at most scenarios. Tracing back variations in archaeal cell division to protein domain architectures The Endosomal Sorting Complex Required for Transport (ESCRT) system is a multi-protein machinery that is involved in cell division of both Eukaryotes and Archaea. This spread across super-phyla suggests that a precursor ESCRT machinery existed already at an evolutionary early stage of life, making it a promising candidate for the (re)construction of a minimal cell division machinery. There are, however, only few experimental data about ESCRT machineries in Archaea, due to high technical challenges in cultivation and microscopy. Here, we analyse the proteins of archaeal ESCRT machineries bioinformatically on a domain level, to enable mechanistical comparison without such challenging experiments. First, we infer that there are at least three different cell division mechanisms utilizing ESCRT proteins in archaea, probably similar in their constriction mechanisms but different in membrane tethering. Second, we show that ESCRT proteins in Asgard archaea are highly similar to eukaryotic ESCRT proteins, strengthening the recently developed idea that all Eukaryotes descended from archaea. Third, we reconstruct a plausible evolutionary development of ESCRT machineries and suggest that a simple ESCRT-based constriction machinery existed in the last archaeal common ancestor. These findings not only give very interesting insights into the evolution of cell division in Archaea and Eukaryotes, but also reveal new research avenues by suggesting hypothesis-driven experiments for both, cell biology and bottom-up synthetic biology. "Neighbourhood watch" model: embryonic epiblast cells assess positional information in relation to their neighbours In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name "neighbourhood watch" model) illuminates the classic "French Flag Problem" and how positional information is interpreted by a sheet of cells in a large developing system.

Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry. IRGM/Irgm1 Aggravates Progression of Atherosclerosis by Inducing Macrophage Apoptosis through the MAPK Signaling Pathway AimsAtherosclerosis underlies most cardiovascular diseases, among which acute coronary syndrome (ACS) caused by plaque rupture (PR) often leads to death. Immune-related GTPases (IRGM/Irgm1) have been extensively studied in inflammatory diseases, but their role in atherosclerosis is unclear. Determining how IRGM/Irgm1 promotes atherosclerotic plaque vulnerability will provide information for new biomarkers and/or therapeutic targets.

Methods and resultsWe identified ruptured and unruptured plaques by optical coherence tomography, and found that serum IRGM was highly expressed in patients with ST-segment elevation myocardial infarction in PR. We used ApoE-/-Irgm1+/+, ApoE-/-Irgm1+/- mice and chimeric mice to establish a model of advanced atherosclerosis. The results of pathological experiments showed that Irgm1 caused plaque necrosis. The ratio of neutral lipids and cholesterol crystals increases, while the content of collagen fibers decreases, aggravating the destabilization of atherosclerotic plaques. In vitro, we used multiple approaches to confirm that Irgm1 promotes macrophage apoptosis by promoting the production of reactive oxygen species and activating the MAPK signaling pathway.

ConclusionsIRGM may be a potential risk factor for PR. Mechanistic studies have shown that IRGM/Irgm1 contributes to the formation and rupture of fragile plaques. This is partly mediated by the induction of macrophage apoptosis via the MAPK signaling pathway. IRGM may offer new strategies for early treatment of ACS.

Translation viewOur findings indicate that IRGM/Irgm1 contributes to formation and rupture of vulnerable plaques. It suggests that IRGM may provide a new target for the early treatment of ACS. 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. 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. Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Time- and cell-dependent atypia and cell death are caused by progressive deficiency in DNA replication Pleiotropy caused by single-gene mutations is common and poorly understood. A zebrafish null mutant of DNA polymerase  subunit B, huli hutu (hht), evolves a complex pleiotropy associated with DNA damage and S phase arrest across multiple organ systems over 5-7 days, including nuclear atypia, a common cellular feature in human cancers and pre-cancers, in gastrointestinal organs, and nuclear fragmentation in the eye and brain. The pleiotropic pattern of hht phenotypes is explained by progressive loss of wild-type maternal pola2 function in homozygous mutant embryos whose pola2 mRNA becomes undetectable by 24 hours post-fertilization (hpf). Inhibition of DNA synthesis by aphidicolin or hydroxyurea in wild-type embryos from 24 hpf phenocopied the pleiotropic pattern of hht. These results are consistent with a model in which time-sensitive, reduced capacity for DNA synthesis results in cell death in fast-replicating cells, and nuclear atypia in tissues with fewer and larger cells. V3 Interneurons are Active and Recruit Spinal Motor Neurons During In Vivo Fictive Swimming in Larval Zebrafish Survival for vertebrate animals is dependent on the ability to successfully find food, locate a mate, and avoid predation. Each of these behaviors requires motor control, which is set by a combination of kinematic properties. For example, the frequency and amplitude of motor output combine in a multiplicative manner to determine features of locomotion such as distance traveled, speed, force (thrust), and vigor. Although there is a good understanding of how different populations of excitatory spinal interneurons establish locomotor frequency, there is a less thorough mechanistic understanding for how locomotor amplitude is established. Recent evidence indicates that locomotor amplitude is regulated in part by a subset of functionally and morphologically distinct V2a excitatory spinal interneurons (type II, non-bursting) in larval and adult zebrafish. Here we provide direct evidence, for the first time, that most V3 interneurons (V3-INs), which are a developmentally and genetically defined population of ventromedial glutamatergic spinal neurons, are active during fictive swimming. We also show that elimination of the spinal V3-IN population reduces the proportion of active MNs during fictive swimming but does not alter the range of locomotor frequencies produced. These data are consistent with V3-INs providing excitatory drive to spinal motor neurons and contributing to the production of locomotor amplitude, but not frequency, during swimming in larval zebrafish.

SIGNIFICANCE STATEMENTCurrently, there is a limited understanding about the cellular and spinal network properties that produce locomotor amplitude, defined as limb displacement in limbed animals or tail-bend in non-limbed animals during locomotion. Here we show, directly for the first time in a vertebrate, that V3 interneurons (V3-INs) in zebrafish larvae are active during in vivo fictive locomotion, and that targeted ablation of the spinal V3-IN population reduces the proportion of active motoneurons during fictive swimming. Importantly, ablation of V3-INs does not affect locomotor frequency (speed), which clarifies their role in motor control rather than rhythm generation. Thus, we propose that the V3-IN population is a source of excitation in the vertebrate locomotor neural circuitry that regulates locomotor amplitude independently of speed. Intrinsic cardiac adrenergic cells contribute to septic cardiomyopathy Occurring independently of cardiac sympathetic nervous system, the intrinsic cardiac adrenergic (ICA) cells have been identified as an important regulator in both of developing and adult cardiac physiological and pathological processes. However, its role in septic cardiomyopathy remains unknown. Herein, we report that lipopolysaccharide (LPS) dose- and time-dependently increased norepinephrine (NE) release from ICA cells, which aggravates myocardial TNF- production and dysfunction. Inhibition of NE synthesis in ICA cells alleviated LPS-elicited cardiac dysfunction as well as TNF- production in Langendorff perfusing hearts. Mechanistically, ICA cell expressed Toll-like receptor 4 (TLR4), activated by LPS, to increase the expression of tyrosine hydroxylase, a key enzyme responsible for NE biosynthesis, via AP-1 binding to its promoter. Surprisingly, LPS-TLR4 signaling triggered no TNF- production in ICA cells due to the elevated Nfkbia and Tnfaip6 expression. In LPS-treated co-culture of ICA cells and cardiomyocytes, the raised NE from ICA cells activated cardiomyocyte {beta}1-adrenergic receptor ({beta}1-AR), driving Ca2+/calmodulin-dependent protein kinase II (CaMKII) to increase the activities of NF-{kappa}B and mitogen-activated protein kinase pathways, which were mimicked by dobutamine. Our findings reveal a cell type-specific TLR4 function triggering NE synthesis, but not TNF- production in inflammatory pathogenesis, and identify ICA cell-derived NE as a paracrine signal in the cross talk among different cardiac cells to enhance myocardial injury during LPS challenge, suggesting that targeting ICA cell-derived NE may be a potential therapeutic strategy for septic cardiomyopathy. Dynamic Phosphorylation of RGS Provides Spatial Regulation of MAP Kinase And Promotes Completion of Cytokinesis during the Yeast Pheromone Response Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces G GTPase activity and subsequent inactivation of all downstream signaling including a MAPK cascade. The MAPK Fus3 phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that RGS phosphorylation peaks early in the pheromone response and diminishes RGS localization to the polarity site and focuses MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS. SPICEMIX: Integrative single-cell spatial modeling of cell identity Spatial transcriptomics technologies promise to reveal spatial relationships of cell-type composition in complex tissues. However, the development of computational methods that can utilize the unique properties of spatial transcriptome data to unveil cell identities remains a challenge. Here, we introduce SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW, a new interpretable method based on probabilistic, latent variable modeling for effective joint analysis of spatial information and gene expression from spatial transcriptome data. Both simulation and real data evaluations demonstrate that SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW markedly improves upon the inference of cell types and their spatial patterns compared with existing approaches. By applying to spatial transcriptome data of brain regions in human and mouse acquired by seqFISH+, STARmap, and Visium, we show that SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW can enhance the inference of complex cell identities, reveal interpretable spatial metagenes, and uncover differentiation trajectories. SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW is a generalizable framework for analyzing spatial transcriptome data to provide critical insights into the cell type composition and spatial organization of cells in complex tissues. 3D Reconstructions of Mouse Skeletal Muscle Reveal a Decrease in the MICOS Complex and Altered Mitochondrial Networks Background: Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. Studies of bioenergetics and protein turnover show that mitochondria mediate this decline in function. Mitochondria cristae are essential for the production of ATP. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging still requires further elucidation Further, it is not completely understood how 3D mitochondrial networks and the specialization of mitochondria are altered during aging. Methods: We measured changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. Nanotunnels were also measured through 3D reconstruction. CRISPR/Cas9 KO was performed to examine changes in mitochondria upon loss of the mitochondrial contact site and cristae organizing system (MICOS) complex and optic atrophy 1 (OPA-1). Metabolomics were used to find key metabolite pathways changed upon off of the MICOS complex. Results: We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle during aging. We also found an association of OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Further, the loss of the MICOS complex was linked with decreased oxidative capacity, and altered mitochondrial metabolism. Conclusions: To our knowledge, studies of mitochondrial changes in skeletal muscle throughout aging remains limited. 3D reconstructions of nanotunnels elucidated novel patterns of mitochondrial aging in skeletal muscle. MICOS proteins decreased with age and mitochondrial morphology was similar between aged skeletal muscle and that of young mice with MICOS protein loss. In tandem, our data suggest a relationship between the MICOS complex and aging, which could be potentially linked to disease states with additional 3D reconstruction. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. High Spatial Resolution MALDI Imaging Mass Spectrometry ofFresh-Frozen Bone Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracking, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 m) MALDI IMS was employed to characterize lipid distributions in fresh-frozen bone, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment. Hematopoietic differentiation is characterized by a transient peak of entropy at a single cell level. Hematopoietic differentiation has been metaphorically represented as linear trajectories with discrete steps from hematopoietic stem cells to mature cells. While the transcriptional state of cells at the beginning or at the end of these trajectories are well described from bulk analysis, what happens in the intermediate states has remained elusive until the use of single cell approaches. Applying Shannon entropy to measure cell-to-cell variability among cells at the same stage of differentiation, we observed a transient peak of gene expression variability in all the hematopoietic differentiation pathways. Strikingly, genes with the highest entropy variation in a given differentiation pathway matched genes known as pathway-specific, whereas genes with the highest expression variation were common to all pathways. Finally, we showed that the level of cell-to-cell variation is increased in the most immature compartment of hematopoiesis in myelodysplastic syndromes. These data suggest that differentiation could be better conceptualized as a dynamical stochastic process with a transient stage of cellular indetermination. HIF-dependent expression of creatine kinase brain isoform (CKB) promotes breast cancer metastasis, whereas cyclocreatine therapy impairs invasion and improves the efficacy of conventional chemotherapies The oxygen-responsive Hypoxia Inducible Factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple negative breast cancers (TNBCs) and correlates with poor patient survival. Since inhibiting the HIF transcription factors with small molecules is challenging, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss and gain of function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, formation of invadopodia, and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth. Fiber orientation downsampling compromises the computation of white matter tract-related deformation Incorporating neuroimaging-revealed structural details into finite element (FE) head models opens vast new opportunities to better understand brain injury mechanisms. Recently, growing efforts have been made to integrate fiber orientation from diffusion tensor imaging (DTI) into FE models to predict white matter (WM) tract-related deformation that is biomechanically characterized by tract-related strains. Commonly used approaches often downsample the spatially enriched fiber orientation to match the FE resolution with one orientation per element (i.e., element-wise orientation implementation). However, the validity of such downsampling operation and corresponding influences on the computed tract-related strains remain elusive. To address this, the current study proposed a new approach to integrate voxel-wise fiber orientation from one DTI atlas (isotropic resolution of 1 mm3) into FE models by embedding orientations from multiple voxels within one element (i.e., voxel-wise orientation implementation). By setting the responses revealed by the newly proposed voxel-wise orientation implementation as the reference, we evaluated the reliability of two previous downsampling approaches by examining the downsampled fiber orientation and the computationally predicted tract-related strains secondary to one concussive impact. Two FE models with varying element sizes (i.e., 6.37 {+/-} 1.60 mm and 1.28 {+/-} 0.55 mm, respectively) were incorporated. The results showed that, for the model with a large voxel-mesh resolution mismatch, the downsampled element-wise fiber orientation, with respect to its voxel-wise counterpart, exhibited an absolute deviation over 30{degrees} across the WM/gray matter interface and the pons regions. Accordingly, this orientation deviation compromised the computation of tract-related strains with normalized root-mean-square errors up to 30% and underestimated the peak tract-related strains up to 10%. For the other FE model with finer meshes, the downsampling-induced effects were lower, both on the fiber orientation and tract-related strains. Taken together, the voxel-wise orientation implementation is recommended in future studies as it leverages the DTI-delineated fiber orientation to a larger extent than the element-wise orientation implementation. Thus, this study yields novel insights on integrating neuroimaging-revealed fiber orientation into FE models and may better inform the computation of WM tract-related deformation, which are crucial for advancing the etiological understanding and computational predictability of brain injury.

Graphic abstract

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org.highwire.dtl.DTLVardef@12ebda9org.highwire.dtl.DTLVardef@1a5d624org.highwire.dtl.DTLVardef@8e068borg.highwire.dtl.DTLVardef@4f8ff5_HPS_FORMAT_FIGEXP  M_FIG C_FIG Host phenology can drive the evolution of intermediate virulence strategies in some obligate-killer parasites AO_SCPLOWBSTRACTC_SCPLOWThe traditional mechanistic trade-offs resulting in a negative correlation between transmission and virulence are the foundation of nearly all current theory on the evolution of parasite virulence. Several ecological factors have been shown to modulate the optimal virulence strategies predicted from mechanistic trade-off models, but these ecological factors have not yet been shown to be sufficient to explain the intermediate virulence strategies observed in any natural system. The timing of seasonal activity, or phenology, is a common factor that influences the types and impact of many ecological interactions but is difficult to incorporate into virulence evolution studies. We develop a mathematical model of a disease system with seasonal host activity to study the evolutionary consequences of host phenology on the virulence of obligate-killer parasite. Results from this model demonstrated that seasonal host activity is sufficient to drive the evolution of intermediate parasite virulence in some types of natural disease systems, even when a traditional mechanistic trade-off between transmission and virulence is not assumed in the modeling framework. The optimal virulence strategy in these systems can be determined by both the duration of the host activity period as well as the variation in the host emergence timing. Parasites with low virulence strategies are favored in environments with long host activity periods and in environments in which hosts emerge synchronously. The results demonstrate that host phenology can be sufficient to select for intermediate optimal virulence strategies, providing an alternative mechanism to account for virulence evolution in some natural systems. The OPAQUE1/DISCORDIA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize Asymmetric divisions produce cells with different fates and are critical for development. Here we show that a maize myosin XI protein, OPAQUE1 (O1), is necessary for asymmetric divisions during maize stomatal development. We analyzed stomatal precursor cells prior to and during asymmetric division to determine why o1 mutants have abnormal division planes. Cell polarization and nuclear positioning occur normally in the o1 mutant, and the future site of division is correctly specified. The defect in o1 occurs during late cytokinesis, when the plant-specific phragmoplast -- made of microtubules, actin and other proteins - forms the nascent cell plate. The phragmoplast becomes misguided and does not meet the previously established division site. Initial phragmoplast guidance is correct in o1. However, as phragmoplast expansion continues, phragmoplasts in o1 stomatal precursor cells become misguided and do not meet the cortex at the established division site. To understand how this myosin protein contributes to phragmoplast guidance, we identified O1-interacting proteins. Other myosins, specific actin-binding proteins, and maize kinesins related to the Arabidopsis thaliana division site markers PHRAGMOPLAST ORIENTING KINESINs (POKs) interact with O1. We propose that different myosins are important at multiple steps of phragmoplast expansion, and the O1 actin motor and POK-like microtubule motors work together to ensure correct late-stage phragmoplast guidance. Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics. Satellitome comparison of two oedipodine grasshoppers highlights the contingent nature of satellite DNA evolution BackgroundThe full catalogue of satellite DNA (satDNA) within a same genome constitutes the satellitome. The Library Hypothesis predicts that satDNA in relative species reflects that in their common ancestor, but the evolutionary mechanisms and pathways of satDNA evolution have never been analyzed for full satellitomes. We compare here the satellitomes of two Oedipodine grasshoppers (Locusta migratoria and Oedaleus decorus) which shared their most recent common ancestor about 22.8 Ma ago.

ResultsWe found that about one-third of their satDNA families (near 60 in every species) showed sequence homology and were grouped into 12 orthologous superfamilies. The turnover rate of consensus sequences was extremely variable among the 20 orthologous family pairs analyzed in both species. The satDNAs shared by both species showed poor association with sequence signatures and motives frequently argued as functional, except for short inverted repeats allowing short dyad symmetries and non-B DNA conformations. Orthologous satDNAs frequently showed different FISH patterns at both intra- and interspecific levels. We defined indices of homogenization and degeneration and quantified the level of incomplete library sorting between species.

ConclusionsOur analyses revealed that satDNA degenerates through point mutation and homogenizes through partial turnovers caused by massive tandem duplications (the so-called satDNA amplification). Remarkably, satDNA amplification increases homogenization, at intragenomic level, and diversification between species, thus constituting the basis for concerted evolution. We suggest a model of satDNA evolution by means of recursive cycles of amplification and degeneration, leading to mostly contingent evolutionary pathways where concerted evolution emerges promptly after lineages split. Metabolic crossfeeding structures the assembly of polysaccharide degrading communities Metabolic processes that fuel the growth of heterotrophic microbial communities are initiated by specialized biopolymer degraders that decompose complex forms of organic matter. It is unclear, however, to what extent degraders control the downstream assembly of the community that follows polymer breakdown. Investigating a model marine microbial community that degrades chitin, we show that chitinases secreted by different degraders produce oligomers of specific chain lengths that not only select for specialized consumers but also influence the metabolites secreted by these consumers into a shared resource pool. Each species participating in the breakdown cascade exhibits unique hierarchical preferences for substrates, which underlies the sequential colonization of metabolically distinct groups as resource availability changes over time. By identifying the metabolic underpinnings of microbial community assembly, we reveal a hierarchical crossfeeding structure that allows biopolymer degraders to shape the dynamics of community assembly.

One sentence summarySpecialized biopolymer degraders direct the trajectory of microbial community assembly through interconnected modes of nutrient crossfeeding.