Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.
Genetic instability from a single S-phase after whole genome duplication Diploid and stable karyotypes are associated with health and fitness in animals. In contrast, whole genome duplications (WGDs) - doubling full chromosome content - are linked to genetic instability (GIN) and frequently found in human cancers 1-3. It has been established that WGDs fuel chromosome instability through abnormal mitosis 4-8, however, the immediate consequences of tetraploidy in the first interphase are not known. This is an essential question because single WGD events such as cytokinesis failure can promote tumorigenesis 9. Here, we found that newly born tetraploid human cells undergo high rates of DNA damage during DNA replication in the first S-phase. Using DNA combing and single cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we found that these defects result from the lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.
The Inflammatory Role of Milk Fat Globule Epidermal Growth Factor VIII in Angiotensin II Induced Arterial Remodeling BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown.

Methods and ResultsIn this study, 30-week-old MFG-E8 knock out (KO) and age-matched wild-type (WT) mice were infused with Ang II or saline. After infusion the with Ang II, the aortic molecular, cellular, and structural remodeling were observed in mice and compared to those infused with saline, but these effects were dependent on the expression of MFG-E8: (1) In the WT mice, Ang II infusion substantially increased intimal-medial thickness, elastic lamina degradation, collagen deposition, and the proliferation of VSMCs; in contrast, in the KO mice, these effects were significantly reduced; (2) In the WT mice, Ang II treatment significantly increased the activation and expression of MMP2, TGF-{beta}1, and its downstream signaling molecule p-SMAD2, and collagen type I production, however, in the KO mice, these molecular effects were significantly reduced; (3) In the WT mice, Ang II treatment increased inflammatory p-NF-{kappa}B p65, MCP1, TNF-, ICAM1, and VCAM1 molecular expression, while conversely, in the KO mice, no significant inflammatory changes were found; (4) Importantly, compared to untreated control mice with a wide range of age from 4-96 weeks, Ang II infused "younger" mice produced an "older" arterial inflammatory phenotype, which was alleviated by MFG-E8 deficiency.

ConclusionsMFG-E8 mediates Ang II associated arterial inflammatory remodeling. Targeting MFG-E8 is a novel molecular approach to curb adverse arterial remodeling during aging and hypertension.

CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Both Ang II and MFG-E8 increases are involved in proinflammatory arterial remodeling mediating the molecular, cellular and tissue events in aging and hypertension.
* MFG-E8 is essential for Ang II induced and age-associated adverse arterial remodeling via the increase of proinflammation, intimal medial thickening, elastin fragmentation, collagen deposition, and VSMC proliferation.


What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension. Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors. 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. A novel Boolean network inference strategy to model early hematopoiesis aging Hematopoietic stem cell (HSC) aging is a multifactorial event that leads to changes in HSC properties and function. These changes are intrinsically coordinated and affect the early hematopoiesis, involving hematopoietic stem and progenitor cells (HSPCs). The objective of this work is to better understand the mechanisms and factors controlling these changes. We have therefore developed an original strategy to construct a Boolean network of genes explaining the priming and homeostasis of HSCs (graphical abstract). Based on our previous scRNA-seq data, we performed an exhaustive analysis of the transcriptional network and identified active transcription modules or regulons along the differentiation trajectory of selected HSPC states. This global view of transcriptional regulation led us to focus on 15 components, 13 selected TFs (Tal1, Fli1, Gata2, Gata1, Zfpm1, Egr1, Junb, Ikzf1, Myc, Cebpa, Bclaf1, Klf1, Spi1) and 2 complexes regulating the ability of HSC to cycle (CDK4/6 - Cyclin D and CIP/KIP). We then defined the connections controlling the differentiation dynamics of HSC states and constructed an influence graph between the TFs involved in the dynamics by mixing observations from our scRNA-seq data and knowledge from the literature. Then, using answer set programming (ASP) and in silico perturbation analysis, we obtained a Boolean model which is the solution of a Boolean satisfiability problem. Finally, perturbation of the model based on age-related changes revealed important regulations, such as the overactivation of Egr1 and Junb or the loss of Cebpa activation by Gata2, which were found to be relevant for the myeloid bias of aged HSC. Our work shows the efficiency of the combination of manual and systematic methods to elaborate a Boolean model. The developed strategy led to the proposal of new regulatory mechanisms underlying the differentiation bias of aged HSCs, explaining the decreased transcriptional priming of HSCs to all mature cell types except megakaryocytes. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Interferon adjusts stem cell output in the young and old brain to meet demand independently of its antiviral function Stem cells show intrinsic interferon signaling, which protects them from viral infections. In the brain, type I interferon signaling increases in stem cells as they age and reduces their ability to become activated for repair. Whether interferons impact stem cell activity only in aging, how, and if it is coupled to the intrinsic immune function remains elusive. Our study shows that interferon-{beta} arrested stem cells in G0 of the cell cycle and transiently increased their TOR signaling before switching it off. This bimodal regulation of TOR is needed to repress translation of Sox2. Single-cell transcriptomics reveals that in the absence of interferon receptors, interferon signaling levels change and its increase with age disappears. Mathematical simulations indicate that interferons are beneficial in the young and harmful in the old brain. Our study identifies interferons as genuine regulators of stem cell homeostasis and potential therapeutic target to repair the aging brain. Agrobacterium mediated transformation and deciphering SNPs in TaLr67 gene homeologs for gene editing in wheat. Diseases adversely affect grain yield of crop plants. Leaf rust is a major disease of wheat. As race-specific resistance breaks down, introduction of newer sources of resistance often from older accessions is necessary. Linkage drag from the donor accessions adversely impacts grain yield. As a result, CIMMYT breeding effort has shifted to using durable resistance, also referred to as race non-specific host resistance, which allows slow rusting but maintains grain yield. One of the key genes for durable resistance is Lr67 and the resistant form of this gene is absent in CIMMYT elite lines. Hence, we have initiated efforts to convert the susceptible copy of Lr67 into its resistant form directly in these elite lines using gene editing. This would eliminate backcrossing and thus save time as well as eliminate linkage drag that would accompany the resistant copy of the gene if it were to be introgressed from an older accession. As first steps, we have isolated and sequenced the genomic copies from each of the A, B, and D genome of the Lr67 gene from three elite lines and an experimental line. Identification of more than 50 single nucleotide polymorphisms (SNPs) in the open reading frames (ORF) among these lines would be useful in designing the guide RNA molecules with precision. Further, we have streamlined genetic transformation of these elite lines, a prerequisite step for gene editing. Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8. The Hippocampus uses cryptographic principles rather than plasticity to enable animals to secretly cache and retrieve their food For animals, the ability to hide and retrieve valuable information, such as the location of food, can mean the difference between life and death. Here, we propose that to achieve this, their brain uses spatial cells similarly to how we utilize encryption for data security. Some animals are able to cache hundreds of thousands of food items annually by each individual and later retrieve most of what they themselves stashed. Rather than memorizing their cache locations as previously suggested, we propose that they use a single cryptographic-like mechanism during both caching and retrieval. The model we developed is based on hippocampal spatial cells, which respond to an animals positional attention, such as when the animal enters a specific region (place-cells) or gazes at a particular location (spatial-view-cells). We know that the region that activates each spatial cell remains consistent across subsequent visits to the same area but not between areas. This remapping, combined with the uniqueness of cognitive maps, produces a persistent crypto-hash function for both food caching and retrieval. We also show that the model stores temporal information that helps animals in food caching order preference as previously observed. This behavior, which we refer to as crypto-taxis, might also explain consistent differences in decision-making when animals are faced with a large number of alternatives such as in foraging. 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. A genomic assessment of the marine-speciation paradox within the toothed whale superfamily Delphinoidea The importance of post-divergence gene flow in speciation has been documented across a range of taxa in recent years, and may have been especially widespread in highly mobile, wide-ranging marine species, such as cetaceans. Here, we studied individual genomes from nine species across the three families of the toothed whale superfamily Delphinoidea (Delphinidae, Phocoenidae, Monodontidae). To investigate the role of post-divergence gene flow in the speciation process, we used a multifaceted approach, including: (i) phylogenomics, (ii) the distribution of shared derived alleles, and (iii) demographic inference. We found the divergence of lineages within Delphinoidea did not follow a process of pure bifurcation, but was much more complex. Sliding-window phylogenomics reveal a high prevalence of discordant topologies within the superfamily, with further analyses indicating these discordances arose due to both incomplete lineage sorting and gene flow. D-statistics, D-foil, and f-branch analyses supported gene flow between members of Delphinoidea, with the vast majority of gene flow occurring as ancient interfamilial events. Demographic analyses provided evidence that introgressive gene flow has likely ceased between all species pairs tested, despite reports of contemporary interspecific hybrids. Our study provides the first steps towards resolving the large complexity of speciation within Delphinoidea; we reveal the prevalence of ancient interfamilial gene flow events prior to the diversification of each family, and suggests that contemporary hybridisation events may be disadvantageous, as hybrid individuals do not appear to contribute to the parental species gene pools. Optimized detection of allelic imbalances specific for homologous recombination deficiency improves the prediction of clinical outcomes in cancer Homologous recombination DNA-repair deficiency (HRD) is a common driver of genomic instability and confers a therapeutic vulnerability in human cancer. Using a systematic pan- cancer characterization, we revealed distinct patterns of somatic allelic imbalances (AIs) in high-grade serous ovarian cancer (HGSC). Using machine learning on a multi-omics dataset, we generated an optimized algorithm to detect HRD in HGSC (ovaHRDscar). ovaHRDscar improved the prediction of clinical outcomes in three independent validation cohorts. Characterization of 98 spatiotemporally distinct tumor samples indicated ovary/adnex as the preferred site to assess HRD. Cancer-specific optimization improved the prediction of clinical outcomes also in triple-negative breast cancer (tnbcHRDscar). In conclusion, our systematic approach based on multi-omics data improves the detection of HRD with the premise to improve patient selection for HR-targeted therapies. Benchmarking of deep learning algorithms for 3D instance segmentation of confocal image datasets Segmenting three dimensional microscopy images is essential for understanding phenomena like morphogenesis, cell division, cellular growth and genetic expression patterns. Recently, deep learning (DL) pipelines have been developed which claim to provide high accuracy segmentation of cellular images and are increasingly considered as the state-of-the-art for image segmentation problems. However, it remains difficult to define their relative performances as the concurrent diversity and lack of uniform evaluation strategies makes it difficult to know how their results compare. In this paper, we first made an inventory of the available DL methods for 3 dimensional (3D) cell segmentation. We next implemented and quantitatively compared a number of representative DL pipelines, alongside a highly efficient non-DL method named MARS. The DL methods were trained on a common dataset of 3D cellular confocal microscopy images. Their segmentation accuracies were also tested in the presence of different image artifacts. A specific method for segmentation quality evaluation was adopted which isolates segmentation errors due to under/over segmentation. This is complemented with a 3D visualization strategy for interactive exploration of segmentation quality. Our analysis shows that the DL pipelines have different levels of accuracy. Two of them, which are end to end 3D and were originally designed for cell boundary detection, show high performance, and offer clear advantages in terms of adaptability to new data.

Author summaryIn recent years a number of deep learning (DL) algorithms based on computational neural networks have been developed which claim to achieve high accuracy and automatic segmentation of 3D microscopy images. Although these algorithms have received considerable attention in the literature, it is difficult to evaluate their relative performances, while it remains unclear whether they really perform better than other, more classical segmentation methods.

To clarify these issues, we performed a detailed, quantitative analysis of a number of representative DL pipelines for cell instance segmentation from 3D confocal microscopy image datasets. We developed a protocol for benchmarking the performances of such DL based segmentation pipelines using common training and test datasets, evaluation metrics and visualizations. Using this protocol, we evaluated and compared four different DL pipelines to identify their strengths and limitations. A high performance non-DL method was also included in the evaluation. We show that DL pipelines may show significant differences in their performances depending on their model architecture and pipeline components but overall show excellent adaptability to unseen data. We also show that our benchmarking protocol could be extended to a variety of segmentation pipelines and datasets. Effect of sublethal prenatal endotoxaemia on murine placental transport systems and lipid homeostasis Infection alters the expression of transporters that mediate the placental exchange of xenobiotics, lipids and cytokines. We hypothesized that lipopolysaccharide (LPS) modifies the expression of placental transport systems and lipid homeostasis. LPS (150 g/kg; i.p.) treatments were administered for 4 h or 24 h, animals were euthanized at gestational days (GD) 15.5 or 18.5, and maternal blood, foetuses and placentae were collected. Increased rates of foetal demise were observed at GD15.5 following LPS treatment, whereas at GD18.5, high rates of early labour occurred and were associated with distinct proinflammatory responses. LPS did not alter ABC transporter mRNA expression but decreased Fabppm at GD15.5 (LPS-4 h) and increased Fat/Cd36 lipid transporter mRNA at GD18.5 (LPS-4 h). At the protein level, breast cancer-related protein (BCRP) and Abcg1 levels were decreased in the placental labyrinth zone (Lz) at GD15.5, whereas P-glycoprotein (P-gp) and Bcrp Lz-immunostaining was decreased at GD18.5. In the placental junctional zone (Jz), P-gp, Bcrp and Abcg1 levels were higher at GD18.5. Specific maternal plasma and placental changes in triacylglycerol, free fatty acid, cholesterol, cholesterol ester and monoacylglycerol levels were detected in a gestational age-dependent manner. In conclusion, LPS-induced foetal death and early labour were associated with altered placental ABC and lipid transporter expression and deranged maternal plasma and placental lipid homeostasis. These changes likely modify foetal xenobiotic exposure and placental lipid exchange in cases of bacterial infection. Cell type-specific inference of differential expression in spatial transcriptomics Spatial transcriptomics enables spatially resolved gene expression measurements at near single-cell resolution. There is a pressing need for computational tools to enable the detection of genes that are differentially expressed (DE) within specific cell types across tissue context. We show that current approaches cannot learn cell type-specific DE due to changes in cell type composition across space and the fact that measurement units often detect transcripts from more than one cell type. Here, we introduce a statistical method, Cell type-Specific Inference of Differential Expression (C-SIDE), that identifies cell type-specific patterns of differential gene expression while accounting for localization of other cell types. We model spatial transcriptomics gene expression as an additive mixture across cell types of general log-linear cell type-specific expression functions. This approach provides a unified framework for defining and identifying gene expression changes in a wide-range of relevant contexts: changes due to pathology, anatomical regions, physical proximity to specific cell types, and cellular microenvironment. Furthermore, our approach enables statistical inference across multiple samples and replicates when such data is available. We demonstrate, through simulations and validation experiments on Slide-seq and MER-FISH datasets, that our approach accurately identifies cell type-specific differential gene expression and provides valid uncertainty quantification. Lastly, we apply our method to characterize spatially-localized tissue changes in the context of disease. In an Alzheimers mouse model Slide-seq dataset, we identify plaque-dependent patterns of cellular immune activity. We also find a putative interaction between tumor cells and myeloid immune cells in a Slide-seq tumor dataset. We make our C-SIDE method publicly available as part of the open source R package https://github.com/dmcable/spacexr. Elucidating Microscopic Events Driven by GTP Hydrolysis Reaction in Ras-GAP System with Semi-reactive Molecular Dynamics Simulation: Alternative Role of Phosphate Binding Loop as Mechanical Energy Storage ATPase and GTPase have been widely found as chemical energy-mechanical work transducers, whereas the physicochemical mechanisms are not satisfactorily understood. We addressed the problem by examining John Ross conjecture that repulsive Coulomb interaction between ADP/GDP and inorganic phosphate (Pi) does the mechanical work upon the system. We effectively simulated the consequence of GTP hydrolysis reaction in a complex system of Rat sarcoma (Ras) and GTPase activation protein (GAP) in the framework of classical molecular dynamics by switching force field parameters between the reactant and product systems. We then observed ca. 5 kcal/mol raise of potential energy about the phosphate-binding loop (P-loop) in Ras protein, indicating that the mechanical work generated via the GTP hydrolysis is converted into the local interaction energy and stored in the P-loop. Interestingly, this local energy storage in the P-loop depends on neither impulsive nor consecutive collisions of GDP and Pi with P-loop. Instead, GTP-GDP conversion itself does work on the Ras system, elevating the potential energy. These observations encourage us to challenge a conjecture previously given by Ross. We assert that triphosphate nucleotide hydrolyses do mechanical work by producing emergent steric interaction accompanied with relaxation, namely, a shift of biomolecular system to non-equilibrium state on the reshaped potential energy landscape. Recalling the universality of the P-loop motif among GTPases and ATPases, the observations that we obtained through this study would progress physicochemical understanding for the operating principles of GTP/ATP hydrolysis-driven biological nano-machines. Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles via lipopolysaccharide signalling Xenopus laevis tadpoles can regenerate functional tails, containing spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around one week of age. At this stage, amputation of the tadpoles tail may either result in scarless wound healing, or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles, and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Editing TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect. Bovine neutrophils release extracellular traps and cooperate with macrophages in Mycobacterium avium subsp. paratuberculosis clearance in vitro Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guerin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1{beta} and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1{beta} and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site. Metabarcoding faecal samples to investigate spatiotemporal variation in the diet of the endangered Westland petrel (Procellaria westlandica) As top predators, seabirds can be indirectly impacted by climate variability and commercial fishing activities through changes in marine communities. However, high mobility and foraging behaviour enables seabirds to exploit prey distributed patchily in time and space. This capacity to adapt to environmental change can be described through the study of their diet. Traditionally, the diet of seabirds is assessed through the morphological identification of prey remains in regurgitates. This sampling method is invasive for the bird and limited in terms of taxonomic resolution. However, the recent progress in DNA-based approaches is now providing a non-invasive means to more comprehensively and accurately characterize animal diets. Here, we used a non-invasive metabarcoding approach to characterize the diet of the Westland petrel (Procellaria westlandica), which is an endangered burrowing species, endemic to the South Island of New Zealand. We collected 99 fresh faecal samples at two different seasons and in two different sub-colonies. Our aims were to describe the diet of the Westland petrel, investigate seasonal and spatial variation in the petrels diet, and assess potential impacts of the New Zealand fishery industry. We found that amphipods were the most common prey, followed by cephalopods and fish. Our results could be the result of natural foraging behaviour, but also suggest a close link between the composition of prey items and New Zealands commercial fishing activities. In particular, the high abundance of amphipods could be the result of Westland petrels feeding on discarded fisheries waste (fish guts). Our results also showed significant differences in diet between seasons (before hatching vs chick-rearing season) and between sampling sites (two sub-colonies 1.5 km apart), indicating plasticity in the foraging strategy of the Westland petrel. Due to its non-invasive nature, metabarcoding of faecal samples can be applied to large numbers of samples to help describe dietary variation in seabirds and indicate their ecological requirements. In our example, dietary DNA (dDNA) provided valuable information regarding the dietary preferences of an iconic species in New Zealands unique biodiversity. dDNA can thus inform the conservation of endangered or at-risk species that have elusive foraging behaviours. Discovery of Aminoglycosides as First in Class, Nanomolar Inhibitors of Heptosyltransferase I A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years and while many have designed novel small-molecule inhibitors, these compounds lack the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. With the help of molecular dynamic simulations, aminoglycoside antibiotics were shown to be putative inhibitors for HepI and in this study, they were experimentally determined to be the first in-class nanomolar inhibitors of HepI with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and MD simulations were used to corroborate kinetic experimental findings. Kinetic analysis methods include Lineweaver-Burk, Dixon, Cornish-Bowden and Mixed-Model of Inhibition which allowed for unambiguous assignment of inhibition mechanism for each inhibitor. In this study, we show that neomycin and kanamycin b are competitive inhibitors against the sugar acceptor substrate while tobramycin exhibits a mixed inhibitory effect and streptomycin is non-competitive. MD simulations also allowed us to suggest that the inhibitors bind tightly and inhibit catalytic dynamics due to a major desolvation penalty of the enzyme active site. While aminoglycosides have long been known as a class of potent antibiotics, they also have been scientifically shown to impact cell membrane stability, and we propose that inhibition of HepI contributes to this effect by disrupting lipopolysaccharide biosynthesis. How do stomata evolve on the road to C4 photosynthesis? C4 photosynthesis optimizes plants carbon and water relations, allowing high photosynthetic rate with low stomatal conductance. Stomata have long been believed as a part of C4 syndrome. However, it remains unclear how stomata traits evolved along the path from C3 to C4. Stomatal patterning was examined in Flaveria genus, a model for studying C4 evolution. Comparative, transgenic and semi-in-vitro experiments were used to study molecular basis that underlies stomatal traits along C4 evolution. Novel results: the evolution from C3 to C4 species through intermediate species is accompanied by a stepwise rather than an abrupt change in the stomatal traits. The initial change occurs near Type II and dramatic change occurs at the C4-like species. On the road to C4, stomata become less in number but bigger in size and changes in stomatal density dominates changes in maximum stomatal conductance (gsmax). The reduction of FSTOMAGEN expression underlies altered gsmax between Flaveria species with different photosynthetic pathways and likely occurs in other C4 lineages. Our study provides insight into the pattern, mechanism and role of stomatal evolution along the road towards C4. This work highlights the stomatal traits in the current C4 evolutionary model and the co-evolution of photosynthetic pathway and stomata. A high content microscopy screening identifies new genes involved in cell width control in Bacillus subtilis How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for Gram-positive bacteria Bacillus subtilis, the average cell length varies as a function of the growth rate but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using high-content screening (HCS) fluorescence microscopy and semi-automated measurement of single-cell dimensions, we screened a library of ~ 4000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control in B. subtilis. wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish Osteoporosis, a disease of bone fragility, and sarcopenia, a condition of reduced muscle mass and strength, commonly occur in the same individual--a condition termed osteosarcopenia. Genetic variants at the CPED1-WNT16 locus have pleiotropic effects on human bone mineral density (BMD) and lean tissue mass. However, our current understanding of Wnt16 and its influence on bone fails to explain effects on lean mass at this locus. In this study, we sought to better understand the biological mechanism underlying pleiotropy at the CPED1-WNT16 locus on BMD and lean mass. Using single cell analysis, microCT imaging, and genetic approaches in zebrafish, we show that wnt16 exerts dual influence on muscle and bone, and provide evidence that this occurs through a function in somite development, a period in which precursors for both of these tissues are specified. We show that mutations in wnt16 alter vertebral size and lean mass during axial skeletogenesis. We show that wnt16+ cells transcriptionally resemble cells within the amniote dermomyotome, and provide evidence they function as myogenic precursors during embryonic development. We also provide evidence that WNT16 is a gene of major effect on lean mass at the CPED-WNT16 locus. Our studies support the potential for wnt16 to exert pleiotropic effects on bone and lean mass through a function in myogenic precursors. New Segmentation and Feature Extraction Algorithm for Classification of White Blood Cells in Peripheral Smear Images This article addresses a new method for the classification of white blood cells (WBCs) using image processing techniques and machine learning methods. The proposed method consists of three steps: detecting the nucleus and cytoplasm, extracting features, and classification. At first, a new algorithm is designed to segment the nucleus. For the cytoplasm to be detected, only a part of it located inside the convex hull of the nucleus is involved in the process. This attitude helps us overcome the difficulties of segmenting the cytoplasm. In the second phase, three shapes and four novel color features are devised and extracted. Finally, by using an SVM model, the WBCs are classified. The segmentation algorithm can detect the nucleus with a dice similarity coefficient of 0.9675. The proposed method can categorize WBCs in Raabin-WBC, LISC, and BCCD datasets with accuracies of 94.65 %, 92.21 %, and 94.20 %, respectively. It is worth mentioning that the hyperparameters of the classifier are fixed only with the Raabin-WBC dataset, and these parameters are not readjusted for LISC and BCCD datasets. The obtained results demonstrate that the proposed method is robust, fast, and accurate.