Hematopoietic-SLC37A2 deficiency accelerates atherosclerosis in LDL receptor-deficient mice Macrophages play a central role in the pathogenesis of atherosclerosis. Our previous study demonstrated that solute carrier family 37 member 2 (SLC37A2), an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter, negatively regulates macrophage Toll-like receptor activation by fine-tuning glycolytic reprogramming in vitro. Whether macrophage SLC37A2 impacts in vivo macrophage inflammation and atherosclerosis under hyperlipidemic conditions is unknown. We generated hematopoietic cell-specific SLC37A2 knockout and control mice in C57Bl/6 Ldlr-/- mice by bone marrow transplantation. Hematopoietic-specific SLC37A2 deletion in Ldlr-/- mice increased plasma lipid concentrations 12-16 wks of Western diet induction, attenuated macrophage anti-inflammatory responses, and resulted in more atherosclerosis compared to Ldlr-/- mice transplanted with wild type bone marrow. Aortic root intimal area was inversely correlated with plasma IL-10 levels, but not total cholesterol concentrations, suggesting inflammation but not plasma cholesterol was responsible for increased atherosclerosis in bone marrow SLC37A2-deficient mice. Our in vitro study demonstrated that SLC37A2 deficiency impaired IL-4-induced macrophage activation, independently of glycolysis or mitochondrial respiration. Importantly, SLC37A2 deficiency impaired apoptotic cell-induced glycolysis, subsequently attenuating IL-10 production. Our study suggests that SLC37A2 expression is required to support alternative macrophage activation in vitro and in vivo. In vivo disruption of hematopoietic SLC37A2 accelerates atherosclerosis under hyperlipidemic pro-atherogenic conditions.
Immunogenicity of SARS-CoV-2 trimetric spike protein associated to Poly(I:C) plus Alum The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (spike protein with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.
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 TRIM28-dependent SUMOylation protects the adult ovary from the testicular pathway Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway. Network Inference from Perturbation Time Course Data Networks underlie much of biology from subcellular to ecological scales. Yet, understanding what experimental data are needed and how to use them for unambiguously identifying the structure of even small networks remains a broad challenge. Here, we integrate a dynamic least squares framework into established modular response analysis (DL-MRA), that specifies sufficient experimental perturbation time course data to robustly infer arbitrary two and three node networks. DL-MRA considers important network properties that current methods often struggle to capture: (i) edge sign and directionality; (ii) cycles with feedback or feedforward loops including self-regulation; (iii) dynamic network behavior; (iv) edges external to the network; and (v) robust performance with experimental noise. We evaluate the performance of and the extent to which the approach applies to cell state transition networks, intracellular signaling networks, and gene regulatory networks. Although signaling networks are often an application of network reconstruction methods, the results suggest that only under quite restricted conditions can they be robustly inferred. For gene regulatory networks, the results suggest that incomplete knockdown is often more informative than full knockout perturbation, which may change experimental strategies for gene regulatory network reconstruction. Overall, the results give a rational basis to experimental data requirements for network reconstruction and can be applied to any such problem where perturbation time course experiments are possible. Dipeptides are minimalistic but sufficient for liquid-liquid phase separation Liquid-liquid phase separation (LLPS) of proteins mediates the assembly of biomolecular condensates involved in physiological and pathological processes. Identifying the minimalistic building blocks and the sequence determinant of protein phase separation is of urgent importance but remains challenging due to the enormous sequence space and difficulties of existing methodologies in characterizing the phase behavior of ultrashort peptides. Here we demonstrate computational tools to efficiently quantify the microscopic fluidity and density of liquid-condensates/solid-aggregates and the temperature-dependent phase diagram of peptides. Utilizing our approaches, we comprehensively predict the LLPS abilities of all 400 dipeptide combinations of coded amino acids based on 492 micro-second molecular dynamics simulations, and observe the occurrences of spontaneous LLPS. We identify 54 dipeptides that form solid-like aggregates and three categories of dipeptides with high LLPS propensity. Our predictions are validated by turbidity assays and differential interference contrast (DIC) microscopy on four representative dipeptides (WW, QW, GF, and VI). Phase coexistence diagrams are constructed to explore the temperature dependence of LLPS. Our results reveal that aromatic moieties are crucial for a dipeptide to undergo LLPS, and hydrophobic and polar components are indispensable. We demonstrate for the first time that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, suggesting that LLPS is a general property of peptides/proteins, independent of their sequence length. This study provides a computational and experimental approach to the prediction and characterization of the phase behavior of minimalistic peptides, and will be helpful for understanding the sequence-dependence and molecular mechanism of protein phase separation.

SignificanceProtein liquid-liquid phase separation (LLPS) is associated with human health and diseases. Identifying the minimalistic building blocks and sequence determinants of LLPS is of urgent importance but remains computationally challenging partially due to the lack of methodologies characterizing the liquid condensates. Herein we provide approaches to evaluate LLPS ability of dipeptides, and screen all 400 dipeptides by MD simulations combined with multi-bead-per-residue models which capture key interactions driving LLPS that are missing in one-bead-per-residue models. Three categories of LLPS dipeptides are identified and the experimentally-verified QW dipeptide is by far the smallest LLPS system. Our results suggest that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, and LLPS is a general property of peptides/proteins, independent of their length. Intramolecular carbon isotope signals reflect metabolite allocation in plants Stable isotopes at natural abundance are key tools to study physiological processes occurring outside the temporal scope of manipulation and monitoring experiments. Whole-molecule carbon isotope ratios (13C/12C) enable assessments of plant carbon uptake yet conceal information about carbon allocation. Here, we identify an intramolecular 13C/12C signal at treering glucose C-5 and C-6 and develop experimentally testable theories on its origin. More specifically, we assess the potential of processes within C3 metabolism for signal introduction based (inter alia) on constraints on signal propagation posed by metabolic networks. We propose that the intramolecular signal reports carbon allocation into major metabolic pathways in actively photosynthesising leaf cells including the anaplerotic, shikimate, and non-mevalonate pathway. We support our theoretical framework by linking it to previously reported whole-molecule 13C/12C increases in cellulose of ozone-treated Betula pendula and a highly significant relationship between the intramolecular signal and tropospheric ozone concentration. Our theory postulates a pronounced preference of leaf-cytosolic triose-phosphate isomerase to catalyse the forward reaction in vivo (dihydroxyacetone phosphate to glyceraldehyde 3-phosphate). In conclusion, intramolecular 13C/12C analysis resolves information about carbon uptake and allocation enabling more comprehensive assessments of carbon metabolism than whole-molecule 13C/12C analysis.

HighlightIntramolecular 13C/12C analysis resolves information about carbon uptake and allocation (and associated environmental controls) enabling more comprehensive assessments of carbon metabolism, plant-environment interactions, and environmental variability than whole-molecule 13C/12C analysis. Impedance scaling for gold and platinum microelectrodes ObjectiveElectrical measurement of the activity of individual neurons is a primary goal for many invasive neural electrodes. Making these "single unit" measurements requires that we fabricate electrodes small enough so that only a few neurons contribute to the signal, but not so small that the impedance of the electrode creates overwhelming noise or signal attenuation. Thus, neuroelectrode design often must strike a balance between electrode size and electrode impedance, where the impedance is often assumed to scale linearly with electrode area.

Approach and main resultsHere we study how impedance scales with neural electrode area and find that the 1 kHz impedance of Pt electrodes (but not Au electrodes) transitions from scaling with area (r-2) to scaling with perimeter (r-1) when the electrode radius falls below 10 microns. This effect can be explained by the transition from planar to spherical diffusion behavior previously reported for electrochemical microelectrodes. Significance: These results provide important intuition for designing small, single unit recording electrodes. Specifically, for materials where the impedance is dominated by a pseudo-capacitance that is associated with a diffusion limited process, the total impedance will scale with perimeter rather than area when the electrode size becomes comparable with the diffusion layer thickness. For Pt electrodes this transition occurs around 10 um radius electrodes. At even lower frequencies (1 Hz) impedance approaches a constant. This transition to r-1 scaling implies that electrodes with a pseudo-capacitance can be made smaller than one might expect before thermal noise or voltage division limits the ability to acquire high-quality single-unit recordings. Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. PRDM9 losses in vertebrates are coupled to those of paralogs ZCWPW1 and ZCWPW2 In most mammals and likely throughout vertebrates, the gene PRDM9 specifies the locations of meiotic double strand breaks; in mice and humans at least, it also aids in their repair. For both roles, many of the molecular partners remain unknown. Here, we take a phylogenetic approach to identify genes that may be interacting with PRDM9, by leveraging the fact that PRDM9 arose before the origin of vertebrates, but was lost many times, either partially or entirely--and with it, its role in recombination. As a first step, we characterize PRDM9 domain composition across 446 vertebrate species, inferring at least thirteen independent losses. We then use the interdigitation of PRDM9 orthologs across vertebrates to test whether it co-evolved with any of 241 candidate genes co-expressed with PRDM9 in mice or associated with recombination phenotypes in mammals. Accounting for the phylogenetic relationship among species, we find two genes whose presence and absence is unexpectedly coincident with that of PRDM9: ZCWPW1, which was recently shown to facilitate double strand break repair, and its paralog ZCWPW2, as well as more tentative evidence for TEX15 and FBXO47. ZCWPW2 is expected to be recruited to sites of PRDM9 binding; its tight coevolution with PRDM9 across vertebrates suggests that it is a key interactor, with a role either in recruiting the recombination machinery or in double strand break repair.

Author SummaryOur understanding of meiotic recombination in mammals has seen great progress over the past 15 years, spurred in part by the convergence of lines of evidence from molecular biology, statistical genetics and evolutionary biology. We now know that in most mammals and likely in many vertebrates, the gene PRDM9 specifies the location of meiotic double strand breaks and that in mice and humans at least, it also aids in their repair. For both roles, however, many of the molecular partners remain unknown. To search for these, we take a phylogenetic approach, leveraging the fact that the complete PRDM9 has been lost at least thirteen times in vertebrates and thus that its presence is interdigitated across species. By this approach, we identify two genes whose presence or absence across vertebrates is coupled to the presence or absence of PRDM9, ZCWPW1 and ZCWPW2, as well as two genes for which the evidence is weaker, TEX15 and FBXO47. ZCWPW1 was recently shown to be recruited to sites of PRDM9 binding and to aid in the repair of double strand breaks. ZCWPW2 is likely recruited to sites of PRDM9 binding as well; its tight coevolution with PRDM9 across vertebrates suggests that it plays an important role either in double strand break formation, potentially as the missing link that recruits the recombination machinery to sites of PRDM9 binding, or in double strand break repair. Structural predictions of the SNX-RGS proteins suggest they belong to a new class of lipid transfer proteins Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins. 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. Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. Simulation of foraging behavior using a decision-making agent with Bayesian and inverse Bayesian inference: Temporal correlations and power laws in displacement patterns It has been stated that in human migratory behavior, the step length series may have temporal correlation and that there is some relationship between this time dependency and the fact that the frequency distribution of step length follows a power-law distribution. Furthermore, the frequency of occurrence of the step length in some large marine organisms has been found to switch between power-law and exponential distributions, depending on the difficulty of prey acquisition. However, to date it has not been clarified how the aforementioned three phenomena arise: the positive correlation created in the step length series, the relation between the positive correlation of the step length series and the form of an individuals step length distribution, and the switching between power-law and exponential distributions depending on the abundance of prey. This study simulated foraging behavior using the Bayesian decision-making agent simultaneously performing both knowledge learning and knowledge-based inference to analyze how the aforementioned three phenomena arise. In the agent with learning and inference, past experiences were stored as hypotheses (knowledge) and they were used in current foraging behavior; at the same time, the hypothesis continued to be updated based on new experiences. The simulation results show that the agent with both learning and inference has a mechanism that simultaneously causes all the phenomena. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knockdown NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Further, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection. Bringing TrackMate into the era of machine-learning and deep-learning. TrackMate is an automated tracking software used to analyze bioimages and distributed as a Fiji plugin. Here we introduce a new version of TrackMate rewritten to improve performance and usability, and integrating several popular machine and deep learning algorithms to improve versatility. We illustrate how these new components can be used to efficiently track objects from brightfield and fluorescence microscopy images across a wide range of bio-imaging experiments. Development of a yeast-based assay for bioavailable phosphorus Preventing eutrophication of inland freshwater ecosystems requires quantifying the phosphorus (P) content of the streams and rivers that feed them. Typical methods for measuring P assess soluble reactive P (SRP) or total P (TP) and require expensive analytical techniques that produce hazardous waste. Here we present a novel method for measuring the more relevant bioavailable P (BAP); this assay utilizes the growth of familiar bakers yeast, avoids production of hazardous waste, and reduces cost relative to measurements of SRP and TP. The yeast BAP (yBAP) assay takes advantage of the observation that yeast density at saturating growth increases linearly with provided P. We show that this relationship can be used to measure P in freshwater in concentration ranges relevant to eutrophication. In addition, we measured yBAP in water containing known amounts of fertilizer and in samples from agricultural waterways. We observed that the majority of yBAP values were between those obtained from standard SRP and TP measurements, demonstrating that the assay is compatible with real-world settings. The cost-effective and nonhazardous nature of the yeast-based assay suggests that it could have utility in a range of settings, offering added insight to identify water systems at risk of eutrophication from excess phosphorus. Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons. Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for clinical sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNA) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell-type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy, especially real-time medical applications. Role of posterodorsal medial amygdala urocortin-3 in pubertal timing in female mice Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD. Sgs1BLM independent role of Dna2DNA2 nuclease at DNA double strand break is inhibited by Nej1XLF The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. The regulation of 5 DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and more recently, it was shown to be important for DNA end-bridging and inhibiting 5 resection mediated by Dna2-Sgs1. Nej1 interacts with Sae2 and this impacts DSB repair in three ways. First, Nej1 inhibits MRX-Sae2 interactions and Sae2 localization to a DSB. Second, Nej1 inhibits Sae2-dependent recruitment of Dna2 in the absence of Sgs1. Third, NEJ1 and SAE2 showed an epistatic relationship for DNA end-bridging, an event that restrains the broken ends and reduces the frequency of genomic deletions from developing at the DSB. Deletion of NEJ1 suppressed the synthetic lethality of sae2{Delta} sgs1{Delta} and was dependent on the nuclease activity of Dna2. These Nej1 functions promote end-joining DSB repair, but could also be relevant for controlling resection initiation during HR repair.

HighlightsO_LINej1 physically interacts with Sae2 and inhibits end-resection at a DSB.
C_LIO_LINej1 inhibits Sae2 interactions with the MRX complex.
C_LIO_LINej1 inhibits Sae2-dependent recruitment of Dna2 to a DSB.
C_LIO_LINEJ1 and SAE2 are epistatic for DNA end-bridging.
C_LIO_LIDeletion of NEJ1 suppresses the synthetic lethality of sae2{Delta} sgs1{Delta}, which is dependent on Dna2 nuclease activity.
C_LI 14-3-3ζ constrains insulin secretion by regulating mitochondrial function in pancreatic β-cells While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3{zeta} in adipocytes, but whether 14-3-3{zeta} has additional metabolic roles in other cell types, such as the pancreatic {beta}-cell, is unclear. We previously documented a pro-survival role of 14-3-3{zeta} in MIN6 insulinoma cells, as depletion of 14-3-3{zeta} induced cell death, but paradoxically, whole-body deletion of 14-3-3{zeta} in mice resulted in significantly enlarged {beta}-cell area with no effects on insulin secretion. To better understand the role of 14-3-3{zeta} in {beta}-cells, we generated {beta}-cell-specific 14-3-3{zeta} knockout ({beta}14-3-3{zeta}KO) mice, and while no differences in {beta}-cell mass were observed, {beta}14-3-3{zeta}KO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3{zeta} led to profound changes to the {beta}-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3{zeta} is a critical isoform in {beta}-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3{zeta}, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3{zeta} and its related isoforms in insulin secretion and mitochondrial function in {beta}-cells. A deeper understanding of how 14-3-3{zeta} influences {beta}-cell function will further advance our knowledge of how insulin secretion from {beta}-cells is regulated. Liver Endothelium Promotes HER3-mediated Cell Survival in KRAS Wild-type and Mutant Colorectal Cancer Cells We previously showed that liver endothelial cells (ECs) secreted soluble factors in a paracrine fashion and activated human epidermal growth factor receptor 3 (HER3, also known as ERBB3) for promoting colorectal cancer (CRC) growth and chemoresistance. However, RAS proteins play a critical role in receptor tyrosine kinase signaling pathways, and KRAS mutations mediate CRC resistance to therapies targeting EGFR, another HER protein. Therefore, the role of KRAS mutation status in EC-induced HER3 activation and CRC survival was investigated as it has therapeutic implications. We used CRC cell lines and patient-derived xenografts harboring KRAS wild-type or mutant genes and demonstrated that liver EC-secreted factors promoted HER3-mediated CRC cell growth independent of KRAS mutation status. Also, blocking HER3 in CRC cells by siRNAs or a HER3 antibody seribantumab blocked EC-induced CRC survival. Our findings highlight the potential of utilizing HER3 targeted therapies for treating patients with mCRC independent of RAS mutational status. Decoding regulatory specificity of human ribosomal proteins Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with little regulatory function. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is still lacking. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We quantified translational and transcriptional changes of gene expression upon deficiency of 75 RPs, including 44 from the large subunit (60S) and 31 from the small subunit (40S), by ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S and the 40S RPs had opposite effects on the two subunits. RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by perturbing P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis and RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity, providing an important resource which can offer novel insights into ribosome regulation in human diseases and cancer.