mTOR driven gene transcription is required for cholesterol production in neurons of the developing cerebral cortex Dysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood.

Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the sterol/cholesterol biosynthesis pathway to be transcriptionally regulated by mTOR complex 1 (mTORC1) signaling in vitro in primary neurons and in vivo in the developing cerebral cortex of the mouse. We find that these genes are shared targets of the transcription factors SREBP, SP1 and NF-Y. Prenatal as well as postnatal mTORC1 inhibition downregulated expression of these genes which directly translated into reduced cholesterol levels pointing towards a substantial metabolic function of the mTORC1 signaling cascade. Altogether, our results indicate that mTORC1 is an essential transcriptional regulator of the expression of sterol/cholesterol biosynthesis genes in the developing brain. Altered expression of these genes may be an important factor contributing to the pathogenesis of neurodevelopmental disorders associated with dysregulated mTOR signaling.
Large scale in vivo acquisition, segmentation, and 3D reconstruction of cortical vasculature using open-source functional ultrasound imaging platform The brain is composed of a dense and ramified vascular network comprising various sizes of arteries, veins, and capillaries. One way to assess the risk of cerebrovascular pathologies is to use computational models to predict the physiological effects of a reduction of blood supply and correlate these responses with observations of brain damage. Therefore, it is crucial to establish a detailed 3D organization of the brain vasculature, which could be used to develop more accurate in silico models. For this purpose, we have adapted our open-access functional ultrasound imaging platform previously designed for recording brain-wide activity that is now capable of fast and reproducible acquisition, segmentation, and reconstruction of the cortical vasculature. For the first time, it allows us to digitize the cortical vasculature in awake rodents with a [~]100 {micro}m3 spatial resolution. Contrary to most available strategies, our approach can be performed in vivo within minutes. Moreover, it is easy to implement since it neither requires exogenous contrast agents nor long post-processing time. Hence, we performed a cortex-wide reconstruction of the vasculature and its quantitative analysis, including i) classification of descending arteries versus ascending veins in more than 1500 vessels/animal, ii) quick estimation of their length. Importantly, we confirmed the relevance of our approach in a model of cortical stroke, which enables quick visualization of the ischemic lesion. This development contributes to extending the capabilities of ultrasound neuroimaging to understand better cerebrovascular pathologies such as stroke, vascular cognitive impairment, and brain tumors and is highly scalable for the clinic.
Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. A Hierarchy of Biomolecular Proportional-Integral-Derivative Feedback Controllers for Robust Perfect Adaptation and Dynamic Performance Proportional-Integral-Derivative (PID) feedback controllers have been the most widely used controllers in industry for almost a century due to their good performance, simplicity, and ease of tuning. Motivated by their success in various engineering disciplines, PID controllers recently found their way into synthetic biology, where the design of feedback molecular control systems has been identified as an important goal. In this paper, we consider the mathematical realization of PID controllers via biomolecular interactions. We propose an array of topologies that offer a compromise between simplicity and high performance. We first demonstrate that different Proportional-Integral (PI) controllers exhibit different capabilities for enhancing the dynamics and reducing variance (cell-to-cell variability). Next, we introduce several derivative controllers that are realized based on incoherent feedforward loops acting in a feedback configuration. Alternatively, we show that differentiators can be realized by placing molecular integrators in a negative feedback loop--an arrangement that can then be augmented by PI components to yield PID feedback controllers. We demonstrate that the derivative component can be exploited for enhancing system stability, dramatically increasing the molecular control systems dynamic performance, and for reducing the noise effect on the output of interest. The PID controller features are established through various deterministic and stochastic analyses as well as numerical simulations. Finally, we provide an experimental demonstration using a recently developed hybrid setup, the cyberloop, where the controller is implemented in silico to control a biological genetic circuit in vivo. The large array of novel biomolecular PID controllers introduced here forms a basis for the design and construction of advanced high-performance biomolecular control systems that robustly regulate the dynamics of living systems. Atomic Force Microscopy Reveals Distinct Protofilament-scale Structural Dynamics in Depolymerizing Microtubule Arrays The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes, implicated in both the complete demolition and fine size control of microtubule-based arrays, are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here we use Atomic Force Microscopy (AFM) to image depolymerizing arrays at single microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-sized cytoskeletal structures.

SIGNIFICANCE STATEMENTOne cannot help but marvel at the precise organization of microtubule polymers in cellular structures such as the axoneme and the spindle. However, our understanding of the biochemical mechanisms that sculpt these arrays comes largely from in vitro experiments with a small number (one or two) of microtubules. This is somewhat akin to studying the architecture of multilane highways by studying one-lane streets. Here we directly visualize depolymerizing microtubule arrays at individual microtubule and protofilament resolution using Atomic Force Microscopy. Our results reveal differences in microtubule depolymerase activity and provide insights into how these differences in enzymatic activity on the nanometer scale can result in the differential remodeling of multi-microtubule arrays on the micron-length scale. Synergy of chemotherapy and macrophage depletion leads to B cell-mediated T cell memory activation and durable triple negative breast cancer regression Immunosuppressive elements within the tumor microenvironment such as Tumor Associated Macrophages (TAMs) can present a barrier to successful anti-tumor responses by cytolytic T cells. We employed preclinical syngeneic p53 null mouse models of triple negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was used to successfully treat several highly aggressive TNBC murine mammary tumors and lung metastasis. Using this regimen and single cell RNA sequencing we characterized tumor infiltrating lymphocytes (TILs) including helper T cells and antigen-presenting B cells that were highly enriched in good responders to combination therapy. Using high dimensional imaging techniques, we identified the close spatial localization of B220+ CD86+ activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks post-treatment in one model that also exhibited long-term tumor regression post-treatment. We also characterized the transcriptional and metabolic heterogeneity of TAMS in these two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model is highly expressed and conserved in human claudin-low breast cancers, and high expression of the T12 signature correlated with reduced overall survival. This T12 tumor TAM signature may help identify human claudin-low breast cancer patients that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rationale combinations of immunotherapy.

SignificanceA treatment regimen that harnessed the immunostimulatory effects of cyclophosphamide coupled with the inhibition of CSF1R was used to successfully treat several highly aggressive claudin-low TNBC murine mammary tumors and lung metastasis. Drug screening at single-organoid resolution via bioprinting and interferometry High-throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms for high-throughput screening use two-dimensional cultures of immortalized cell lines which do not accurately reflect the biology of human tumors. More clinically relevant model systems, such as three-dimensional tumor organoids, can be difficult to screen and scale. For example, manually seeded organoids coupled to destructive endpoint assays allow for the characterization of response to treatment, but do not capture the transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We therefore developed a pipeline to generate bioprinted tumor organoids linked to label-free, real-time imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D organoid structures that preserve tumor histology and gene expression. HSLCI imaging in tandem with machine learning-based image segmentation and organoid classification tools enables accurate, label-free parallel mass measurements for thousands of bioprinted organoids. We demonstrate that our method quantitatively identifies individual organoids as insensitive, transiently sensitive, or persistently sensitive to specific treatments. This opens new avenues for rapid, actionable therapeutic selection using automated tumor organoid screening. Late-acting self-incompatible system, preferential allogamy and delayed selfing in the heteromorphic invasive populations of Ludwigia grandiflora subsp. hexapetala AO_SCPLOWBSTRACTC_SCPLOWBreeding system influences local population genetic structure, effective size, offspring fitness and functional variation. Determining the respective importance of self- and cross-fertilization in hermaphroditic flowering plants is thus important to understand their ecology and evolution. The worldwide invasive species, Ludwigia grandiflora subsp. hexapetala (Lgh) presents two floral morphs: one self-compatible short-styled morph (S-morph) and one self-incompatible long-styled morph (L-morph). In this study, we identified the breeding systems of western European experimental and natural populations of Lgh by comparing structural characteristics of pollen and style, by studying self- and cross-pollen tube elongations and the viability of the resulting seeds and seedlings in both floral morphs. Our results showed no differences in pollen shape and stigma surfaces no matter the floral morph. In the self-incompatible L-morph flowers, self-pollen tubes were stopped tardily, in the ovarian area, and were unable to fertilize the ovules. This first formal identification of a late-acting, prezygotic self-incompatible system (LSI) in Ludwigia genus contributes a case of LSI in an additional family within the Myrtales order. In the self-compatible S-morph flowers, self-pollen always succeeded to self-fertilize the ovules that nearly all developed into viable seedlings. However, cross-pollen tubes always elongated faster than self-pollen tubes in both morphs. S-morph individuals may thus advantage preferential allogamy over selfing when cross-pollen is available despite its self-compatibility. As expected in late-acting self-incompatible systems, L-morph flowers authorised 0.2{per thousand} of selfed seeds during the uppermost flowering season, that increased to 1{per thousand} at the end of the flowering season. Such delayed selfing resulted in a significant quantity of viable floating seeds. They may contribute to the local regeneration, seed bank and propagation of the L-morph, which may contribute to explain its invasion success worldwide. Management plans of Lgh would gain to consider the breeding systems we identified. 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. 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. 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. The origins and drivers of Neotropical diversity The origins and evolution of the outstanding Neotropical biodiversity are still debated. A comprehensive understanding is hindered by the lack of deep-time comparative data across wide phylogenetic and ecological contexts. Here, we evaluate four evolutionary scenarios assuming different diversification trajectories and drivers of Neotropical diversification, and assess their variation across Neotropical regions and taxa. Our analysis of 150 phylogenies (12,512 species) of seed plants and tetrapods reveals that Neotropical diversity has mostly expanded through time (70% of the clades), while scenarios of saturated and declining diversity also account for 21% and 9% of Neotropical diversity, respectively. We identify five biogeographic areas that represent distinctive units of long-term Neotropical evolution (Pan-Amazonia, Dry Diagonal, Bahama-Antilles, Galapagos, and an  elsewhere region) and find that diversity dynamics do not differ across these areas, suggesting no geographic structure in long-term Neotropical diversification. In contrast, diversification dynamics differ substantially across taxa: plant diversity mostly expanded through time (88%), while a substantial fraction (43%) of tetrapod diversity accumulated at a slower pace or declined toward the present. These opposite evolutionary patterns may reflect different capacities for plants and tetrapods to cope with climate change, with potential implications for future adaptation and ecosystem resilience. Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies Dementia with Lewy bodies is pathologically defined by the cytoplasmic accumulation of alpha-synuclein within neuronal cells in the brain. Alpha-synuclein is predominately pre-synaptic, but has been reported present in various subcellular compartments in cell and animal models. In particular, nuclear alpha-synuclein is evident in-vitro and in disease models and has been associated with altered DNA integrity, gene transcription, nuclear homeostasis. However, owing to various factors, the presence of alpha-synuclein in the nuclei of human brain cells remains controversial, as does its role in synucleinopathies.

Here, we close this gap and provide a unique demonstration confirming the presence of nuclear alpha-synuclein in post-mortem brain tissue obtained from cases of dementia with Lewy bodies as well as from controls via immunohistochemistry, immunoblot, and label-free mass-spectrometry.

Discrete intra-nuclear alpha-synuclein puncta reactive against phosphorylated serine 129-alpha-synuclein and pan-alpha-synuclein antibodies were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations from Dementia with Lewy bodies cases and matched controls. Subsequent biochemical analysis of subcellular fractionated tissue confirmed alpha-synuclein as present in a nuclear fraction at levels ~ 10-fold lower than in the cytoplasm. Critically, however, an increase in monomeric nuclear alpha-synuclein phosphorylated as serine 129 was observed in cases of dementia with Lewy bodies alongside higher molecular weight pan- and phosphorylation reactive alpha-synuclein species, consistent with the formation of intranuclear phosphorylated alpha-synuclein oligomers. Furthermore, the presence of nuclear alpha-synuclein was confirmed via label free mass spectrometry, as 6 unique alpha-synuclein derived peptide sequences were identified in nuclear fractions (71.4% sequence coverage).

Collectively, our data confirm the presence of nuclear alpha-synuclein in human brain tissue and describe nuclear pathology associated with dementia with Lewy bodies. These findings address a major controversy in the synucleinopathy field by confirming the presence of nuclear alpha-synuclein in autoptic human brain tissue and, for the first time, identify that alpha-synuclein is aggregated into novel and potentially pathological assemblies in the nucleus as part of the disease process associated with dementia with Lewy bodies and thus may contribute to the disease phenotype. Social integration predicts survival in female white-faced capuchin monkeys Across multiple species of social mammals, a growing number of studies have found that that individual sociality is associated with survival. In long-lived species, like primates, lifespan is one of the main components of fitness. We used 18 years of data from the Lomas Barbudal Monkey project to quantify social integration in 11 capuchin (Cebus capucinus) groups and tested whether female survivorship was associated with females tendencies to interact with three types of partners: (1) all group members, (2) adult females, (3) adult males. We found strong evidence that females who engaged more with other females in affiliative interactions and foraged in close proximity experienced increased survivorship. We found some weak evidence that females might also benefit from engaging in more support in agonistic contexts with other females. These benefits were evident in models that account for the females rank and group size. Female interactions with all group members also increased survival, but the estimates of the effects were more uncertain. In interactions with adult males, only females who provided more grooming to males survived longer. The results presented here suggest that social integration may result in survival-related benefits. Females might enjoy these benefits through exchanging grooming for other currencies, such as coalitionary support or tolerance. Pathological LSD1 mutations repress enhancer-mediated gene regulatory networks in early differentiation Lysine-specific demethylase 1 (LSD1/KDM1A) demethylates both histone and non-histone substrates, recruits repressive chromatin complexes, and is increased in cancers. De novo LSD1 mutations impairing protein function lead to a rare developmental disorder, but the molecular details of the pathology remains unclear. Using patient-derived fibroblasts, reprogrammed pluripotent stem cells, and differentiated cells, we found over 4000 differentially expressed genes and 68 transcription factors (TFs) whose motif accessibilities changed upon LSD1 mutation. An enhancer-mediated gene regulatory network approach identified transcriptional repressors with impaired activity in fibroblast and stem cells, leading to erroneous activation of their target genes. We also revealed overall decreases in TF target gene expression during early lineage differentiation of LSD1 mutant stem cells, likely caused by increased activity of repressive histone deacetylases (HDACs), co-factors of LSD1. Indeed, an HDACs inhibitor restored changes in gene expression including downregulation phenotype. Our findings characterize the molecular pathogenesis of LSD1 mutations and suggest potential therapeutic strategies for the developmental disorder and cancers caused by LSD1 dysregulations. The Clade Displacement Index: how to detect horizontal gene transfers in unrooted gene trees While most genes of any organism are inherited vertically - i.e. from its parent organisms - sometimes they can be exchanged between unrelated species in a process known as the horizontal gene transfer (HGT). Studies of HGT contribute to our knowledge about the mechanisms of evolution, including the emergence of new pathogens, and a great deal of effort has been put into different methods of finding transferred genes. The golden standard of HGT detection is the analysis of the incongruence between the gene and the species trees. Those methods typically require rooted trees, in which the direction of evolution is known. Gene trees are typically unrooted, and rooting them is yet another step in HGT analysis, prone to errors which may lead to wrong conclusions. A natural question arises: can HGTs be detected in gene trees without rooting them at all?

It turns out that, for a particular, yet broad, class of transfers, the answer to this question is: yes. It also turns out that the same methodology can be applied to complement the bootstrap support in assessing the stability of gene tree topology. In this article, we present the Clade Displacement Index, a measure of shift of a given clades location between two trees. We derive algorithms to compute it and give several examples of its applications to HGT detection and gene tree stability analysis. We finish by pointing out directions for further studies and an example that shows that not all HGTs are detectable without knowing the location of the root of the gene tree.

A Jupyter Notebook with the implementation and applications of CDI described in this paper is available at https://github.com/mciach/CDI Mathematical Modeling and Analysis of Mitochondrial Retrograde Signaling Dynamics Mitochondria, semi-autonomous eukaryotic organelles, participate in energy production and metabolism, making mitochondrial quality control crucial. As most mitochondrial proteins are encoded by nuclear genes, quality control depends on proper mitochondria-nucleus communication, designated mitochondrial retrograde signaling. Early studies focused on retrograde signaling participants and specific gene knockouts. However, mitochondrial signal modulation remains elusive. Using yeast, we simulated signal propagation following mitochondrial damage and proposed a mathematical model based ordinary differential equations. Mitochondrial retrograde signaling decisions were described using a Boolean model. Dynamics were analyzed through an ordinary differential equation-based model and extended to evaluate the model response to noisy damage signals. Simulation revealed localized protein concentration dynamics, including waveforms, frequency response, and robustness under noise. Retrograde signaling is bistable with three localized steady states, and increased damage compromises robustness. We elucidated mitochondrial retrograde signaling, thus providing a basis for drug design against yeast and fungi.

Author SummaryThe yeast RTG pathway regulates mitochondrial metabolism and mitochondrial quality through passing mitochondrial signal to the nucleus to modulate gene expressions. Using microscopic data of RTG proteins translocation from cytosol to nucleus, the parameters were found by fitting 16 knockout conditions simulated from the proposed differential equation-based model. Further, through dose response, ultrasensitivity, frequency response and noise interference, we demonstrated a switch-like property of RTG pathway activation, a capacity charging property of RTG protein translocation, a low pass filter property of signals in different frequencies, and noise amplification due to mitochondrial signal. The proposed mathematical model enables us to understand the dynamics and mechanisms in mitochondrial retrograde signaling in yeast and provides potential antifungal treatment strategies. O-specific antigen-dependent surface hydrophobicity mediates aggregate assembly type in Pseudomonas aeruginosa Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here we show that in an in vitro model of cystic fibrosis (CF) containing the polymers eDNA and mucin, O-specific antigen is a major factor in determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs.

ImportanceDuring chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways, however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways. A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in five hours. The screen tested 1280 FDA-approved drugs and identified Pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacologic and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with Pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt-signaling. In contrast, HTR2C induced epithelial to mesenchymal transition (EMT) through activation of Wnt-signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs. MG53 slows neuromuscular junction loss and prolongs survival in ALS Respiratory failure from progressive respiratory muscle weakness is the most common cause of death in amyotrophic lateral sclerosis (ALS). Defects in neuromuscular junctions (NMJs) and progressive NMJ loss occur at early stages, thus stabilizing and preserving NMJs represents a potential therapeutic strategy to slow ALS disease progression. Here we demonstrate that NMJ damage is repaired by MG53, an intrinsic muscle protein involved in plasma membrane repair. Compromised diaphragm muscle membrane repair and NMJ integrity are early pathological findings in ALS. Diaphragm muscles from ALS mouse models show increased susceptibility to injury and intracellular MG53 aggregation, which is also a hallmark of human muscle samples from ALS patients. We show that systemic administration of recombinant human MG53 protein (rhMG53) in ALS mice protects against injury to diaphragm muscle, preserves NMJ integrity, and slows ALS disease progression. As MG53 is present in circulation in rodents and humans under physiological conditions, our findings provide proof-of-concept data supporting MG53 as a potentially safe and effective therapy to mitigate ALS progression. Inhibition of microbial deconjugation of micellar bile acids protects against intestinal permeability and liver injury Altered host-microbe interactions and increased intestinal permeability have been implicated in the pathogenesis of a range of diseases. However, the mechanisms by which gut microbes affect epithelial barrier integrity remain unclear. Few host-produced metabolites that protect against epithelial damage have been identified, and whether microbial metabolism of host factors alters intestinal barrier function is unknown. Here, we investigate the effects of bacterial metabolism of host-produced bile acid (BA) metabolites on epithelial barrier integrity. We observe that rats fed a choline-deficient, high-fat diet (CDAHFD) exhibit reduced abundance of host-produced conjugated BAs in the intestine at early timepoints coinciding with increased permeability. We show that in vitro, conjugated BAs protect gut epithelial monolayers from damage caused by bacterially produced unconjugated BAs through micelle formation. We then demonstrate that inhibition of BA deconjugation using a small molecule inhibitor of gut bacterial bile salt hydrolase (BSH) enzymes prevents development of pathologic intestinal permeability and hepatic inflammation in CDAHFD-fed rats. Finally, we show that the predominant conjugated BAs in humans protect against epithelial barrier disruption in vitro. Our study identifies a protective role for conjugated BAs in intestinal epithelial barrier function and suggests that rational manipulation of microbial BA metabolism could be leveraged to regulate gut barrier integrity. Loss of the DYRK1A protein kinase results in reduction of ribosomal protein genes expression, ribosome mass and translation defects Ribosomal proteins (RPs) are evolutionary conserved proteins that are essential for protein translation. RP expression must be tightly regulated to ensure the appropriate assembly of ribosomes and to respond to the growth demands of cells. The elements regulating the transcription of RP genes (RPGs) have been characterized in yeast and Drosophila, yet how cells regulate the production of RPs in mammals is less well understood. Here, we show that a subset of RPG promoters is characterized by the presence of the palindromic TCTCGCGAGA motif and marked by the recruitment of the protein kinase DYRK1A. The presence of DYRK1A at these promoters is associated with enhanced binding of the TATA-binding protein, TBP, and it is negatively correlated with the binding of the GABP transcription factor, establishing at least two clusters of RPGs that could be coordinately regulated. However, DYRK1A silencing leads to a global reduction of RPGs, mRNA pointing at DYRK1A activities beyond those dependent on its chromatin association. Significantly, cells in which DYRK1A is depleted have reduced RP levels, fewer ribosomes, reduced global protein synthesis and smaller size. We therefore propose a novel role for DYRK1A in coordinating the expression of genes encoding RPs, thereby controlling cell growth in mammals. Citation needed?Wikipedia bibliometrics during the first waveof the COVID-19 pandemic. BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Distinct SARS-CoV-2 antibody reactivity patterns elicited by natural infection and mRNA Vaccination We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8,476 finger stick blood specimens were collected before and after an aggressive mRNA vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing 10 SARS-CoV-2 antigens, 4 SARS, 3 MERS, 12 Common CoV, and 8 Influenza antigens. Twenty-six percent of 3,347 specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive. The Ab response was predominantly against nucleocapsid (NP), full length spike and the spike S2 domain. Anti-receptor binding domain (RBD) reactivity was low and there was no cross-reactivity against SARS S1 or SARS RBD. An aggressive mRNA vaccination campaign at the UCI Medical Center started on December 16, 2020 and 6,724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% in December to 79% in January, 93% in February and 99% in March. mRNA vaccination induced much higher Ab levels especially against the RBD domain and significant cross-reactivity against SARS RBD and S1 was also observed. Nucleocapsid protein Abs can be used to distinguish individuals in a population of vaccinees to classify those who have been previously infected and those who have not, because nucleocapsid is not in the vaccine. Previously infected individuals developed higher Ab titers to the vaccine than those who have not been previously exposed. These results indicate that mRNA vaccination rapidly induces a much stronger and broader Ab response than SARS-CoV-2 infection. Ovule siRNAs methylate protein-coding genes in trans 24-nt small interfering siRNAs maintain asymmetric DNA methylation at thousands of euchromatic transposable elements in plant genomes in a process call RNA-directed DNA Methylation (RdDM). RdDM is dispensable for growth and development in Arabidopsis, but is required for reproduction in other plant species, such as Brassica rapa. 24-nt siRNAs are particularly abundant in maternal reproductive tissue, due largely to overwhelming expression from a small number of loci in the ovule and developing seed coat, termed siren loci. Recently it was shown that abundantly expressed 24-nt siRNAs produced in the tapetal tissue of anthers can methylate male meiocyte genes in trans (Long et al., 2021). Here we show that a similar process takes place in female tissue. siRNAs are produced from gene fragments embedded in some siren loci, and these siRNAs can trigger methylation in trans at related protein-coding genes. This trans-methylation is associated with silencing of some target genes and may be responsible for seed abortion in RdDM mutants. Furthermore, we demonstrate that a consensus sequence in at least two families of DNA transposons is associated with abundant siren expression, most likely through recruitment of the CLSY3 putative chromatin remodeller. This research describes a new mechanism whereby RdDM influences gene expression and sheds light on the role of RdDM during plant reproduction. Natural variations in reproductive timing of brood size and X-chromosome nondisjunction in Caenorhabditis elegans Reproductive timing underlies self-sperm production and X-chromosome nondisjunction rate in Caenorhabditis elegans. These traits can be measured as brood size and male-production rate, and brood size decreases and male-production rate increases as worms age. These phenotypic changes occur simultaneously; however, whether these changes share common underlying genetic architectures still remains unclear. To enhance our understanding of reproductive timing in C. elegans, we measured and compared these two reproductive timing-related traits of 96 wild strains during early, late and total reproductive periods. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. The brood size and the male-production rate were not genetically correlated, implying that they might have different genetic architectures and that meiosis quality can be maintained despite increased progeny-production in the late reproductive period caused by more self-sperm. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life history traits in wild C. elegans strains.