Cell-type specific circadian bioluminescence rhythms in Dbp reporter mice Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.
Robust and Remote Measurement of Heart Rate Based on A Surveillance Camera Arrhythmia is a marked symptom of many cardiovascular diseases (CVDs). The accurate and timely detection of heart rate can greatly reduce the harm of arrhythmia to people. However, it is still a challenge to robustly and remotely measure heart rate in daily life due to the changing environmental conditions during measurement, such as the varying light intensity, the movement of people, and the uncertain distance between the sensor to people. In this study, we propose a method to accurately measure human heart rate within a distance of 4.5 meters under different light intensities by simply using a surveillance camera. After a 20-second color video of a persons hand is captured by the camera, a method based on the Fast Fourier Transform (FFT) algorithm is designed to extract the blood volume pulse wave to calculate the heart rate. According to the comparison between the real heart rate and results measured by electrocardiography (ECG), the proposed method achieves an accuracy of 95.8% when the measurement is performed within a distance of 4.5 meters and 90% when within 5.0 meters. Our experiments show that when the illuminance varies between 100-1000 lux (lighting level indoor), we still get the correct results. Our experiments also demonstrate that the proposed method accurately obtain heart rate even when the light intensity is below 32 lux (300-500 lux in a workplace environment). The methods strong adaptability to changing environmental conditions makes it applicable to many scenarios, such as homes of the elderly, classrooms, and other public spaces.
Genome of the estuarine oyster provides insights into climate impact and adaptive plasticity Understanding the roles of genetic divergence and phenotypic plasticity in adaptation is central to evolutionary biology and important for assessing adaptive potential of species under climate change. Analysis of a chromosome-level assembly and resequencing of individuals across wide latitude distribution in the estuarine oyster (Crassostrea ariakensis) revealed unexpectedly low genomic diversity and population structures shaped by historical glaciation, geological events and oceanographic forces. Strong selection signals were detected in genes responding to temperature and salinity stress, especially of the expanded solute carrier families, highlighting the significance of gene expansion in environmental adaptation. Genes exhibiting high plasticity showed strong selection in upstream regulatory regions that modulate transcription, indicating selection favoring plasticity. Our findings suggest that genomic variation and population structure in marine bivalves are heavily influenced by climate history and physical forces, and gene expansion and selection may enhance phenotypic plasticity that is critical for the adaptation to rapidly changing environments. A liquid +TIP-network regulates microtubule dynamics through tubulin condensation Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here we show, that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid-networks. When CLIP-170 and EB3 act together the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells these liquid networks can condense tubulin. In vitro in the presence of microtubules, EB3/CLIP-170 phase separation can co-condense tubulin all along the microtubule. At this condition microtubule growth speed increases up to two-fold and depolymerization events are strongly reduced, compared to conditions with phase separation deficient networks. Our data show that phase separated EB3/CLIP-170 networks impact microtubule growth dynamics beyond direct protein-microtubule interactions. Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications. Bone marrow haematopoietic stem cells influence liver homeostatic networks and cancer development In fatty liver disease, systemic homeostasis is perturbed. While pre-clinical models are used to understand its pathogenesis, translating this knowledge to patients is difficult. However, by focusing on the most preserved homeostasis systems between species and models, novel disease dimensions can be unearthed. We interrogated core liver gene co-expression networks in a mouse model of liver cancer following dietary challenge. Immunometabolic modules showed temporal changes under the influence of diet duration and aging. The behaviour of immune network in tumours mirrored their counterparts in non-tumour liver. A high immune response network was associated with a lower tumour burden in mice and humans. This module in mice was enriched for genes related to haematopoietic cell differentiation. Consistently, the bone marrow haematopoietic stem and progenitor cells response was reflective of the liver immune response. Linking haematopoiesis to hepatic homeostasis uncovers a hitherto unexplored dimension of tissue crosstalk that can inform pathogenesis. ASURAT: functional annotation-driven unsupervised clustering of single-cell transcriptomes MotivationSingle-cell RNA sequencing (scRNA-seq) analysis reveals heterogeneity and dynamic cell transitions. However, conventional gene-based analyses require intensive manual curation to interpret the biological implications of computational results. Hence, a theory for efficiently annotating individual cells is necessary.

ResultsWe present ASURAT, a computational pipeline for simultaneously performing unsupervised clustering and functional annotation of disease, cell type, biological process, and signaling pathway activity for single-cell transcriptomic data, using correlation graph-based decomposition of genes based on database-derived functional terms. We validated the usability and clustering performance of ASURAT using scRNA-seq datasets for human peripheral blood mononuclear cells, which required fewer manual curations than existing methods. Moreover, we applied ASURAT to scRNA-seq and spatial transcriptome datasets for small cell lung cancer and pancreatic ductal adenocarcinoma, identifying previously overlooked subpopulations and differentially expressed genes. ASURAT is a powerful tool for dissecting cell subpopulations and improving biological interpretability of complex and noisy transcriptomic data.

AvailabilityA GPLv3-licensed implementation of ASURAT is on GitHub (https://github.com/keita-iida/ASURAT). Hidden Talents: Poly (I:C)-induced maternal immune activation improves mouse visual discrimination performance and reversal learning in a sex-dependent manner. While there is a strong focus on the negative consequences of maternal immune activation (MIA) on developing brains, very little attention is directed towards potential advantages of early life challenges. In this study we utilized a polyinosine-polycytidylic acid (poly(I:C)) MIA model to test visual discrimination (VD) and reversal learning (RL) in mice using touchscreen technology. Significant sex differences emerged in that MIA reduced the latency for males to make a correct choice in the VD task while females reached criterion sooner, made fewer errors, and utilized fewer correction trials in RL compared to saline controls. These surprising improvements were accompanied by the sex-specific upregulation of several genes critical to cognitive functioning, indicative of compensatory plasticity in response to MIA. In contrast, when exposed to a  two-hit stress model (MIA + loss of the social component of environmental enrichment (EE)), mice did not display anhedonia but required an increased number of PD and RL correction trials. These animals also had significant reductions of CamK2a mRNA in the prefrontal cortex. Appropriate functioning of synaptic plasticity, via mediators such as this protein kinase and others, are critical for behavioral flexibility. Although EE has been implicated in delaying the appearance of symptoms associated with certain brain disorders, these findings are in line with evidence that it also makes individuals more vulnerable to its loss. Overall, with the right  dose, early life stress exposure can confer at least some functional advantages, which are lost when the number or magnitude of these exposures become too great. Cellular feedback to organotelluranes displays modulation of antioxidant proteins gene expression Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds.



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org.highwire.dtl.DTLVardef@52bb82org.highwire.dtl.DTLVardef@19476d9org.highwire.dtl.DTLVardef@6aa99org.highwire.dtl.DTLVardef@1aa605e_HPS_FORMAT_FIGEXP  M_FIG C_FIG Evidence for a hydrogen sulfide-sensing E3 ligase in yeast In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor which activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the transsulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30s WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the transsulfuration pathway to regulate synthesis of these special amino acids. From GWAS Variant to Function: a Study of ~148,000 Variants for Blood Cell Traits. Genome-wide association studies (GWAS) have identified hundreds of thousands of genetic variants associated with complex diseases and traits. However, most variants are noncoding and not clearly linked to genes, making it challenging to interpret these GWAS signals. We present a systematic variant-to-function study, prioritizing the most likely functional elements of the genome for experimental follow-up, for >148,000 variants identified for hematological traits. Specifically, we developed VAMPIRE: Variant Annotation Method Pointing to Interesting Regulatory Effects, an interactive web application implemented in R Shiny (http://shiny.bios.unc.edu/vampire/). This tool efficiently integrates and displays information from multiple complementary sources, including epigenomic signatures from blood cell relevant tissues or cells, functional and conservation summary scores, variant impact on protein and gene expression, chromatin conformation information, as well as publicly available GWAS and phenome-wide association study (PheWAS) results. Leveraging data generated from independently performed functional validation experiments, we demonstrate that our prioritized variants, genes, or variant-gene links are significantly more likely to be experimentally validated. This study not only has important implications for systematic and efficient revelation of functional mechanisms underlying GWAS variants for hematological traits, but also provides a prototype that can be adapted to many other complex traits, paving the path for efficient variant to function (V2F) analyses. Design, Mutate, Screen: High-throughput creation of genetic clocks with different period-amplitude characteristics Directed evolution has become an invaluable tool in protein engineering and has also greatly influenced the construction of synthetic gene circuits. The ability to generate diversity at precise targets for directed evolution approaches has improved vastly, allowing researchers to create large, specific mutant libraries with relative ease. Screening approaches for large mutant libraries have similarly come a long way, especially when the desired behavior can easily be tested for with static, single time-point assays. For more complex gene circuits with dynamic phenotypes that change over time, directed evolution approaches to controlling and tuning circuit behavior have been hindered by the lack of sufficiently high-throughput screening methods to isolate variants with desired characteristics. Here we utilize directed mutagenesis and multiplexed microfluidics to develop a workflow for creating, screening and tuning dynamic gene circuits that operate at the population level. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and tune its dynamics while uncovering principles regarding its behavior. Lastly, we utilize this directed evolution workflow to construct a new synchronized genetic oscillator that exhibits robust dynamics over long time scales. Game of theories: an agent-based model of how variation in contest cost-benefits determines the evolution of contest resolution rules Game-theory based models are used to understand rules that animals use to settle contests over indivisible resources. However, the empirical literature of contests indicates controversial support to models, with some species supporting different models and other species showing no support to any model. Since strategies used to resolve contests may have different associated costs, it is possible that different conditions have determined the evolution of distinct assessment strategies used by animals. We used an agent-based model to explore the importance of the following conditions: resource availability, probability of reproduction with resource, and damage costs on evolution of assessment strategies. We used self- and mutual-assessment models as a heurist framework to build agents with different assessment strategies. In our model, agents competed for resources in scenarios with different combinations of resource availability, probability of reproduction with resource, and damage costs. We found that agents following the self-assessment with damage strategy were prevalent in scenarios with no probability of reproduction without the resource, independently of other variables. We also found that agents following the non-aggressive strategy occurred in all scenarios. However, agents using the non-aggressive strategy were prevalent only in scenarios with probability of reproduction with the resource. Finally, we observed that agents using mutual-assessment occurred only in a scenario with high risk of damage, low availability of resources, and with probability of reproduction without the resource. These results indicate that agents following the self-assessment with damage and non-aggressive strategies may be able to stay at most scenarios. Chemomechanically voxelated niches for programmable histogenesis Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Youngs moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models. Experimental evolution in TP53 deficient human gastric organoids recapitulates tumorigenesis The earliest events during human tumor initiation are poorly characterized but may hold clues as to how to detect and prevent malignancy. Here we model occult pre-neoplasia by engineering TP53 deficiency, a common and early event in gastric cancer, into human gastric organoids. By performing experimental evolution in multiple clonally derived cultures over two years we define causal relationships between this common initiating genetic lesion and resulting phenotypes. TP53 loss elicited progressive aneuploidy, including copy number alterations and complex structural variants that are common in gastric cancers and which follow preferred temporal orders. Longitudinal single cell sequencing of TP53 deficient gastric organoids similarly indicates progression towards malignant transcriptional programs. Moreover, lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programs repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a striking degree of phenotypic convergence in pre-malignant epithelial organoids, implying that the earliest stages of tumorigenesis may be predictable while illuminating evolutionary constraints and barriers to malignant transformation.

Graphical abstract

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org.highwire.dtl.DTLVardef@122debborg.highwire.dtl.DTLVardef@4cd2daorg.highwire.dtl.DTLVardef@1a720d6org.highwire.dtl.DTLVardef@15ccaa7_HPS_FORMAT_FIGEXP  M_FIG C_FIG 2,3-cAMP treatment mimics stress molecular response in Arabidopsis thaliana The role of the RNA degradation product 2,3-cyclic adenosine monophosphate (2,3-cAMP) is poorly understood. Recent studies have identified 2,3-cAMP in plant material and determined its role in stress signaling. The level of 2,3-cAMP increases upon wounding, dark, and heat, and 2,3-cAMP by binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insight into 2,3-cAMP function, we used a multi-omics approach combining transcriptomics, metabolomics, and proteomics to dissect Arabidopsis response to 2,3-cAMP treatment. We demonstrated that 2,3-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway from human cells might also exist in plants. Transcriptomic analysis revealed only minor overlap between 2,3-cAMP-and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2,3-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses including protein and RNA degradation products, glucosinolates, chaperones and SG components. Finally, we demonstrated that 2,3-cAMP treatment influences the movement of processing bodies, supporting the role of 2,3-cAMP in the formation and motility of membraneless organelles. High-plex Multiomic Analysis in FFPE at Subcellular Level by Spatial Molecular Imaging The Spatial Molecular Imaging platform (CosMxTM SMI, NanoString, Seattle, WA) utilizes a high-plex in-situ imaging chemistry for both RNA and protein detection. This automated instrument provides 1000s of plex, at high sensitivity (1 to 2 copies/cell), very low error rate (0.0093 false calls/cell), and background ([~]0.04 counts/cell). The imaging system generates three-dimensional super-resolution localization of analytes (< 50 nm in the X-Y plane), at [~] 1 million cells per sample, four samples per run. Cell segmentation is antibody-based, compatible with FFPE samples. Multiomic data (980 RNAs, 108 proteins) were measured at subcellular resolution using FFPE tissues (non-small cell lung (NSCLC) and breast cancer) and allowed identification of over 18 distinct cell types, 10 unique tumor microenvironments, and 100 pairwise ligand-receptor interactions. Over 800,000 single cells and [~]260 million transcripts data are released into the public domain allowing extended data analysis by the entire spatial biology research community. Divergent Acyl Carrier Protein Decouples Mitochondrial Fe-S Cluster Biogenesis from Fatty Acid Synthesis in Malaria Parasites Plasmodium falciparum malaria parasites are early-diverging eukaryotes with many unusual metabolic adaptations. Understanding these adaptations will give insight into parasite evolution and unveil new parasite-specific drug targets. Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis. In yeast and humans, mACP also functions to biochemically couple FASII activity to electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast to most eukaryotes, the Plasmodium mitochondrion lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC followed by broader cellular defects. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of both Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other Apicomplexan pathogens, including Toxoplasma and Babesia. This discovery also highlights the ancient, fundamental role of ACP in mitochondrial Fe-S cluster biogenesis and unveils an evolutionary driving force to retain this interaction with ACP independent of its eponymous function in FASII.

Significance StatementPlasmodium malaria parasites are single-celled eukaryotes that evolved unusual metabolic adaptations. Parasites require a mitochondrion for blood-stage viability, but essential functions beyond the electron transport chain are sparsely understood. Unlike yeast and human cells, the Plasmodium mitochondrion lacks fatty acid synthesis enzymes but retains a divergent acyl carrier protein (mACP) incapable of tethering acyl groups. Nevertheless, mACP is essential for parasite viability by binding and stabilizing the core mitochondrial Fe-S cluster biogenesis complex via a divergent molecular interface lacking an acyl-pantetheine group that contrasts with other eukaryotes. This discovery unveils an essential metabolic adaptation in Plasmodium and other human parasites that decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis and evolved at or near the emergence of Apicomplexan parasitism. RNA-guided Cas12a and Cas9 knockout and knockin of the omega-1 locus of Schistosoma mansoni We compared the efficiency of gene knockout (KO) and precision of insertion (knock-in, KI) of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with that of SpCas9 from Streptococcus pyogenes, aiming to enhance the functional genomics toolkit for Schistosoma mansoni. Programmed DNA cleavages catalyzed by Cas12a and Cas9 result in staggered and blunt ended strand breaks, respectively. TTTV, the optimal protospacer adjacent motif for AsCas12a would occur frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key enzyme termed omega-1 that is secreted by the schistosome egg. AsCas12a was more efficient than SpCas9 for gene knockout of omega-1 as determined by tracking of indels by decomposition (P < 0.001). Resulting from CRISPREsso2 analysis, most mutations were deletions; SpCas9 induced short deletions of 3 nt in length whereas AsCas12a induced deletions of 2 to 26 nt. Knockout efficiency of both nucleases markedly increased in the presence of short, single stranded oligodeoxynucleotide (ssODN) donor templates. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands of the targeted gene were tested, resulting in KO efficiencies of 15.67, 28.71 and 21.43% in the SpCas9 plus donor ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans cleavage activity against the ssODNs by activated AsCas12a was not apparent in vitro. Programmed SpCas9 editing led to more precise transgene insertion than AsCas12a, with KI efficiencies of 17.07% for the KI_SpCas9 group, 14.58% for KI_AsCas12a-NT-ssODN and 12.37% for KI_AsCas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases. These findings revealed that AsCas12a and SpCas9 both provide tractable routes for RNA-guided programmed mutation of the genome of the schistosome egg. Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption Human skin functions as a physical barrier, preventing the entry of foreign pathogens while also accommodating a myriad of commensal microorganisms. A key contributor to the skin landscape is the sebaceous gland. Mice devoid of sebocytes are prone to skin infection, yet our understanding of how sebocytes function in host defense is incomplete. Here we show that the small proline-rich proteins, SPRR1 and SPRR2 are bactericidal in skin. SPRR1B and SPPR2A were induced in human sebocytes by exposure to the bacterial cell wall component lipopolysaccharide (LPS). Further, LPS injected into mouse skin triggered the expression of the mouse SPRR orthologous genes, Sprr1a and Sprr2a, through stimulation of MYD88. Both mouse and human SPRR proteins displayed potent bactericidal activity against MRSA (methicillin-resistant Staphylococcus aureus), Pseudomonas aeruginosa and skin commensals. Thus, Sprr1a-/-;Sprr2a-/- mice are more susceptible to MRSA and Pseudomonas aeruginosa skin infection. Lastly, mechanistic studies demonstrate that SPRR proteins exert their bactericidal activity through binding and disruption of the bacterial membrane. Taken together, these findings provide insight into the regulation and antimicrobial function of SPRR proteins in skin and how the skin defends the host against systemic infection. If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context. LPS-TLR4 pathway exaggerates alcohol-induced acute-on-chronic liver failure via provoking NETs formation Backgrounds: Intrahepatic infiltration of neutrophils is a character of alcoholic acute-on-chronic liver failure (AACLF) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. Intestinal bacteria and the gut-liver axis have been thought to play a key role in many liver diseases also including AACLF. However, whether NETs appear in AACLF and play a role in AACLF is still unsure. Methods: WT, NE KO, and TLR4 KO mice were used to build the AACLF model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AACLF related markers were detected. Results: The serum MPO-DNA and LPS concentration was increased in AACLF patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AACLF mice by testing MPO-DNA, Cit H3, and NE. These markers decreased with gut detergent and restored markers with gut detergent plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AACLF related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with combined treatment with antibiotics plus LPS. But the liver injury, intrahepatic fat, fibro deposition, and liver inflammation-related markers did show a significant difference in TLR4 KO mice when they received the same treatment. Conclusion: Intestinal-derived LPS promotes NETs formation in AACLF through the TLR4 pathway and further accelerates the AACLF process by NETs. The Digital 3D-Atlas MAKER (DAMAKER): a dynamic and expandable digital 3D-tool for monitoring the temporal changes in tissue growth during hindbrain morphogenesis Reconstruction of prototypic three-dimensional (3D) atlases at the scale of whole tissues or organs requires specific methods to be developed. We have established a digital 3D-atlas maker (DAMAKER) and built a digital 3D-temporal atlas to monitor the changes in the growth of the tissue and neuronal differentiation domain in the zebrafish hindbrain. DAMAKER integrates spatial and temporal data from cell populations, neuronal differentiation and brain morphogenesis, through in vivo imaging techniques paired with image analyses and segmentation tools. First, we generated a 3D-reference from several imaged hindbrains and segmented them using a trainable tool; these were aligned using rigid registration, revealing distribution of neuronal differentiation growth patterns along the axes. Second, we quantified the dynamic growth of the neuronal differentiation domain vs. the progenitor domain, and by in vivo neuronal birthdating experiments we generated a digital 3D-temporal map of the neuronal growth in the whole hindbrain, revealing the spatiotemporal dynamics of neuronal differentiation upon morphogenesis. Last, we applied it to glutamatergic and GABAergic neurons, as proof-of-concept that the digital 3D-temporal map could be used as a proxy to infer neuronal birthdate. As this protocol uses open-access tools and algorithms, it can be shared for standardized, accessible, tissue-wide cell population atlas construction. 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. Resolving when (and where) the Thylacine went extinct Like the Dodo and Passenger Pigeon before it, the predatory marsupial Thylacine (Thylacinus cynocephalus), or  Tasmanian tiger, has become an iconic symbol of human-caused extinction. The last captive animal died in 1936, but even today reports of the Thylacines possible ongoing survival in remote regions of Tasmania are newsworthy and capture the publics imagination. Extirpated from mainland Australia in the mid-Holocene, the large island of Tasmania became the species final stronghold. Following European settlement in the 1800s, the Thylacine was heavily persecuted and pushed to the margins of its range, although many sightings were reported thereafter--even well beyond the 1930s. To gain a new depth of insight into the extinction of the Thylacine, we assembled an exhaustive database of 1,237 observational records from Tasmania (from 1910 onwards), quantified their uncertainty, and charted the patterns these revealed. We also developed a new method to visualize the species 20th-century spatio-temporal dynamics, to map potential post-bounty refugia and pinpoint the most-likely location of the final persisting subpopulation. A direct reading of the high-quality records (confirmed kills and captures, in combination with sightings by past Thylacine hunters and trappers, wildlife professionals and experienced bushmen) implies a most-likely extinction date within four decades following the last capture (i.e., 1940s to 1970s). However, uncertainty modelling of the entire sighting record, where each observation is assigned a probability and the whole dataset is then subject to a sensitivity analysis, suggests that extinction might have been as recent as the late 1980s to early 2000s, with a small chance of persistence in the remote south-western wilderness areas. Beyond the intrinsically fascinating problem of reconstructing the final fate of the Thylacine, the new spatio-temporal mapping of extirpation developed herein would also be useful for conservation prioritization and search efforts for other rare taxa of uncertain status. Multilevel multinomial models as a tool to extract population spatial networks from capture-recapture data O_LIEstimating the impacts of anthropogenic disturbances requires an understanding of the habitat use patterns of individuals within a population. This is especially the case when disturbances are localized within a populations spatial range, as variation in habitat-use within a population can drastically alter the distribution of impacts.
C_LIO_LIHere, we illustrate the potential for multilevel multinomial models to generate spatial networks from capture-recapture data, a common data source use in wildlife studies to monitor population dynamics and habitat use. These spatial networks capture which regions of a populations spatial distribution share similar/dissimilar individual usage patterns, and can be especially useful for detecting structured habitat use within the populations spatial range.
C_LIO_LIUsing simulations and 18 years of capture-recapture data from St. Lawrence Estuary (SLE) beluga, we show that this approach can successfully estimate the magnitude of similarities/dissimilarities in individual usage patterns across sectors, and identify sectors that share similar individual usage patterns that differ from other sectors, i.e., structured habitat use. In the case of SLE beluga, this method identified multiple clusters of individuals, each preferentially using restricted areas within their summer range of the SLE.
C_LIO_LISynthesis and applications. Multilevel multinomial models can be effective at estimating spatial structure in habitat use within wildlife populations sampled by capture-recapture of individuals. Our finding of a structured habitat use within the SLE beluga summer range has direct implications for estimating individual exposures to localized stressors, such as underwater noise from shipping or other activities.
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