SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists The imaging of plant seedlings, fungal mycelia and bacterial colonies grown on Petri plates is common in phenotyping assays, and is typically done manually despite the procedures being time-consuming and laborious. The main reason for this is the still limited availability of existing automated phenotyping tools and facilities. Additionally, constructing a custom-made automated solution is a daunting task for most research groups specializing in biology.

Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment. SPIRO was designed for biologists by biologists; thus, its assembly does not require experience in engineering or programming and its operation is sufficiently intuitive to be carried out without training. SPIRO has a small footprint optimal for fitting into standard incubators for plants and microbes, and is equipped with an LED light source for imaging in the dark, thus allowing acquisition of photos under optimal growth conditions. SPIROs web-based user interface allows setting up experiments and downloading data remotely, without interfering with samples growth. SPIROs excellent image quality is optimal for automated image processing, which we demonstrate with two semi-automated assays for analysis of commonly used phenotypic traits: seed germination and root growth.

Moreover, the robot can be easily customized for a specific use, as all information about SPIRO, including the models for 3D-printed structural components, control software, and scripts for image analysis, are released under permissive open-source licenses.
High-pressure sprayed siRNAs trigger influence the efficiency but not the profile of transitive silencing In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED-RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex (RISC) reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the GFP-expressing Nicotiana benthamiana 16C line, high pressure spraying protocol (HPSP), and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3 of the GFP transgene was less efficient in inducing silencing when compared to the siRNAs targeting the 5 and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3 end of the target RNA.

SIGNIFICANCE STATEMENTThis work answers a long-standing question about the role of siRNA triggers in initiating transitive silencing. By using high-pressure spraying-mediated delivery of synthetic siRNAs, we provided experimental evidence that target position of 22-nt-long primary siRNAs influences the efficiency of RDR6-driven double stranded RNA (dsRNA) synthesis, but it does not change the profile of accumulating secondary siRNAs originating from processing of RDR6-produced dsRNA.
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. In vivo profiling of site-specific human cancer cell states in zebrafish Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a significant challenge. We present a quantitative high-resolution imaging assay of cancer cell morphology in zebrafish xenografts to probe functional adaptation to variable cell extrinsic cues and molecular interventions. We focus on Ewing Sarcoma, a pediatric cancer driven by a single oncogenic fusion protein EWSR1-FLI1, and with little to no additional somatic mutations, making it a prototypical form of cancer whose adaptation to microenvironments is likely driven by acute, non-genomic mechanisms. Using computer vision analysis of 3D cell shapes, we find systematic shifts in the distribution of cell morphotypes between distinct sites in the fish embryo. We also find site-specific morphological responses to differential expression of EWSR1-FLI1. Combining these data we propose a model where Ewing Sarcoma cancer cell plasticity is sensitive both to expression fluctuation of EWSR1-FLI1 and signals from the surrounding tissue microenvironment, with either or both factors possibly contributing to the oncogenic potential of these cells. Behavioural analysis of swarming mosquitoes reveals higher hearing sensitivity than previously measured with electrophysiology methods Mosquitoes of many species mate in station-keeping swarms. Mating chases ensue as soon as a male detects the flight tones of a female with his auditory organs. Previous studies of hearing thresholds have mainly used electrophysiological methods that prevent the mosquito from flying naturally. The main aim of this study was to quantify behaviourally the sound-level threshold at which males can hear females. Free-flying male Anopheles coluzzii were released in a large arena (~2 m high x 2 m x 1 m) with a conspicuous object on the ground that stimulates swarming behaviour. Males were exposed to a range of natural and synthetic played-back sounds of female flight. We monitored the responses of males and their distance to the speaker by recording changes in their wingbeat frequency and angular speed. We show that the mean male behavioural threshold of particle-velocity hearing lies between 13-20 dB SVL (95%-CI). A conservative estimate of 20 dB SVL (i.e., < 0.5 m/s particle velocity) is already 12 to 26 dB lower than most of the published electrophysiological measurements from the Johnstons organ. In addition, we suggest that 1) the first harmonic of female flight-sound is sufficient for males to detect her presence, 2) males respond with a greater amplitude to single-female sounds than to the sound of a group of females and 3) the response of males to the playback of the flight sound of a live female is the same as that of a recorded sound of constant frequency and amplitude. Predictive uncertainty in mechanistic models of cellular processes calibrated to experimental data Mathematical models are often used to explore network-driven cellular processes from a systems perspective. However, a dearth of quantitative data suitable for model calibration leads to models with parameter unidentifiability and questionable predictive power. Here we introduce a Bayesian and Machine-Learning based Measurement Model approach to explore how quantitative and non-quantitative data constrain models of apoptosis execution within a missing data context. We find two orders of magnitude more ordinal (e.g. immunoblot) data are necessary to achieve accuracy comparable to quantitative (e.g. fluorescence) data. Notably, ordinal and nominal (e.g. immunostain) non-quantitative data synergize to reduce model uncertainty and improve accuracy. Further, model prediction accuracy and certainty strongly depend on rigorous data-driven formulations of the measurement, and the size and make-up of the datasets. Finally, we demonstrate the potential of a data-driven Measurement Model approach to identify model features that could lead to informative experimental measurements and improve model predictive power. Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants. ALLOPURINOL BLOCKS THE AORTIC ANEURYSM IN A MOUSE MODEL OF MARFAN SYNDROME VIA REDUCING AORTIC OXIDATIVE STRESS BackgroundThe pathogenesis and progression of aortic aneurysm in Marfan syndrome (MFS) involves dysregulated TGF-{beta} and nitric oxide signaling, altered hemodynamics, and biomechanical forces. Increasing evidence indicates that redox stress participates in MFS aortopathy, though its contribution is not well established. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mice aortic samples. Here we address the contribution of xanthine oxidoreductase (XOR), which is transcribed as dehydrogenase (XDH) and then post-translationally modified to oxidase (XO), catabolizing mainly purines into uric acid and ROS.

Methods and ResultsWe report that in MFS patients, XOR protein levels increased in aortic samples. In MFS mice (Fbn1C1041G/+), XOR mRNA transcripts augmented in the aorta of young but not older MFS mice. Moreover, the enzymatic activity of XO form increased with respect to XDH. The administration of the XOR inhibitor allopurinol (ALO) blocked the progression of aortic root aneurysm in MFS mice. ALO was also protective when administrated before the appearance of aneurysm. MFS-induced elastic fiber fragmentation, fibrotic remodeling, nuclear translocation of pNRF2, and increased 3-nitrotyrosine levels in the aortic tunica media as well as endothelial dysfunction were all prevented by allopurinol. The large overproduction of H2O2 in MFS aorta did not occur in allopurinol-treated mice whose uric acid plasma levels remained unaltered.

ConclusionsThis study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and supports a clinical trial for ALO in the pharmacological treatment of MFS aortopathy.

GRAPHICAL ABSTRACT

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org.highwire.dtl.DTLVardef@eab02aorg.highwire.dtl.DTLVardef@1d3b39eorg.highwire.dtl.DTLVardef@1b54d0forg.highwire.dtl.DTLVardef@1d61ad0_HPS_FORMAT_FIGEXP  M_FIG C_FIG Quantifying the potential for red blood cell β-adrenergic sodium-proton exchangers to protect oxygen transport in hypoxic and hypercapnic white seabass White seabass (Atractoscion nobilis) are increasingly experiencing periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically-stimulated sodium-proton-exchangers ({beta}-NHE). We hypothesized that RBC {beta}-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the response of RBCs to adrenergic stimulation, and quantified the protective effect of {beta}-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (PO2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). The presence of RBC {beta}-NHEs was confirmed by functional, molecular and bioinformatic data and super-resolution imaging revealed, for the first time, the subcellular location of {beta}-NHE protein in vesicle-like structures and on the RBC membrane, and its translocation after adrenergic stimulation. The activation of RBC {beta}-NHEs increased Hb-O2 saturation by [~]8% in normoxia at 1 kPa PCO2, and by up to 20% in hypoxia. Our results confirm that RBC {beta}-NHE activity in white seabass has great potential to protect arterial O2 transport in environmentally relevant conditions of hypoxia and hypercapnia, but also reveal a potential vulnerability of fish to combinations of these stressors. Deep Heterogeneous Dilation of LSTM for Transient-phase Gesture Prediction through High-density Electromyography: Towards Application in Neurorobotics Deep networks have been recently proposed to estimate motor intention using conventional bipolar surface electromyography (sEMG) signals for myoelectric control of neurorobots. In this regard, Deepnets are generally challenged by long training times (affecting practicality and calibration), complex model architectures (affecting the predictability of the outcomes), and a large number of trainable parameters (increasing the need for big data). Capitalizing on our recent work on homogeneous temporal dilation in a Recurrent Neural Network (RNN) model, this paper proposes, for the first time, heterogeneous temporal dilation in an LSTM model and applies that to high-density surface electromyography (HD-sEMG), allowing for the decoding of dynamic temporal dependencies with tunable temporal foci. In this paper, a 128-channel HD-sEMG signal space is considered due to the potential for enhancing the spatiotemporal resolution of human-robot interfaces. Accordingly, this paper addresses a challenging motor intention decoding problem of neurorobots, namely, transient intention identification. Our approach uses only the dynamic and transient phase of gesture movements when the signals are not stabilized or plateaued, which can significantly enhance the temporal resolution of human-robot interfaces. This would eventually enhance seamless real-time implementations. Additionally, this paper introduces the concept of "dilation foci" to modulate the modeling of temporal variation in transient phases. In this work a high number (e.g., 65) of gestures is included, which adds to the complexity and significance of the understudied problem. Our results show state-of-the-art performance for gesture prediction in terms of accuracy, training time, and model convergence. Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen While mutation is often deleterious, it can also be adaptive. Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study we investigate two factors that are predicted to influence the trade-off between the costs and benefits of mutation: ecology and genome size. To investigate the relationship between these factors and mutation rate we conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact ecology can have on the adaptive potential of bacterial pathogens. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Development of a Novel PROTAC using the Nucleic Acid Aptamer as a Targeting Ligand for Tumor Selective Degradation of Nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell Endothelial and skeletal muscle lineages arise from common embryonic progenitors. Despite their shared developmental origin, adult endothelial cells (ECs) and muscle stem cells (MuSCs) (satellite cells) have been thought to possess distinct gene signatures and signaling pathways. Here we shift this paradigm by uncovering how adult MuSC behavior is affected by the expression of a subset of EC transcripts. We used several computational analyses including single-cell RNAseq to show that MuSCs express low levels of canonical EC markers. We demonstrate that MuSC survival is regulated by one such prototypic endothelial signaling pathway (VEGFA-FLT1). Using pharmacological and genetic gain- and loss-of-function studies, we identify the FLT1-AKT1 axis as the key effector underlying VEGFA-mediated regulation of MuSC survival. All together, our data support that the VEGFA-FLT1-AKT1 pathway promotes MuSC survival during muscle regeneration, and highlights how the minor expression of select transcripts is sufficient for affecting cell behavior. On co-activation pattern analysis and non-stationarity of resting brain activity The non-stationarity of resting-state brain activity has received increasing attention in recent years. Functional connectivity (FC) analysis with short sliding windows and coactivation pattern (CAP) analysis are two widely used methods for assessing the dynamic characteristics of brain activity observed with functional magnetic resonance imaging (fMRI). However, the statistical nature of the dynamics captured by these techniques needs to be verified. In this study, we found that the results of CAP analysis were similar for real fMRI data and simulated stationary data with matching covariance structures and spectral contents. We also found that, for both the real and simulated data, CAPs were clustered into spatially heterogeneous modules. Moreover, for each of the modules in the real data, a spatially similar module was found in the simulated data. The present results suggest that care needs to be taken when interpreting observations drawn from CAP analysis as it does not necessarily reflect non-stationarity or a mixture of states in resting brain activity.

HighlightsO_LICAP was conducted for real fMRI data and a stationary null model.
C_LIO_LIThe results of CAP analysis were similar for the real and simulated data.
C_LIO_LISimilar results for real and simulated data in both ROI- and voxel-based analyses.
C_LIO_LIResults of CAP analysis may not reflect non-stationarity of resting brain activity.
C_LI Ribosome actuator via the SpoT/ppGpp pathway to mitigate gene overexpression burden Gene overexpression causes a burden on ribosomes, which decreases the expression of other genes and growth rate. Here, we engineer a genetic ribosome actuator that increases gene expression rate and growth rate by over 150% and 80%, respectively. The actuator expresses the hydrolysis domain of the SpoT enzyme (SpoTH) in a bacterial strain with high basal levels of ppGpp, thus increasing ribosome availability. We use this actuator in a feedforward control arrangement, wherein SpoTH is co-expressed with a gene of interest (GOI). Without SpoTH co-expression, activation of the GOI decreases the expression of a constitutive gene and growth rate by over 80% and 40%, respectively. By contrast, with SpoTH co-expression, the ribosome binding site (RBS) of SpoTH can be tuned to achieve inappreciable change of either the expression of the constitutive gene or growth rate when the GOI is activated to the same level. We finally employ the actuator to keep growth rate constant while expressing dCas9 to a level that would otherwise decrease growth rate by more than 40% due to toxicity. This actuator will be relevant in applications where high cellular protein concentrations are desired without growth defects or global reduction of gene expression. Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA BackgroundNon-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear.

MethodsUsing network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPAR agonist, fenofibrate, by MIR20B and the synergic effect of combination of fenofibrate with anti-MIR20B.

ResultsWe revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)- treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In MIR20B-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of MIR20B significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-MIR20B exhibited the synergic effect on improvement of NAFLD in MCD-fed mice.

ConclusionsTaken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD.

FundingThis research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940), and the Future-leading Project Research Fund (1.210034.01) of UNIST. Highly variable response of hard coral taxa to successive coral bleaching events (2019-2020) and rising ocean temperatures in Northeast Peninsular Malaysia. Due to current greenhouse gas emissions, Malaysian coral reefs are predicted to experience severe annual coral bleaching events by 2043, threatening the survival of coral reefs within this century. However, there is no field data on how Malaysian coral reefs respond to successive events of coral bleaching. Despite the notion that many scleractinian taxa exhibit increased thermal tolerance over the last decade, it remains unresolved whether these changes are a result of  weeding out thermally susceptible species and actually ameliorate accelerating warming rates and increasing frequencies of heat disturbances. Moreover, complex interaction of environmental and biological factors that underlie differences in the bleaching response necessitate conducting studies at the within-reef scale (i.e., leeward shallow, windward shallow). Here, we studied two successive thermal stress events starting during the 2019 El Nino Southern Oscillation (ENSO), and determined bleaching trajectories of 29 hard coral taxa across fine spatio-temporal gradients in Northeast Peninsular Malaysia. Analysis of climate trajectories affirms accelerating warming rates (0.17{degrees}C per decade) and higher return-frequency of heat disturbance. Despite high annual maximum temperatures above the putative bleaching threshold (31.07{degrees}C and 31.74{degrees}C, respectively), accumulated thermal stress was low during both bleaching episodes (Degree Heating Weeks of 1.05{degrees}C-weeks and 0.18{degrees}C-weeks, respectively), suggesting widespread thermal sensitivity of hard coral taxa (55.21% and 26.63% bleaching incidence in 2019 and 2020, respectively). However, significant discrepancies between satellite and in-situ temperature data were found (0.63{degrees}C; SD{+/-}0.26). Bleaching severity was highly taxon-specific, varied across and within reef scales due to wind exposure and depth (e.g., less bleaching at shallow windward sites), and partially contrasted historical bleaching observations (e.g., Acropora and Montipora were less susceptible, Cyphastrea, Echinopora, Goniastrea, Heliopora and Porites were highly susceptible). While bleaching severity was higher in 2019, Galaxea and Leptastrea were bleaching more in 2020 despite lower heat stress, suggesting negative legacy effects of the 2019 bleaching event on these taxa. In conclusion, hard corals were subjected to more frequent heat stress during the last decade and remain highly vulnerable to marine heatwaves across all biophysical reef scales. Annual coral bleaching impacted all hard coral taxa and reduced thermal tolerance in at least two taxa. The biochemical resolving power of fluorescence lifetime imaging: untangling the roles of the instrument response function and photon-statistics A deeper understanding of spatial resolution has led to innovations in microscopy and the disruption of biomedical research, as with super-resolution microscopy. To foster similar advances in time-resolved and spectral imaging, we have previously introduced the concept of  biochemical resolving power in fluorescence microscopy. Here, we apply those concepts to investigate how the instrument response function (IRF), sampling conditions, and photon-statistics limit the biochemical resolution of fluorescence lifetime microscopy. Using Fisher information analysis and Monte Carlo simulations, we reveal the complex dependencies between photon-statistics and the IRF, permitting us to quantify resolution limits that have been poorly understood (e.g., the minimum resolvable decay time for a given width of the IRF and photon-statistics) or previously underappreciated (e.g., optimization of the IRF for biochemical detection). With this work, we unravel common misunderstandings on the role of the IRF and provide theoretical insights with significant practical implications on the design and use of time-resolved instrumentation. F1F0 ATP Hydrolysis is a Determinant of Metabolic Rate, a Correlate of Lifespan, and a Weakness of Cancer Dramatic rectal temperature decrease, in mice administered with a drug that specifically inhibits F1F0 ATP hydrolysis, suggests that F1F0 ATP hydrolysis is a major determinant of metabolic rate in vivo. Across twelve investigated species, less F1F0 ATP hydrolysis correlates with greater maximal lifespan. Specific drug inhibition of F1F0 ATP hydrolysis exerts potent anti-cancer activity in vitro. The "dark matter" of protein variants carries a distinct DNA signature and predicts damaging variant effects AO_SCPLOWBSTRACTC_SCPLOWSignatures of DNA motifs associated with distinct mutagenic exposures have been defined for somatic variants, but little is known about the consequences different mutational processes pose to the cell, especially how mutagens exert damage on specific proteins and their three-dimensional structures. Here we identify a DNA mutational signature which corresponds to damaging protein variants. We show that this mutational signature is under-sampled in sequencing data from tumour cohorts, constituting the "dark matter" of the mutational landscape which could only be accessed using deep mutational scanning (DMS) data. By training a set of gradient boosting classifiers, we illustrate that DMS data from only a handful ({approx} 10) of experiments can accurately predict variant impact, and that DNA mutational signatures embed information about the protein-level impact of variants. We bridge the gap between DNA sequence variations and protein-level consequences, discuss the significance of this signature in informing protein design and molecular principles of protein stability, and clarify the relationship between disease association and the true impact mutations bring to protein function. Tunable Cellular Localization and Extensive Cytoskeleton-Interplay of Reflectins Reflectin proteins are nature copolymers consist of repeated canonical domains, locating in a biophotonic system called Bragg lamellae and manipulating the dynamic structural coloration of iridocytes. Their biological functions are intriguing, but the underlying mechanism is not fully understood. In this study, reflectin A1, A2, B1 and C were found to present distinguished cyto-/nucleoplasmic localization preferences. Comparable intracellular localization can be reproduced by truncated reflectin variants, suggesting a conceivable evolutionary order among reflectin proteins. Secondly, the size-dependent access of reflectin variants into nucleus demonstrate a potential model of how reflectins get into Bragg lamellae. Thirdly, RfA1 was found to extensively interact with cytoskeleton, including its binding to actin and microtubule minus-end-directed movement. This implies cytoskeleton system plays fundamental role during the organization and transportation of reflectin proteins. Findings presented here recommend reflectins as programmable biomaterials which can be used to decipher their evolution processes, to delineate their biological mechanism, and to achieve tunable intracellular targeting as editable tags. Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra-hepatic environment Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immuno-pathological biomarkers and underlying mechanisms remain unclear. Here we present a comprehensive single cell-transcriptomic atlas together with immune-phenotyping of disease-associated cell states (DACS) isolated from intra-hepatic tissue and matched PBMCs of either CHB or FC patients. We find that the intra-hepatic environment displays specific cell identities and molecular signatures that are distinct from PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells. Overall, these findings provide novel insights into immuno-pathological cell states associated with FC that could serve as prognostic biomarkers. Host Chitinase 3-like-1 is a Universal Therapeutic Target for SARS-CoV-2 Viral Variants in COVID 19 COVID 19 is the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2; SC2) which has caused a world-wide pandemic with striking morbidity and mortality. Evaluation of SC2 strains demonstrated impressive genetic variability and many of these viral variants are now defined as variants of concern (VOC) that cause enhanced transmissibility, decreased susceptibility to antibody neutralization or therapeutics and or the ability to induce severe disease. Currently, the delta ({delta}) and omicron (o) variants are particularly problematic based on their impressive and unprecedented transmissibility and ability to cause break through infections. The delta variant also accumulates at high concentrations in host tissues and has caused waves of lethal disease. Because studies from our laboratory have demonstrated that chitinase 3-like-1 (CHI3L1) stimulates ACE2 and Spike (S) priming proteases that mediate SC2 infection, studies were undertaken to determine if interventions that target CHI3L1 are effective inhibitors of SC2 viral variant infection. Here we demonstrate that CHI3L1 augments epithelial cell infection by pseudoviruses that express the alpha, beta, gamma, delta or omicron S proteins and that the CHI3L1 inhibitors anti-CHI3L1 and kasugamycin inhibit epithelial cell infection by these VOC pseudovirus moieties. Thus, CHI3L1 is a universal, VOC-independent therapeutic target in COVID 19. An evolutionary ecological mechanism of CyanoHABs: unavoidable consequences when low nutrient requirements meet eutrophication The mechanism of cyanobacterial harmful algal blooms (CyanoHABs) is complicated and confusing. One major reason is they are studied primarily from an ecological perspective and on bloom-forming species only. This narrow angle loses a broader evolutionary and ecological context in which CyanoHABs occur and fails to provide information on relevant components to achieve a wholistic understanding.

To derive a comprehensive mechanism of CyanoHABs, we examine CyanoHABs through the overlooked evolutionary and ecological lenses: evolutionary radiation, ecological comparison with co-living algae, and recently identified genomic functional repertoire between blooming and non-blooming species. We found four key factors: (1) elaborate diverse functional repertoire and low nutrient requirement in cyanobacteria molded by early adaptive evolution, (2) cyanobacteria having lower nutrient requirements than co-living algae, (3) there is no directed evolution in biological functions toward water eutrophication in cyanobacteria, (4) the CyanoHAB-associated functional repertoire are more abundant and complete in blooming than non-blooming species. These factors lead us to postulate a preliminary mechanism of CyanoHABs as a synergistic quad: superior functional repertoire, established with long adaptive radiation under nutrient-deficient conditions and not evolved toward eutrophic conditions, enables cyanobacteria to efficiently utilize elevated nutrients under current eutrophic levels for excess growth and CyanoHABs thereof, due to their lower nutrient requirements than co-living algae. This working comprehensive mechanism without doubt needs further empirical testing, which can be undertaken with more comparative studies of multiple species using integrated systems biology approaches.