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.
Differential phase register of Hes1 oscillations with mitoses underlies cell-cycle heterogeneity in ER+ breast cancer cells. Here, we study the dynamical expression of endogenously labelled Hes1, a transcriptional repressor implicated in controlling cell proliferation, to understand how cell-cycle length heterogeneity is generated in ER+ breast cancer cells. We find that Hes1 shows oscillatory expression with approximately 25h periodicity and during each cell-cycle has a variable peak in G1, a trough around G1-S transition and a less variable second peak in G2/M. Compared to other subpopulations, the cell-cycle in CD44HighCD24Low cancer stem cells is longest and most variable. Most cells divide around the peak of the Hes1 expression wave but preceding mitoses in slow dividing CD44HighCD24Low cells appear phase-shifted, resulting in a late-onset Hes1 peak in G1. The position, duration and shape of this peak, rather than the Hes1 expression levels, are good predictors of cell-cycle length. Diminishing Hes1 oscillations by enforcing sustained expression slows down the cell-cycle, impairs proliferation, abolishes the dynamic expression of p21, and increases the percentage of CD44HighCD24Low cells. Reciprocally, blocking the cell-cycle causes an elongation of Hes1 periodicity, suggesting a bidirectional interaction of the Hes1 oscillator and the cell-cycle. We propose that Hes1 oscillations are functionally important for the efficient progression of the cell-cycle and that the position of mitosis in relation to the Hes1 wave underlies cell-cycle length heterogeneity in cancer cell subpopulations.

Significance statementTumours exhibit heterogeneities that are not due to mutations, including Cancer Stem Cells with different potencies. We show that the cancer stem cell state predisposed to dormancy in vivo has a highly variable and long cell-cycle. Using single-cell live-imaging for the transcriptional repressor Hes1 (a key molecule in cancer), we show a new type of circadian-like oscillatory expression of Hes1 in all cells in the population. The most potent cancer stem cells tend to divide around the trough of the Hes1 oscillatory wave, a feature predictive of a long cell-cycle. A novel concept proposed here is that the position of cell division with respect to the Hes1 wave is predictive of its prospective cell-cycle length and cancer cellular sub-state.
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 Face dissimilarity judgements are predicted by representational distance in morphable and image-computable models Human vision is attuned to the subtle differences between individual faces. Yet we lack a quantitative way of predicting how similar two face images look, or whether they appear to show the same person. Principal-components-based 3D morphable models are widely used to generate stimuli in face perception research. These models capture the distribution of real human faces in terms of dimensions of physical shape and texture. How well does a "face space" defined to model the distribution of faces as an isotropic Gaussian explain human face perception? We designed a behavioural task to collect dissimilarity and same/different identity judgements for 232 pairs of realistic faces. The stimuli densely sampled geometric relationships in a face space derived from principal components of 3D shape and texture (Basel Face Model, BFM). We then compared a wide range of models in their ability to predict the data, including the BFM from which faces were generated, a 2D morphable model derived from face photographs, and image-computable models of visual perception. Euclidean distance in the BFM explained both similarity and identity judgements surprisingly well. In a comparison against 14 alternative models, we found that BFM distance was competitive with representational distances in state-of-the-art image-computable deep neural networks (DNNs), including a novel DNN trained on BFM identities. Models describing the distribution of facial features across individuals are not only useful tools for stimulus generation. They also capture important information about how faces are perceived, suggesting that human face representations are tuned to the statistical distribution of faces. The LOTUS Initiative for Open Natural Products Research: Knowledge Management through Wikidata Contemporary bioinformatic and chemoinformatic capabilities hold promise to reshape knowledge management, analysis and interpretation of data in natural products research. Currently, reliance on a disparate set of non-standardized, insular, and specialized databases presents a series of challenges for data access, both within the discipline and for integration and interoperability between related fields. The fundamental elements of exchange are referenced structure-organism pairs that establish relationships between distinct molecular structures and the living organisms from which they were identified. Consolidating and sharing such information via an open platform has strong transformative potential for natural products research and beyond. This is the ultimate goal of the newly established LOTUS initiative, which has now completed the first steps toward the harmonization, curation, validation and open dissemination of 750,000+ referenced structure-organism pairs. LOTUS data is hosted on Wikidata and regularly mirrored on https://lotus.naturalproducts.net. Data sharing within the Wikidata framework broadens data access and interoperability, opening new possibilities for community curation and evolving publication models. Furthermore, embedding LOTUS data into the vast Wikidata knowledge graph will facilitate new biological and chemical insights. The LOTUS initiative represents an important advancement in the design and deployment of a comprehensive and collaborative natural products knowledge base.

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org.highwire.dtl.DTLVardef@184136borg.highwire.dtl.DTLVardef@16e0d6org.highwire.dtl.DTLVardef@30ae1org.highwire.dtl.DTLVardef@1bf4825_HPS_FORMAT_FIGEXP  M_FIG C_FIG Darwin's small and medium ground finches might have taste preferences, but not for human foods Urbanization is rapidly changing ecological niches. On the inhabited Galapagos Islands, Darwins finches consume human-introduced foods preferentially; however, it remains unclear why. Here we presented pastry with flavour profiles typical of human foods (oily, salty, sweet) to small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis) to test if latent taste preferences might drive selection of human foods. If human-food flavours were consumed more than a neutral or bitter control only at sites with human foods, then we predicted tastes were acquired after urbanization; however, if no site-differences were found then this would indicate latent taste preferences. Contrary to both predictions, we found little evidence that human-food flavours were preferred compared to control flavours at any site. Instead, finches showed a weak aversion to oily foods, but only at remote (no human foods present) sites. This was further supported by behavioural responses, with beak wiping occurring more often at remote sites after finches tasted flavours associated with human foods. Our results suggest, therefore, that while Darwins finches may have acquired a tolerance to the flavours of human food, latent taste preferences are unlikely to have played a major role in their dietary response to increased urbanization. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. 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. NUCLEAR RETENTION OF LEPTIN/UPD2 REGULATES ORGANISMAL RESILIENCE TO NUTRIENT EXTREMES Adipokines released from the adipocytes function as a systemic adipometer; they impinge on neural circuits to signal nutrient status. On starvation, adipokines must be retained to signal energy deficit; else, it significantly reduces starvation survival. But how fat cells retain adipokines is unclear. Here, we demonstrate that Atg8, a cell-intrinsic autophagy factor, regulates the starvation-induced acute retention of the Leptin Drosophila ortholog Upd2. We show that on starvation, as a direct consequence of Atg8s lipidation, Upd2 accumulates in the nucleus. We illustrate that Upd2s nuclear retention is critical to fat mobilization and increased starvation resilience. Furthermore, nuclear Upd2 promotes the expression of a secreted innate immune gene signature. This hints at an unanticipated connection between adipokine nuclear retention and increased innate immunity. In conclusion, we propose that, during starvation, Atg8s role is not just limited to autophagy but is critical for withholding adipokines in the nucleus to promote starvation resilience.

GRAPHICAL ABSTRACT

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org.highwire.dtl.DTLVardef@1826425org.highwire.dtl.DTLVardef@17cca52org.highwire.dtl.DTLVardef@1c70ac7org.highwire.dtl.DTLVardef@94a024_HPS_FORMAT_FIGEXP  M_FIG C_FIG O_LIIn fed state Upd2 requires Atg8 for nuclear exit and cytosolic localization.
C_LIO_LIAtg8s lipidation on starvation results in Upd2s nuclear accumulation.
C_LIO_LIUpd2 nuclear retention on starvation increases fat mobilization and post-starvation hunger.
C_LIO_LIOn starvation Upd2 nuclear retention increases expression of a secreted innate immune signature.
C_LI Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation and cytokine responses in COVID-19 Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19), with effective versus dysregulated responses influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the diverse genetic backgrounds of the Collaborative Cross founder strains crossed to K18-hACE2 transgenic mice that confers high susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 F1 progeny resulted in a spectrum of weight loss, survival, viral replication kinetics, histopathology, and cytokine profiles, some of which were sex-specific. Importantly, survival was associated with early type I interferon (IFN) expression and a phased proinflammatory response distinct from mice with severe disease. Thus, dynamics of inflammatory responses observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding antiviral immunity and evaluating countermeasures.

One Sentence SummaryGenetically diverse mice display a broad spectrum of clinically relevant responses to SARS-CoV-2 infection, reflecting variability in COVID-19 disease. DNA Strand Breaks and Gaps Direct Retroviral Integration Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and reverse transcribed (RT) copy DNA (cDNA). Previous studies demonstrated DNA site recognition limited intasome integration. Using single molecule Forster resonance energy transfer (smFRET), we show Prototype Foamy Virus (PFV) intasomes pause at DNA strand breaks and gaps. The break/gap discontinuities are similar to base excision repair (BER) lesion-processing intermediates, which affect retrovirus integration in vivo. Pausing targeted site-directed integration at the break/gap without inducing intasome conformational alterations. An 8-oxo-guanine lesion normally processes by BER and a G/T mismatch or a +T nucleotide insertion that induce flexibility or a bend in the DNA backbone did not promote intasome pausing or targeted integration. These results suggest that repair intermediates can modulate dynamic intasome-DNA interactions which target retroviral integration. A Multi-omics Method Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem Cell-derived Cardiomyocytes Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show immense promise for patient-specific disease modeling, cardiotoxicity screening, and regenerative therapy development. However, hPSC-CMs in culture have not recapitulated the structural or functional properties of adult CMs in vivo thus far. To gain global insight into hPSC-CM biology, we established a multi-omics method for analyzing the hPSC-CM metabolome and proteome from the same cell culture, creating multi-dimensional profiles of hPSC-CMs. Specifically, we developed a sequential extraction to capture metabolites and proteins from the same hPSC-CM monolayer cultures, and analyzed these extracts using high-resolution mass spectrometry (MS). Using this method, we annotated 205 metabolites/lipids and 4,008 proteins from 106 cells with high reproducibility. We further integrated the proteome and metabolome measurements to create network profiles of molecular phenotypes for hPSC-CMs. Out of 310 pathways identified using metabolomics and proteomics, 40 pathways were considered significantly overrepresented (FDR-corrected p [&le;] 0.05). Highly populated pathways included those involved in protein synthesis (ribosome, spliceosome), ATP generation (oxidative phosphorylation), and cardiac muscle contraction. This multi-omics method achieves deep coverage of metabolites and proteins, creating a multidimensional view of the hPSC-CM phenotype, which provides a strong technological foundation to advance the understanding of hPSC-CM biology. Nuclear Progestin Receptor Mediated Linkage of Blood Coagulation and Ovulation Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates. Atrazine induced In vivo immunotoxicity in Bivalves Atrazine is ubiquitously used broad-spectrum herbicide to control the weeds in agriculture. The present study aimed to evaluate the acute toxicity and immunotoxicity of Atrazine in two ecologically and economically important bivalves. Acute toxicity of atrazine evaluated in triplicates by taking control and six experimental groups each comprising of 30 animals and treated with a range of atrazine from 2 PPM to 12 PPM for 96 hours. Mortalities were recorded for every 24 hours until 96 hours and data analyzed by one-way ANOVA and Dunnett T-test. The results indicated a significant increase in mortalities with increase in dose and time of exposure in both species. The values of LC50 were determined as 6.10 PPM and 4.90 PPM respectively for Perna viridis and Paphia malabarica. Furthermore, the immunotoxic potential of atrazine assessed by treating mussels and clams with the five sub-lethal doses of atrazine for 14 days and quantifying the viability of hemocytes by using simple yet reliable Tryphan blue exclusion assay. The results of the present study suggest atrazine-induced immunotoxicity by decreasing the number of viable hemocytes in bivalves. Hemocytes with phagocytic function are indispensable to confer innate immunity in bivalves, decreased viability of these cells leads to compromised immunity. This study is first of its kind to implicate atrazine with the immunotoxicity in bivalves. Fast ATP-dependent Subunit Rotation in reconstituted FoF1-ATP synthase trapped in Solution FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. Single-molecule Forster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

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org.highwire.dtl.DTLVardef@1bd800org.highwire.dtl.DTLVardef@228aeorg.highwire.dtl.DTLVardef@1bce39forg.highwire.dtl.DTLVardef@3f01a_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1.C_FLOATNO C_FIG Monitoring fish communities through environmental DNA metabarcoding in the fish pass system of the second largest hydropower plant in the world The Itaipu Hydroelectric Power Plant is the second largest in the world in power generation. The artificial barrier created by its dam imposes an obstacle for fish migration. Thus, in 2002, a fish pass system, named Piracema Channel, was built to allow fish to access areas upstream of the reservoir. We tested the potential of environmental DNA metabarcoding to monitor the impact of both the dam and associated fish pass system in the Parana River fish communities and to compare it with traditional monitoring methods. Using a fragment of the 12S gene, we characterized richness and community composition based on amplicon sequence variants, operational taxonomic units, and zero-radius OTUs. We combined GenBank and in-house data for taxonomic assignment. We found that different bioinformatics approaches showed similar results. Also, we found a decrease in fish diversity from 2019 to 2020 probably due to the recent extreme drought experienced in southeastern Brazil. The highest alpha diversity was recorded in the mouth of the fish pass system, located in a protected valley with the highest environmental heterogeneity. Despite the clear indication that the reference databases need to be continuously improved, our results demonstrate the analytical efficiency of the metabarcoding to monitor fish species. Distinct mutations and lineages of SARS-CoV-2 virus in the early phase of COVID-19 pandemic and subsequent global expansion A novel coronavirus, SARS-CoV-2, has caused over 190 million cases and over 4 million deaths worldwide since it occurred in December 2019 in Wuhan, China. Here we conceptualized the temporospatial evolutionary and expansion dynamics of SARS-CoV-2 by taking a series of cross-sectional view of viral genomes from early outbreak in January 2020 in Wuhan to early phase of global ignition in early April, and finally to the subsequent global expansion by late December 2020. Based on the phylogenetic analysis of the early patients in Wuhan, Wuhan/WH04/2020 is supposed to be a more appropriate reference genome of SARS-CoV-2, instead of the first sequenced genome Wuhan-Hu-1. By scrutinizing the cases from the very early outbreak, we found a viral genotype from the Seafood Market in Wuhan featured with two concurrent mutations (i.e. M type) had become the overwhelmingly dominant genotype (95.3%) of the pandemic one year later. By analyzing 4,013 SARS-CoV-2 genomes from different continents by early April, we were able to interrogate the viral genomic composition dynamics of initial phase of global ignition over a timespan of 14-week. 11 major viral genotypes with unique geographic distributions were also identified. WE1 type, a descendant of M and predominantly witnessed in western Europe, consisted a half of all the cases (50.2%) at the time. The mutations of major genotypes at the same hierarchical level were mutually exclusive, which implying that various genotypes bearing the specific mutations were propagated during human-to-human transmission, not by accumulating hot-spot mutations during the replication of individual viral genomes. As the pandemic was unfolding, we also used the same approach to analyze 261,323 SARS-CoV-2 genomes from the world since the outbreak in Wuhan (i.e. including all the publicly available viral genomes) in order to recapitulate our findings over one-year timespan. By 25 December 2020, 95.3% of global cases were M type and 93.0% of M-type cases were WE1. In fact, at present all the four variants of concern (VOC) are the descendants of WE1 type. This study demonstrates the viral genotypes can be utilized as molecular barcodes in combination with epidemiologic data to monitor the spreading routes of the pandemic and evaluate the effectiveness of control measures. Moreover, the dynamics of viral mutational spectrum in the study may help the early identification of new strains in patients to reduce further spread of infection, guide the development of molecular diagnosis and vaccines against COVID-19, and help assess their accuracy and efficacy in real world at real time. Living with high potassium: a balance between nutrient acquisition and stress signaling during K-induced salt stress O_LIHigh potassium (K) in the growth medium is more toxic to plants than Na at similar concentrations. However, the molecular mechanisms underlying plant responses to K-induced salt stress are virtually unknown.
C_LIO_LIWe examined Arabidopsis thaliana and its extremophyte relative Schrenkiella parvula, using a comparative multi-omics approach to identify cellular processes affected by excess K and understand which deterministic regulatory pathways are active to avoid tissue damage while sustaining growth.
C_LIO_LIA. thaliana showed limited capacity to curb excess K accumulation and prevent nutrient depletion contrasting to S. parvula which could limit excess K accumulation without restricting nutrient uptake. Facilitated by a targeted transcriptomic response, promoting nitrogen uptake along with other key nutrients and uninterrupted N assimilation into primary metabolites during excess K-stress allowed S. parvula to boost its antioxidant and osmolyte pools concurrently leading to sustained growth. Antithetically, A. thaliana showed transcriptional responses indicative of a poor balance between stress signaling, increased ROS levels, and reduced photosynthesis, subsequently leading to inhibited growth.
C_LIO_LIThe ability to regulate independent nutrient uptake and a coordinated transcriptomic response to avoid non-specific stress signaling are two main deterministic steps towards building stress resilience to excess K+-induced salt stress.
C_LI 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. Alpha satellite insertion close to an ancestral centromeric region Human centromeres are mainly composed of alpha satellite DNA hierarchically organized as higher-order repeats (HORs). Alpha satellite dynamics is shown by sequence homogenization in centromeric arrays and by its transfer to other centromeric locations, for example during the maturation of new centromeres. We identified during prenatal aneuploidy diagnosis by FISH a de novo insertion of alpha satellite DNA from the centromere of chromosome 18 (D18Z1) into cytoband 15q26. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by lack of constriction and absence of CENP-A binding. The insertion was associated with a 2.8 kbp deletion and likely occurred in the paternal germline. The site was enriched in long terminal repeats (LTRs) and located ~10 Mbp from the location where a centromere was ancestrally seeded and became inactive in the common ancestor of humans and apes 20-25 million years ago. Long read mapping to the T2T-CHM13 human genome assembly revealed that the insertion derives from a specific region of chromosome 18 centromeric 12-mer HOR array in which the monomer size follows a regular pattern. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the methylation status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name  alpha satellite insertion. It also expands our knowledge on alphoid DNA dynamics and conveys the possibility that alphoid arrays can relocate near vestigial centromeric sites. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Exploring the natural origins of SARS-CoV-2 in the light of recombination The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (Sarbecoviruses) have been identified, is fuelling speculation on the natural origins of SARS-CoV-2. We have comprehensively analysed phylogenetic relations between SARS-CoV-2, and the related bat and pangolin Sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Clustering of the inferred recombination events is non-random with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift in the ancestry of bat Sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm horseshoe bats, Rhinolophus, are the likely SARS-CoV-2 progenitor reservoir species. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and co-circulation of these viruses ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years or so. We confirm that a direct proximal ancestor to SARS-CoV-2 is yet to be sampled, since the closest relative shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for more wildlife sampling to (i) pinpoint the exact origins of SARS-CoV-2s animal progenitor, and (ii) survey the extent of the diversity in the related Sarbecoviruses phylogeny that present high risk for future spillover.

HighlightsO_LIThe origin of SARS-CoV-2 can be traced to horseshoe bats, genus Rhinolophus, with ranges in both China and Southeast Asia.
C_LIO_LIThe closest known relatives of SARS-CoV-2 exhibit frequent transmission among their Rhinolophus host species.
C_LIO_LISarbecoviruses have undergone extensive recombination throughout their evolutionary history.
C_LIO_LIAccounting for the mosaic patterns of these recombinants is important when inferring relatedness to SARS-CoV-2.
C_LIO_LIBreakpoint patterns are consistent with recombination hotspots in the coronavirus genome, particularly upstream of the pike open reading frame with a coldspot in S1.
C_LI An exploratory study on the role of the stiffness of breast cancer cells in their detachment from spheroids and migration in 3D collagen matrices BackgroundTumour-cell detachment is a critical early event in the metastatic cascade. However, the role of the cells mechanical properties in detachment and migration is not well understood. This exploratory study aimed to assess how intracellular stiffness changes these processes.

MethodsMDA-MB-231 cells were embedded as 10,000-cell spheroids in 2 and 4 mg/ml collagen matrices. Intracellular stiffness was assessed with mitochondria tracking microrheology of cells that migrated distances equivalent to four and six times the cell diameter (dC) from the spheroid and compared to cells at the spheroid surface (0dC), representing medium, high and no migration, respectively.

Results and discussionThe mitochondrial mean square displacement and intracellular stiffness decreased during detachment and migration for both collagen concentrations (i.e. rigidities). The mean square displacement of 4dC and 6dC cells was similar, whereas cell stiffness was lower for 4dC than for 6dC cells. With increasing matrix rigidity, the intracellular stiffness decreased for 0dC cells and did not change for 4dC and 6dC cells. It is proposed that decreased cell stiffness drives detachment and migration and increased matrix rigidity physically hinders migration, and cells need to become softer or remodel the environment to migrate. The independence of the stiffness of migrated cells from matrix rigidity suggests that cells remodel their environment through matrix proteins cleavage to migrate.

ConclusionsThe study revealed the collective effects of enhanced migratory conditions and increased matrix rigidity on the mechanical properties of the cells. The expression of matrix metalloproteinases and transforming growth factor {beta} and the role of cell volume on detachment and migration in matrices with varying pore sizes are proposed targets for further studies on metastatic cancer cells. Proinflammatory innate cytokines and metabolomic signatures predict T cell response in active COVID-19 The underlying factors contributing to the evolution of SARS-CoV-2-specific T cell responses during COVID-19 infection remain unidentified. To address this, we characterized innate and adaptive immune responses with metabolomic profiling longitudinally at three different time points (0-3, 7-9, and 14-16 days post-COVID-19 positivity) from young mildly symptomatic active COVID-19 patients infected with the ancestral virus in mid-2020. We observed that anti-RBD IgG and viral neutralization are significantly reduced against the Delta variant compared to the ancestral strain. In contrast, compared to the ancestral strain, T cell responses remain preserved against the delta and omicron variants. We determined innate immune responses during the early stage of active infection in response to TLR 3/7/8 mediated activation in PBMCs and serum metabolomic profiling. Correlation analysis indicated PBMCs-derived proinflammatory cytokines, IL-6, IL-1{beta}, and IL-23, and the abundance of plasma metabolites, pyroglutamate, itaconate, and azelaic acid, were predictive of a robust SARS-CoV-2-specific Th1 response at a later stage (two weeks after PCR positivity). These observations may contribute to designing effective vaccines and adjuvants that promote innate immune responses and metabolites to induce long-lasting anti-SARS-CoV-2 specific T cells response.

Key MessageO_LIEarly proinflammatory innate cytokine response is critical for a robust T cell response during COVID-19.
C_LIO_LIWe report a set of plasma metabolites that significantly correlate with the innate and adaptive immune responses.
C_LI

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/483930v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1bdb656org.highwire.dtl.DTLVardef@dd23aorg.highwire.dtl.DTLVardef@164e404org.highwire.dtl.DTLVardef@b5f389_HPS_FORMAT_FIGEXP  M_FIG C_FIG Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. OrganoID: a versatile deep learning platform for tracking and analysis of single-organoid dynamics Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses, however these metrics are difficult and labor-intensive to obtain for high-throughput image datasets. Here, we present OrganoID, a robust image analysis platform that automatically recognizes, labels, and tracks single organoids, pixel-by-pixel, in brightfield and phase-contrast microscopy experiments. The platform was trained on images of pancreatic cancer organoids and validated on separate images of pancreatic, lung, colon, and adenoid cystic carcinoma organoids, which showed excellent agreement with manual measurements of organoid count (96%) and size (95%) without any parameter adjustments. Single-organoid tracking accuracy remained above 89% over a four-day time-lapse microscopy study. Automated single-organoid morphology analysis of a chemotherapy dose-response experiment identified decreased organoid circularity as an important morphological feature reflecting drug response. OrganoID enables straightforward, detailed, and accurate image analysis to accelerate the use of organoids in high-throughput, data-intensive biomedical applications.