Spatial and Texture Analysis of Root System Distribution with Earth Mover's Distance (STARSEED) PurposeRoot system architectures are complex, multidimensional, and challenging to characterize effectively for agronomic and ecological discovery.

MethodsWe propose a new method, Spatial and Texture Analysis of Root SystEm distribution with Earth movers Distance (STARSEED), for comparing root system distributions that incorporates spatial information through a novel application of the Earth Movers Distance (EMD).

ResultsWe illustrate that the approach captures the response of sesame root systems for different genotypes and soil moisture levels. STARSEED provides quantitative and visual insights into changes that occur in root architectures across experimental treatments.

ConclusionSTARSEED can be generalized to other plants and provides insight into root system architecture development and response to varying growth conditions not captured by existing root architecture metrics and models. The code and data for our experiments are publicly available: https://github.com/GatorSense/STARSEED.
Arabidopsis AtPME2 has a pH-dependent processivity and control cell wall mechanical properties Pectin methylesterases (PMEs) modify homogalacturonans (HG) chemistry and play a key role in regulating primary cell wall mechanical properties. How PME activity can fine-tune pectin structure in the growing plant has remained elusive. Here we report on the Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum suitable for detecting recombinant and native enzyme independent of species source. At neutral pH AtPME2 is preferentially active on pectins with a degree of 55-70% methylesterification and can be inhibited by PME inhibitor protein (PMEI). We show that the mode of action for AtPME2 can switch from full processivity (at pH 8), creating large blocks of unmethylated galacturonic acid, to low processivity (at pH 5) and relate these observations to the differences in electrostatic potential of the protein at acidic and alkaline pH. To assess the role of AtPME2 in development, we characterized two knock-out lines. We show that in the context of acidified apoplast, low-processive demethylesterification by AtPME2 can loosen the cell wall, with consequent increase in cell elongation and etiolated hypocotyl length. Our study brings insights into how the pH-dependent regulation by PME activity could affect pectin structure and associated cell wall mechanical properties in expansion.

One sentence summaryThe processivity of AtPME2, a pectin methylesterase that fine-tunes cell wall pectins is modulated by pH in vitro and impacts the mechanical properties of the wall, affecting development in planta.
Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible. Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. The important choice of reference environment in microevolutionary climate response predictions It is well documented that individuals of wild populations can adjust to climate change by means of phenotypic plasticity, but few reports on adaptation by means of genetically based microevolution caused by selection. Disentanglement of these separate effects requires that the reference environment (the environmental zero-point) is defined, and this should not be done arbitrarily. The problem is that an error in the reference environment may lead to large errors in predicted microevolution. Together with parameter values and initial mean trait values, the reference environment can be estimated from environmental, phenotypic and fitness data. A prediction error method for this purpose is described, with the feasibility shown by simulations. As shown in a toy example, an estimated reference environment may have large errors, especially for small populations. This may still be a better choice than use of an initial environmental value in a recorded time series, or the mean value, which is often used. Another alternative may be to use the mean value of a past and stationary stochastic environment, which the population is judged to have been fully adapted to, in the sense that the expected geometric mean fitness was at a global maximum. Exceptions are cases with constant phenotypic plasticity, where the microevolutionary changes per generation follow directly from phenotypic and environmental data, independent of the chosen reference environment. Citation needed? Wikipedia and 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. Probing Patterning in Microbial Consortia with picCASO: a Cellular Automaton for Spatial Organisation Microbial consortia exhibit spatial patterning across diverse environments. Since probing the self-organization of natural microbial communities is limited by their inherent complexity, synthetic models have emerged as attractive alternatives. In this study, we develop novel frameworks of bacterial communication and explore the emergent spatiotemporal organization of microbes. Specifically, we build quorum sensing-mediated models of microbial growth that are utilized to characterize the dynamics of communities from arbitrary initial configurations and establish the effectiveness of our communication strategies in coupling the growth rates of microbes. Our simulations indicate that the behavior of quorum sensing-coupled consortia can be most effectively modulated by the rates of secretion of AHLs. Such a mechanism of control enables the construction of desired relative populations of constituent species in spatially organized populations. Our models accurately recapitulate previous experiments that have investigated pattern formation in synthetic multi-cellular systems. Additionally, our software tool enables the easy implementation and analysis of our frameworks for a variety of initial configurations and simplifies the development of sophisticated gene circuits facilitating distributed computing. Overall, we demonstrate the potential of spatial organization as a tunable parameter in synthetic biology by introducing a communication paradigm based on the location and strength of coupling of microbial strains.

Author SummaryInteracting microorganisms that coexist in a given environment tend to have well-defined spatial arrangements. While the emergence of such organization is seen across different microbiomes in nature, it is hitherto not well understood. Decoding the inherent spatial patterning of microbes is constrained by the complexity of their natural habitats. Here, we take advantage of synthetic models of microbial communities to study the dynamics of emergent spatial organization. Our framework of bacterial communication utilizes modular synthetic devices to couple growth rates. In addition to uncovering potential principles of spatial organization, this work enables the construction of complex genetic circuits distributed across communicating strains. Additionally, we have developed a software tool, picCASO, that streamlines the investigation of microbial systems communicating through such frameworks. Large Bi-ethnic Study of Plasma Proteome Leads to Comprehensive Mapping of cis-pQTL and Models for Proteome-wide Association Studies Improved understanding of genetic regulation of proteome can facilitate the identification of causal mechanisms for complex traits. We analyzed data on 4,657 plasma proteins from 7,213 European American (EA) and 1,871 African American (AA) individuals from the ARIC study, and further replicated findings on 467 AA individuals from the AASK study. Here we identified 2,004 proteins in EA and 1,618 in AA, with majority overlapping, which showed associations with common variants in cis-regions. Availability of AA samples led to smaller credible sets and significant number of population-specific cis-pQTLs. Elastic-net produced powerful models for protein prediction in both populations. An application of proteome-wide association studies (PWAS) to serum urate and gout, implicated several proteins, including IL1RN, revealing the promise of the drug anakinra to treat acute gout flares. Our study demonstrates the value of large and diverse ancestry study for genetic mechanisms of molecular phenotypes and their relationship with complex traits. Enhancing CDK4/6 inhibitor therapy for medulloblastoma using nanoparticle delivery and scRNA-seq-guided combination with sapanisertib CDK4/6 inhibitors hold promise for brain tumor treatment, but efficacy has been limited by recurrence in both preclinical models and clinical trials. To address recurrence, we tested a nanoparticle formulation of the CDK4/6 inhibitor palbociclib (POx-palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma. We then analyzed medulloblastomas in mice receiving palbociclib treatment, and compared the efficacy of combining palbociclib with specific inhibitors suggested by our analysis. POx-Palbo showed reduced toxicity compared to conventional palbociclib, was tolerable in parenteral administration, improved CNS pharmacokinetics, and extended survival of mice with medulloblastoma. Recurrence, however, remained problematic as fractions of tumor cells proliferated during therapy. ScRNA-seq identified a gene expression pattern unique to proliferating medulloblastoma cells in POx-Palbo-treated mice, marked by up-regulation of the glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes. Reduced mTORC1 signaling, suggested by ribosomal suppression in POx-Palbo-treated tumors was confirmed by decreased 4EBP1 phosphorylation (p4EBP1). Further reducing mTORC1 activity by combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects, with increased suppression of both pRB and p4EBP1, and prolonged mouse survival compared to either agent alone. In contrast, targeting cell cycle progression by combining POx-Palbo with the SHH-pathway inhibitor vismodegib, or with the replication-targeting agents gemcitabine or etoposide, failed to enhance efficacy. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment, and identify POx-palbo plus sapanisertib as effective combinatorial therapy for SHH medulloblastoma. This combination may be appropriate for testing in patients with recurrence, who need new options. Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific pre-hydrogel digests. ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities which were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissuespecific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. ECM conditioned media had only a minor effect on upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, the extracellular matrix protein type XXII collagen had similar expression patterns as paxillin, with smaller effect sizes. Using tissue specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscletendon junctions to study the expression of MTJ specific proteins.

Impact StatementThe muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, it is overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction specific features and markers that should be included in tissue models of the muscletendon unit, while gaining a deeper understanding of the natural junction. This research aims to inform future methods to engineer a more relevant multi-tissue platform to study the muscletendon unit. Identification of a Golgi-localized altORF peptide reveals a minimal Golgi targeting motif Prior work has identified signal sequences and motifs that are necessary and sufficient to target proteins to specific subcellular regions and organelles such as the plasma membrane, nucleus, Endoplasmic Reticulum, and mitochondria. In contrast, minimal sequence motifs that are sufficient for Golgi localization remain largely elusive. In this work, we identified a 37 amino acid alternative open reading frame (altORF) within the mRNA of the centromere protein, CENP-R. This altORF peptide localizes specifically to the cytoplasmic surface of the Golgi apparatus. Through mutational analysis, we identify a minimal 10 amino acid sequence and a critical cysteine residue that are necessary and sufficient for Golgi localization. Pharmacological perturbations suggest that this peptide is modified through palmitoylation to promote its localization. Together, our work defines a minimal sequence that is sufficient for Golgi targeting and provide a valuable Golgi marker for live cell imaging. Task-independent metrics of computational hardness predict human cognitive performance The survival of human organisms depends on our ability to solve complex tasks in the face of limited cognitive resources. However, little is known about the factors that drive the complexity of those tasks. Here, building on insights from computational complexity theory, we quantify the computational hardness of cognitive tasks using a set of task-independent metrics related to the computational resource requirements of individual instances of a task. We then examine the relation between those metrics and human behavior and find that they predict both time spent on a task as well as accuracy in three canonical cognitive tasks. Our findings demonstrate that performance in cognitive tasks can be predicted based on generic metrics of their inherent computational hardness.

TeaserThe ability of humans to solve cognitive tasks is affected by generic mathematical properties of problems related to their computational complexity. Distribution and molecular evolution of the anti-CRISPR family AcrIF7 Anti-CRISPRs are proteins capable of blocking CRISPR-Cas systems and typically encoded in mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPRs function and evolution. Here we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered five sub-clusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families. Membrane-catalyzed aggregation of islet amyloid polypeptide is dominated by secondary nucleation Type II diabetes is characterized by the loss of pancreatic {beta}-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, i.e. the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent {beta}-cell death in type II diabetes. Differential metabolism of arsenicals regulates Fps1-mediated arsenite transport Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1, but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins; whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1, but also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs.

Summary (for JCB table of contents)

Jongmin Lee, David E. Levin

Lee and Levin investigate the question of how different stressors can drive divergent outputs from an active SAPK. The work describes the mechanism by which two forms of arsenic that both activate the yeast SAPK Hog1 differentially regulate the glycerol channel Fps1. Detection and functional resolution of soluble multimeric immune complexes by a comprehensive FcγR reporter cell panel Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings. Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. 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. Metabarcoding faecal samples to investigate spatiotemporal variation in the diet of the endangered Westland petrel (Procellaria westlandica) As top predators, seabirds can be indirectly impacted by climate variability and commercial fishing activities through changes in marine communities. However, high mobility and foraging behaviour enables seabirds to exploit prey distributed patchily in time and space. This capacity to adapt to environmental change can be described through the study of their diet. Traditionally, the diet of seabirds is assessed through the morphological identification of prey remains in regurgitates. This sampling method is invasive for the bird and limited in terms of taxonomic resolution. However, the recent progress in DNA-based approaches is now providing a non-invasive means to more comprehensively and accurately characterize animal diets. Here, we used a non-invasive metabarcoding approach to characterize the diet of the Westland petrel (Procellaria westlandica), which is an endangered burrowing species, endemic to the South Island of New Zealand. We collected 99 fresh faecal samples at two different seasons and in two different sub-colonies. Our aims were to describe the diet of the Westland petrel, investigate seasonal and spatial variation in the petrels diet, and assess potential impacts of the New Zealand fishery industry. We found that amphipods were the most common prey, followed by cephalopods and fish. Our results could be the result of natural foraging behaviour, but also suggest a close link between the composition of prey items and New Zealands commercial fishing activities. In particular, the high abundance of amphipods could be the result of Westland petrels feeding on discarded fisheries waste (fish guts). Our results also showed significant differences in diet between seasons (before hatching vs chick-rearing season) and between sampling sites (two sub-colonies 1.5 km apart), indicating plasticity in the foraging strategy of the Westland petrel. Due to its non-invasive nature, metabarcoding of faecal samples can be applied to large numbers of samples to help describe dietary variation in seabirds and indicate their ecological requirements. In our example, dietary DNA (dDNA) provided valuable information regarding the dietary preferences of an iconic species in New Zealands unique biodiversity. dDNA can thus inform the conservation of endangered or at-risk species that have elusive foraging behaviours. Modeling miRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses. 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. 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. Cross-dataset reproducibility of human retinotopic maps Population receptive field (pRF) models fit to fMRI data are used to non-invasively measure retinotopic maps in human visual cortex, and these maps are a fundamental component of visual neuroscience experiments. Here, we examined the reproducibility of retinotopic maps across two datasets: a newly acquired retinotopy dataset from New York University (NYU) (n=44) and a public dataset from the Human Connectome Project (HCP) (n=181). Our goal was to assess the degree to which pRF properties are similar across datasets, despite substantial differences in their experimental protocols. The two datasets simultaneously differ in their stimulus apertures, participant pool, fMRI protocol, MRI field strength, and preprocessing pipeline. We assessed the cross-dataset reproducibility of the two datasets in terms of the similarity of vertex-wise pRF estimates and in terms of large-scale polar angle asymmetries in cortical magnification. Within V1, V2, V3, and hV4, the group-median NYU and HCP vertex-wise polar angle estimates were nearly identical. Both eccentricity and pRF size estimates were also strongly correlated between the two datasets, but with a slope different from 1; the eccentricity and pRF size estimates were systematically greater in the NYU data. Next, to compare large-scale map properties, we quantified two polar angle asymmetries in V1 cortical magnification previously identified in the HCP data. The NYU dataset confirms earlier reports that more cortical surface area represents horizontal than vertical visual field meridian, and lower than upper vertical visual field meridian. Together, our findings show that the retinotopic properties of V1, V2, V3, and hV4 can be reliably measured across two datasets, despite numerous differences in their experimental design. fMRI-derived retinotopic maps are reproducible because they rely on an explicit computational model of the fMRI response. In the case of pRF mapping, the model is grounded in physiological evidence of how visual receptive fields are organized, allowing one to quantitatively characterize the BOLD signal in terms of stimulus properties (i.e., location and size). The new NYU Retinotopy Dataset will serve as a useful benchmark for testing hypotheses about the organization of visual areas and for comparison to the HCP 7T Retinotopy Dataset. Minimizing spectral overlap in multicolor flow cytometry experiments Selecting fluorochromes for polychromatic panels for flow cytometry is complex and time-consuming. Poorly designed panels can result in overlap between the emission spectra of the different fluorochromes, making their signals difficult to separate. While assessing all possible panels is simple to do programmatically, the combinatorial complexity of most real world problems renders brute force computation impractical. Here we present a novel complexity optimization algorithm for fast design of minimal spectral overlap fluorochrome combinations. To aid researchers in designing fluorochrome panels we implemented the algorithm in a web server, Spectracular, that also considers instrument laser configuration and allows users to define various constraints related to antibody-fluorochrome availability and marker co-expression. A Hierarchy of Biomolecular Proportional-Integral-Derivative Feedback Controllers for Robust Perfect Adaptation and Dynamic Performance Proportional-Integral-Derivative (PID) feedback controllers have been the most widely used controllers in industry for almost a century due to their good performance, simplicity, and ease of tuning. Motivated by their success in various engineering disciplines, PID controllers recently found their way into synthetic biology, where the design of feedback molecular control systems has been identified as an important goal. In this paper, we consider the mathematical realization of PID controllers via biomolecular interactions. We propose an array of topologies that offer a compromise between simplicity and high performance. We first demonstrate that different Proportional-Integral (PI) controllers exhibit different capabilities for enhancing the dynamics and reducing variance (cell-to-cell variability). Next, we introduce several derivative controllers that are realized based on incoherent feedforward loops acting in a feedback configuration. Alternatively, we show that differentiators can be realized by placing molecular integrators in a negative feedback loop--an arrangement that can then be augmented by PI components to yield PID feedback controllers. We demonstrate that the derivative component can be exploited for enhancing system stability, dramatically increasing the molecular control systems dynamic performance, and for reducing the noise effect on the output of interest. The PID controller features are established through various deterministic and stochastic analyses as well as numerical simulations. Finally, we provide an experimental demonstration using a recently developed hybrid setup, the cyberloop, where the controller is implemented in silico to control a biological genetic circuit in vivo. The large array of novel biomolecular PID controllers introduced here forms a basis for the design and construction of advanced high-performance biomolecular control systems that robustly regulate the dynamics of living systems. Toxicity Profile of Artesunate in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel.