Mitochondrial pyruvate supports lymphoma proliferation by fueling a non-canonical glutamine metabolism pathway The fate of pyruvate, which is modulated by the activity of the mitochondrial pyruvate carrier (MPC), is a defining metabolic feature in many cancers. Diffuse large B-cell lymphomas (DLBCLs) are a genetically and metabolically heterogeneous cancer. Although MPC expression and activity differed between DLBCL subgroups, mitochondrial pyruvate was uniformly consumed by glutamate pyruvate transaminase 2 (GPT2) to support -ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid (TCA) cycle in DLBCLs. Furthermore, we found that MPC inhibition unexpectedly leads to decreased glutaminolysis, which is contrary to previous observations in other cell types. We also discovered that MPC inhibition and depletion only decreased DLBCL proliferation in an extracellular matrix (ECM) environment and in vivo xenografts, but not in the typical DLBCL suspension environment. We also have found that the metabolic profile of DLBCL cells in ECM is markedly different from cells in suspension environment. Thus, we report that besides the canonical glutamate dehydrogenase (GDH)-mediated glutaminolysis, the non-canonical GPT2 mediated consumption and assimilation of glutamine and pyruvate in DLBCLs enables their proliferation in an extracellular environment-dependent manner.

HIGHLIGHTSO_LIGlutamine, but not glucose, is a major carbon source for the tricarboxylic acid cycle in diffuse large B-cell lymphomas.
C_LIO_LIMitochondrial pyruvate supports glutaminolysis in diffuse large B-cell lymphomas by supplying pyruvate for glutamate pyruvate transaminase 2-mediated  -ketoglutarate production.
C_LIO_LIMitochondrial pyruvate carrier inhibition leads to decreased glutaminolysis in diffuse large B-cell lymphomas.
C_LIO_LI -ketoglutarate production is important for diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment.
C_LIO_LIMitochondrial pyruvate carrier activity supports diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment and in mouse xenografts.
C_LI
Heterogeneity of circulating epithelial cells in breast cancer at single-cell resolution: identifying tumor and hybrid cells Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. Circulating tumor cells and hybrid cells are identified as CD45-negative and CD45-positive epithelial cells. However, such an approach is challenging because epithelial cells are observed in the blood of healthy individuals. In this study, we applied single-cell RNA sequencing to profile CD45-negative and CD45-positive circulating epithelial cells (CECs) in 20 breast cancer patients and one healthy donor. DNA ploidy analysis was used to identify the tumor and hybrid cells among CD45[boxh] and CD45+ CECs in patients, respectively. Functional enrichment analysis was applied to characterize aneuploid and diploid cells. Diploid cells were also annotated to generate cell-type candidates and analyzed for copy-number aberrations (CNAs) to confirm or refute their tumor origin. CD45[boxh] and CD45+ CECs were found in cancer patients (25.5 (range 0-404) and median 6.5 (0-147)) and the healthy donor (8 and 11 cells) and divided into three clusters. Two CD45[boxh] CEC clusters were predominantly aneuploid (97% and 98%), but one cluster contained more diploid (59%) than aneuploid cells. CD45+ CECs were mostly diploid: only clusters 1 and 2 had aneuploid cells (16% and 2%). Diploid CD45[boxh] and CD45+ CECs were annotated as different immune cells and surprisingly harbored many CNAs. Cancer-associated signaling pathways were found only in aneuploid cells of CD45[boxh] CEC cluster 1 and diploid cells of CD45+ CEC cluster 1. Thus, our findings suggest that CECs in breast cancer patients are a highly heterogeneous population comprising aneuploid (tumor and hybrid) and diploid (normal) cells. DNA ploidy analysis is an effective instrument for identifying tumor and hybrid cells among CD45[boxh] and CD45+ CECs, respectively.
Three membrane fusion pore families determine the pathway to pore dilation During exocytosis secretory vesicles fuse with a target membrane and release neurotransmitters, hormones or other bioactive molecules through a membrane fusion pore. The initially small pore may subsequently dilate for full contents release, as commonly observed in amperometric traces. The size, shape and evolution of the pore is critical to the course of contents release, but exact fusion pore solutions accounting for membrane tension and bending energy constraints have not been available. Here we obtained exact solutions for fusion pores between two membranes. We find three families: a narrow pore, a wide pore and an intermediate tether-like pore. For high tensions these are close to the catenoidal and tether solutions recently reported for freely hinged membrane boundaries. We suggest membrane fusion initially generates a stable narrow pore, and the dilation pathway is a transition to the stable wide pore family. The unstable intermediate pore is the transition state that sets the energy barrier for this dilation pathway. Pore dilation is mechanosensitive, as the energy barrier is lowered by increased membrane tension. Finally, we study fusion pores in nanodiscs, powerful systems for the study of individual pores. We show that nanodiscs stabilize fusion pores by locking them into the narrow pore family.

SignificanceDuring neurotransmission, hormone release and other fundamental processes, secretory vesicles fuse their membranes with target membranes to release contents through an initially small membrane fusion pore that subsequently dilates. Dilation is assisted by proteins such as SNAREs and synaptotagmin. While macroscopic soap film shapes are well characterized, finding exact solutions for microscopic cellular membrane surfaces is made more complex by bending energy constraints. Here, computational analysis revealed three families of fusion pores between two membranes. Our work suggests membrane fusion generates a member of the narrow pore family, and pore dilation is a transition to the wide pore family. The energy barrier that SNAREs or synaptotagmin must surmount to achieve dilation is set by a third unstable intermediate pore family. Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model. Standardized genome-wide function prediction enables comparative functional genomics: a new application area for Gene Ontologies in plants BackgroundGenome-wide gene function annotations are useful for hypothesis generation and for prioritizing candidate genes potentially responsible for phenotypes of interest. We functionally annotated the genes of 18 crop plant genomes across 14 species using the GOMAP pipeline.

ResultsBy comparison to existing GO annotation datasets, GOMAP-generated datasets cover more genes, contain more GO terms, and produce datasets similar in quality (based on precision and recall metrics using existing gold standards as the basis for comparison). From there, we sought to determine whether the datasets across multiple species could be used together to carry out comparative functional genomics analyses in plants. To test the idea and as a proof of concept, we created dendrograms of functional relatedness based on terms assigned for all 18 genomes. These dendrograms were compared to well-established species-level evolutionary phylogenies to determine whether trees derived were in agreement with known evolutionary relationships, which they largely are. Where discrepancies were observed, we determined branch support based on jack-knifing then removed individual annotation sets by genome to identify the annotation sets causing unexpected relationships.

ConclusionsGOMAP-derived functional annotations used together across multiple species generally retain sufficient biological signal to recover known phylogenetic relationships based on genome-wide functional similarities, indicating that comparative functional genomics across species based on GO data hold promise for generating novel hypotheses about comparative gene function and traits. Computational modeling and quantitative cell physiology reveal central parameters for the brassinosteroid-regulated cell growth of the Arabidopsis root Brassinosteroids (BR) are key hormonal regulators of plant development. However, whereas the individual components of BR perception and signaling are well characterized experimentally, the question of how they can act and whether they are sufficient to carry out the critical function of cellular elongation remains open. Here, we combined computational modeling with quantitative cell physiology to understand the dynamics of the plasma membrane (PM)-localized BR response pathway during the initiation of cell elongation in the epidermis of the Arabidopsis root tip. The model, consisting of ordinary differential equations, comprises the BR induced hyperpolarization of the PM, the acidification of the apoplast and subsequent cell wall swelling. We demonstrated that the competence of the root epidermal cells for the BR response predominantly depends on the amount and activity of H+-ATPases in the PM. The model further predicted that an influx of cations is required to compensate for the shift of positive charges caused by the apoplastic acidification. A potassium channel was subsequently identified and experimentally characterized, fulfilling this function. Thus, we established the landscape of components and parameters capable of triggering and guiding cellular elongation through the fast response to BRs, a central process in plant development. Thermal preconditioning in a reef-building coral alleviates oxidative damage through a BI-1 mediated antioxidant response Global coral reef decline is largely driven by the breakdown of the coral-algal symbiosis during temperature stress. Corals can acclimatize to higher temperatures, but the cellular processes underlying this ability are poorly understood. We show that preconditioning-based improvements in thermal tolerance in Pocillopora acuta are accompanied by increases in host glutathione reductase (GR) activity and expression, which prevents DNA damage. A strong correlation between GR and BI-1 expression in heat-stressed preconditioned corals and the presence of an antioxidant response element (ARE) in the GR promoter suggest BI-1 could regulate GR expression through Nrf2/ARE pathway. To fortify this link, we developed an siRNA- mediated gene knockdown protocol and targeted the coral BI-1 gene. BI-1 knock-down specifically decreased GR expression and activity and increased oxidative DNA damage in heat- stressed preconditioned corals, showing that BI-1-mediated enhanced antioxidant response during acute heat stress is a key mechanism that prevents oxidative DNA damage after preconditioning.

TeaserPreconditioning improves redox homeostasis and prevents oxidative stress in a thermally stressed reef-building coral Resolving when (and where) the Thylacine went extinct Like the Dodo and Passenger Pigeon before it, the predatory marsupial Thylacine (Thylacinus cynocephalus), or  Tasmanian tiger, has become an iconic symbol of human-caused extinction. The last captive animal died in 1936, but even today reports of the Thylacines possible ongoing survival in remote regions of Tasmania are newsworthy and capture the publics imagination. Extirpated from mainland Australia in the mid-Holocene, the large island of Tasmania became the species final stronghold. Following European settlement in the 1800s, the Thylacine was heavily persecuted and pushed to the margins of its range, although many sightings were reported thereafter--even well beyond the 1930s. To gain a new depth of insight into the extinction of the Thylacine, we assembled an exhaustive database of 1,237 observational records from Tasmania (from 1910 onwards), quantified their uncertainty, and charted the patterns these revealed. We also developed a new method to visualize the species 20th-century spatio-temporal dynamics, to map potential post-bounty refugia and pinpoint the most-likely location of the final persisting subpopulation. A direct reading of the high-quality records (confirmed kills and captures, in combination with sightings by past Thylacine hunters and trappers, wildlife professionals and experienced bushmen) implies a most-likely extinction date within four decades following the last capture (i.e., 1940s to 1970s). However, uncertainty modelling of the entire sighting record, where each observation is assigned a probability and the whole dataset is then subject to a sensitivity analysis, suggests that extinction might have been as recent as the late 1980s to early 2000s, with a small chance of persistence in the remote south-western wilderness areas. Beyond the intrinsically fascinating problem of reconstructing the final fate of the Thylacine, the new spatio-temporal mapping of extirpation developed herein would also be useful for conservation prioritization and search efforts for other rare taxa of uncertain status. Antibiotic inhibition of the Plasmodium apicoplast decreases haemoglobin degradation and antagonises dihydroartemisinin action The World Health Organisation (WHO) recommends artemisinin (ART) combinations for treatment of uncomplicated Plasmodium falciparum malaria. Understanding the interaction between co-administered drugs within combination therapies is clinically important to prevent unintended consequences. The WHO guidelines recommend second line treatments that combine artesunate with tetracycline, doxycycline, or clindamycin--antibiotics that target the Plasmodium relict plastid, the apicoplast. In addition, antibiotics can be used simultaneously against other infectious diseases, leading to their inadvertent combination with ARTs. One consequence of apicoplast inhibition is a perturbation to haemoglobin uptake and trafficking--a pathway required for activation of ART derivatives. Here, we show that apicoplast-targeting antibiotics reduce the abundance of the catalyst of ART activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between ART and these antibiotics, suggesting that apicoplast inhibitors reduce ART activation. These data have potential clinical implications due to the reliance on--and widespread use of--both ARTs and these antibiotics in malaria endemic regions. Prediction of survival after partial hepatectomy using a physiologically based pharmacokinetic model of indocyanine green liver function tests The evaluation of hepatic function and functional capacity of the liver are essential tasks in hepatology as well as in hepatobiliary surgery. Indocyanine green (ICG) is a widely applied test compound that is used in clinical routine to evaluate hepatic function. Important questions for the functional evaluation with ICG in the context of hepatectomy are how liver disease such as cirrhosis alters ICG elimination, and if postoperative survival can be predicted from preoperative ICG measurements. Within this work a physiologically based pharmacokinetic (PBPK) model of ICG was developed and applied to the prediction of the effects of a liver resection under various degrees of cirrhosis. For the parametrization of the computational model and validation of model predictions a database of ICG pharmacokinetic data was established. The model was applied (i) to study the effect of liver cirrhosis and liver resection on ICG pharmacokinetics; and (ii) to evaluate the model-based prediction of postoperative ICG-R15 as a measure for postoperative outcome. Key results are the accurate prediction of changes in ICG pharmacokinetics caused by liver cirrhosis and postoperative changes of ICG-elimination after liver resection, as validated with a wide range of data sets. Based on the PBPK model, individual survival after liver resection could be classified, demonstrating its potential value as a clinical tool. High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8. Microvesicles Transfer Mitochondria and Increase Mitochondrial Function in Brain Endothelial Cells We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage- derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.

HighlightsO_LIMicrovesicles transfer mitochondria to endothelial cells and brain slice neurons
C_LIO_LIMitochondrial transfer increased ATP in ischemic brain endothelial cells (BECs)
C_LIO_LITransfer of microvesicles increased mitochondrial function in hypoxic BECs
C_LIO_LITransfer of exosomes did not affect mitochondrial function in hypoxic BECs
C_LIO_LIHomotypic BEC-derived EVs result in greater ATP levels in the recipient BECs
C_LI Communities of tropical soil fungi differ between burned and unburned forest, with corresponding changes in plant community composition, litter and soil chemistry Fire is predicted to be more severe and frequent in forests of the Australian Monsoon Tropics over the coming decades. The way in which groups of ecologically important soil fungi respond to disturbance caused by fire has not been studied in Australian tropical forest ecosystems. Ectomycorrhizal (EM) fungi are important tree symbionts and saprotrophic fungi drive soil nutrient cycles. We analysed both publicly-available environmental DNA sequence data as well as soil chemistry data to test a hypothesis that burned areas in a contiguous tropical forest have different community composition and diversity of EM and saprotrophic soil fungi relative to nearby unburned sites. We tested this hypothesis by measuring community-level taxonomic composition, fungal diversity, species richness and evenness. We determined whether changes in fungal communities were associated with fire-altered soil chemical/physical properties, vegetation types, or the direct effect of fire. Soil fungi differed in abundance and community phylogenetic structure between forest sites that had experienced fire, and sites dominated by unburned forest. EM community composition differed between unburned and burned sites, which had more mycorrhizal hosts including Corymbia intermedia, Acacia flavescens and Acacia midgleyi. Lower diversity of saprotrophic fungi was correlated with lower soil nutrient levels and different litter composition at burned sites. Pyrophilic, truffle-like EM fungi that rely on mycophagous mammals for dispersal were abundant at recently burned sites. We conclude that EM fungi show different patterns of diversity in burned tropical forest, likely driven by changing plant communities, whereas differences in saprotrophic fungal communities of burned sites may be driven by by reduced soil nutrient levels and altered litter composition.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells with Consistent Place Fields Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge) in vivo calcium imaging from CA1 hippocampus in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3{+/-}5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements on in vivo imaging and place field literature are discussed.

Significance statementIn vivo calcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the first in vivo Ca2+ recording from CA1 hippocampus in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3{+/-}5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the hippocampus is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization. Supergene formation: evidence for recombination suppression among multiple functional loci within inversions. Supergenes are genetic architectures associated with discrete and concerted variation in multiple traits. It has long been suggested that supergenes control these complex polymorphisms by suppressing recombination between set of coadapted genes. However, because recombination suppression hinders the dissociation of the individual effects of genes within supergenes, there is still little evidence that supergenes evolve by tightening linkage between coadapted genes. Here, combining an landmark-free phenotyping algorithm with multivariate genome wide association studies, we dissected the genetic basis of wing pattern variation in the butterfly Heliconius numata. We showed that the supergene controlling the striking wing-pattern polymorphism displayed by this species contains many independent loci associated with different features of wing patterns. The three chromosomal inversions of this supergene suppress recombination between these loci, supporting the hypothesis that they may have evolved because they captured beneficial combinations of alleles. Some of these loci are associated with colour variations only in morphs controlled by inversions, indicating that they were recruited after the formation of these inversions. Our study shows that supergenes and clusters of adaptive loci in general may form via the evolution of chromosomal rearrangements suppressing recombination between co-adapted loci but also via the subsequent recruitment of linked adaptive mutations. Estimation of cell cycle kinetics in higher plant root meristem links organ position with cellular fate and chromatin structure Plant root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To shape the organs structure signaling between the different cells and cell files must be highly coordinated. Thereby, diverging kinetics of chromatin remodeling and cell growth in these files need to be integrated and balanced by differential cell growth and local differences in cell proliferation frequency. Understanding the local differences in cell cycle duration in the RAM and its correlation with chromatin organization is crucial to build a holistic view on the different regulatory processes and requires a quantitative estimation of the chromatin geometry and underlying mitotic cell cycle phases timing at every cell file and every position. Unfortunately, so far precise methods for such analysis are missing.

This study presents a robust and straightforward pipeline to determine in parallel the duration of cell cycles key stages in all cell layers of a plants root and their nuclei organization. The methods combine marker-free techniques based on the detection of the nucleus, deep analysis of the chromatin phase transition, incorporation of 5-ethynyl-2'-deoxyuridine (EdU), and mitosis with a deep-resolution plant phenotyping platform to analyze all key cell cycle events kinetics.

In the Arabidopsis thaliana L. RAM S-phase duration was found to be as short as 20-30 minutes in all cell files. The subsequent G2-phase duration however depends on the cell type/position and varies from 3.5 hours in the pericycle to more than 4.5 hours in the epidermis. Overall, S+G2+M duration in Arabidopsis under our condition is 4 hours in the pericycle and up to 5.5 hours in the epidermis.

Endocycle duration was determined as the time required to achieve 100% EdU index in the transition zone and estimated to be in the range of 3-4 hours.

Besides Arabidopsis, we show that the presented technique is applicable also to root tips of other dicot and monocot plants (tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.) and wheat (Triticum aestivum L.). In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes. Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were obtained from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. High-coverage sequencing revealed that adaptation of the parasites to culture was accompanied by changes in gene copy numbers. T. brucei has so far been considered to be uniformly diploid, but we also found trisomy of chromosome 5 not only in one Lister 427 culture, but also in the MAK98 field isolate. Trisomy of chromosome 6, and increased copies of other chromosome segments, were also seen in established cultured lines. The two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. Initial results suggested that the two strains have differing infection patterns in rodents. MAK65 is uniformly diploid and grew more reproducibly in bloodstream-form culture than MAK98. 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. A pilot study on the home range and movement patterns of the Andean Fox Lycalopex culpaeus (Molina 1782) in Cotopaxi National Park, Ecuador This study reports movement patterns and home range estimates of an Andean fox (Lycalopex culpaeus) in Cotopaxi National Park in Ecuador, representing the first GPS-tagging of the species. The GPS functioned well during the 197-day tracking period. Home range sizes ranged between 4.9 - 8.1 km2, depending on the estimation method. Movement speeds averaged 0.17 km/hr at day vs. 0.87 km/hr. at night, and distance traveled averaged 0.23 km at day vs. 0.89 km at night. These preliminary results highlight the importance of collecting unbiased, high-quality data which enables an enhanced understanding on mammal behavior and human/animal interaction. An interplay between cellular growth and atypical fusion defines morphogenesis of a modular glial niche Neural stem cells (NSCs) are embedded in a multi-layered, intricate cellular microenvironment supporting their activity, the niche. Whilst shape and function are inseparable, the morphogenetic aspects of niche development are poorly understood. Here, we use the formation of the glial network of a NSC niche to investigate acquisition of architectural complexity. Cortex glia (CG) in Drosophila regulate neurogenesis and build a reticular structure around NSCs. We first show that individual CG cells grow tremendously to ensheath several NSC lineages, eventually spanning the entire tissue while partitioning the NSC population. Elaborate proliferative mechanisms convert these cells into syncytia rich in cytoplasmic bridges. Unexpectedly, CG syncytia further undergo homotypic cell-cell fusion, relying on defined molecular players of cell fusion such as cell surface receptors and actin regulators. Exchange of cellular components is however dynamic in space and time, a previously unreported unique mechanism. This atypical cell fusion remodels cellular borders, restructuring the CG syncytia. Ultimately, the coordination of growth and fusion builds the multi-level architecture of the niche, and creates a modular, spatial partition of the NSC population. Our findings provide novel insights into how a niche forms and organises while developing intimate contacts with a stem cell population. 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. Localization of signaling receptors maximizes cellular informationacquisition in spatially-structured natural environments Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization maximizes information acquisition in simulated natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold. Broadly, our work shows how cells can maximize the fidelity of information transfer by adapting the spatial organization of signaling molecules to the spatial structure of the environment. Immunogenicity of SARS-CoV-2 trimetric spike protein associated to Poly(I:C) plus Alum The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (spike protein with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route. Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins The human proteome is replete with short linear motifs (SLiMs) of 4-6 residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to systematically examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of ENAH, an actin regulator that is upregulated in invasive cancers, we screened 36-residue proteome-derived peptides for binding. We discovered a pocket on the ENAH EVH1 domain that diverged from its orthologs to recognize extended SLiMs, and we found that proteins with two EVH1-binding SLiMs can wrap around a single domain. We also found that the ciliary protein PCARE uses an extended 23-residue region to obtain higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of broader proteomic context on motif-mediated interactions, revealing diverse mechanisms of contextual control over EVH1 interactions and establishing that SLiMs cant be fully understood outside of their native context.