Contingency and chance erase necessity in the experimental evolution of ancestral proteins To understand why evolution produced the biological systems that exist today, we must know how important chance, contingency, and necessity were during history. Previous observations suggest that each of these modes of causality affects evolution in various settings, but their relative roles and interactions are not well characterized because they have never been systematically assessed in a single system or on a timescale relevant to evolutionary history. To this end, we reconstructed ancestral B-cell-lymphoma-2-family proteins and developed a continuous evolution method to select for defined protein-protein interaction specificities. By repeatedly evolving a series of ancestral proteins to acquire specificities that occurred during history, we show that contingency steadily overwhelms chance and erases necessity as the primary cause of sequence variation in proteins over long phylogenetic timescales. As a result, evolutionary trajectories launched from distant starting points are essentially unpredictable, even under strong and identical selection pressures. Genetic dissection of the outcomes shows that chance arises because numerous sets of mutations can alter specificity at any point in time, while contingency arises because historical substitutions change these sets. Patterns of variation in extant protein sequences are therefore largely the idiosyncratic product of a particular course of unpredictable historical events.
An Evolutionary Process Without Variation and Selection Natural selection successfully explains how organisms accumulate adaptive change despite that traits acquired over a lifetime are eliminated at the end of each generation. However, in some domains that exhibit cumulative, adaptive change--e.g., cultural evolution, and earliest life--acquired traits are retained; these domains do not face the problem that Darwins theory was designed to solve. Lack of transmission of acquired traits occurs when germ cells are protected from environmental change, due to a self-assembly code used in two distinct ways: (i) actively interpreted during development to generate a soma, and (ii) passively copied without interpretation during reproduction to generate germ cells. Early life and cultural evolution appear not to involve a self-assembly code used in these two ways. We suggest that cumulative, adaptive change in these domains is due to a lower-fidelity evolutionary process, and model it using Reflexively Autocatalytic and Foodset-generated networks. We refer to this more primitive evolutionary process as Self-Other Reorganisation (SOR) because it involves internal self-organising and self-maintaining processes within entities, as well as interaction between entities. SOR encompasses learning but in general operates across groups. We discuss the relationship between SOR and Lamarckism, and illustrate a special case of SOR without variation.
Spindle position dictates division site during asymmetric cell division in moss Asymmetric cell division (ACD) underlies the development of multicellular organisms. It is considered that the division site in land plants is predetermined prior to mitosis and that the localization of the mitotic spindle does not govern the division plane. This contrasts with animal ACD, in which the division site is defined by active spindle-positioning mechanisms. Here, we isolated a hypomorphic mutant of the conserved microtubule-associated protein TPX2 in the moss Physcomitrium patens and observed abnormal spindle motility during cell division. This defect compromised the position of the division site and produced inverted daughter cell sizes in the first ACD of gametophore (leafy shoot) development. The phenotype was rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we have identified an active spindle-positioning mechanism that, reminiscent of acentrosomal ACD in animals, involves microtubules and actin filaments and sets the division site in plants. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. 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. Transmembrane helices are an overlooked and evolutionarily conserved source of major histocompatibility complex class I and II epitopes Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most haplotypes of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHCI and MHC-II. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells respond more to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs therefore are less prone to escape mutations that enable pathogens to evade T cell responses. Hierarchical autoencoder-based integration improves performance in multi-omics cancer survival models through soft modality selection With decreasing costs of high-throughput sequencing, more and more datasets providing omics profiles of cancer patients become available. Thus, novel survival analysis approaches integrating these differently sized and heterogeneous molecular and clinical groups of variables start being developed. Due to the difficulty of the task of multi-omics data integration, the Cox Proportional-Hazards (PH) model using clinical data has remained one of the best-performing techniques, barely outperformed by models using molecular data modalities. There is therefore a need for methods that can successfully perform multi-omics integration in survival analysis and outperform the clinical Cox PH model. Moreover, while certain deep learning methods have been shown to provide state-of-the-art accuracy of cancer survival prediction, most of them show no benefit or even decay in performance when integrating a larger number of modalities, further motivating a need to investigate how modality-specific representations should be integrated when using neural networks for multi-omics integration. We benchmarked multiple integration techniques for a neural network architecture, revealing that hierarchical autoencoder-based integration of modality-specific representations outperformed other methods such as max-pooling and was comparable with state-of-the-art statistical approaches for multi-omics integration. Further, we showed that the hierarchical autoencoder-based integration of modality-specific representations achieved increased performance through a soft modality selection mechanism, focusing on the most informative modalities for each cancer. We thus framed multiomics integration as a partial group-wise feature selection problem, highlighting that only those models performed well that could adequately weight important modalities in the presence of the high noise imposed by less important modalities. Regulation of DNA damage response by trimeric G-protein Signaling Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DNA damage hinges on the guanine nucleotide-exchange modulator of heterotrimeric G-protein, Gi*{beta}{gamma}, GIV/Girdin. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1s BRCT-modules via both phospho-dependent and -independent mechanisms. GIV promotes NHEJ, and binds and activates Gi and enhances the  free G{beta}{gamma}[-&gt;]PI-3-kinase[-&gt;]Akt pathway, thus revealing the enigmatic origin of prosurvival Akt signals during dsDNA repair. Absence of GIV, or the loss of either of its two functions impaired DNA repair, and induced cell death when challenged with numerous cytotoxic agents. That GIV selectively binds few other BRCT-containing proteins suggests convergent signaling such that heterotrimeric G-proteins may finetune sensing, repair, and outcome after DNA damage.

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org.highwire.dtl.DTLVardef@3d5202org.highwire.dtl.DTLVardef@1d32d89org.highwire.dtl.DTLVardef@152a85dorg.highwire.dtl.DTLVardef@1ab9953_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LINon-receptor G protein modulator, GIV/Girdin binds BRCA1
C_LIO_LIBinding occurs in both canonical and non-canonical modes
C_LIO_LIGIV sequesters BRCA1 away from dsDNA breaks, suppresses HR
C_LIO_LIActivation of Gi by GIV enhances Akt signals, favors NHEJ
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IN BRIEFIn this work, the authors show that heterotrimeric G protein signaling that is triggered by non-receptor GEF, GIV/Girdin, in response to double-stranded DNA breaks is critical for decisive signaling events which favor non-homologous end-joining (NHEJ) and inhibit homologous recombination (HR). Preprints in motion: tracking changes between preprint posting and journal publication during a pandemic Amidst the COVID-19 pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press and policymakers for the first time. This phenomenon has sharpened longstanding questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through 30th April 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper. Honeycomb crystallography: comb formation under geometric frustrations 1As honeybees build their nests in pre-existing tree cavities, they must deal with dealing with the presence of geometric constraints, resulting in non-regular hexagons and topological defects in the comb. In this work, we study how bees adapt to their environment in order to regulate the comb structure. Specifically, we identify the irregularities in honeycomb structure in the presence of various geometric frustrations. We 3D-print experimental frames with a variety of constraints imposed on the imprinted foundations. The combs constructed by the bees show clear evidence of reoccurring patterns built by bees in response to specific geometric frustrations on these starter frames. Furthermore, using an experimental-modeling framework, we demonstrate that these patterns can be successfully modeled and replicated through a simulated annealing process, in which the minimized potential is a variation of the Lennard-Jones potential that only considers first-neighbor interactions according to a Delaunay triangulation. Our simulation results not only confirm the connection between honeycomb structures and other crystal systems such as graphene, but also show that irregularities in the honeycomb structure can be explained as the result of local interactions between honeybees and their immediate surroundings, leading to emergent global order. Additionally, our computational model can be used to describe specific strategies that bees use to effectively solve each geometric mismatch problem while minimizing cost of comb building. Inter-tissue convergence of gene expression during ageing suggests age-related loss of tissue and cellular identity Developmental trajectories of gene expression may reverse in their direction during ageing, a phenomenon previously linked to cellular identity loss. Our analysis of cerebral cortex, lung, liver and muscle transcriptomes of 16 mice, covering development and ageing intervals, revealed widespread but tissue-specific ageing-associated expression reversals. Cumulatively, these reversals create a unique phenomenon: mammalian tissue transcriptomes diverge from each other during postnatal development, but during ageing, they tend to converge towards similar expression levels, a process we term Divergence followed by Convergence, or DiCo. We found that DiCo was most prevalent among tissue-specific genes and associated with loss of tissue identity, which is confirmed using data from independent mouse and human datasets. Further, using publicly available single-cell transcriptome data, we showed that DiCo could be driven both by alterations in tissue cell type composition and also by cell-autonomous expression changes within particular cell types. Major transcriptomic, epigenetic and metabolic changes underly the pluripotency continuum in rabbit preimplantation embryos Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit pre-implantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic, and metabolic map of the pluripotency continuum in rabbit preimplantation embryos and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with that of humans and non-human primates.

Summary StatementRabbit preimplantation embryos share characteristics with human and monkey embryos with respect to timing of early lineage segregation and expression of marker genes for naive and primed pluripotency. 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. ConVarT: a search engine for orthologous variants and functional inference of human genetic variants The availability of genetic variants, together with phenotypic annotations from model organisms, facilitates comparing these variants with equivalent variants in humans. However, existing databases and search tools do not make it easy to scan for equivalent variants, namely "matching variants" (MatchVars) between humans and other organisms. Therefore, we developed an integrated search engine called ConVarT (http://www.convart.org/) for matching variants between humans, mice, and C. elegans. ConVarT incorporates annotations (including phenotypic and pathogenic) into variants, and these previously unexploited phenotypic MatchVars from mice and C. elegans can give clues about the functional consequence of human genetic variants. Our analysis shows that many phenotypic variants in different genes from mice and C. elegans, so far, have no counterparts in humans, and thus, can be useful resources when evaluating a relationship between a new human mutation and a disease. Ribosome actuator via the SpoT/ppGpp pathway to mitigate gene overexpression burden Gene overexpression causes a burden on ribosomes, which decreases the expression of other genes and growth rate. Here, we engineer a genetic ribosome actuator that increases gene expression rate and growth rate by over 150% and 80%, respectively. The actuator expresses the hydrolysis domain of the SpoT enzyme (SpoTH) in a bacterial strain with high basal levels of ppGpp, thus increasing ribosome availability. We use this actuator in a feedforward control arrangement, wherein SpoTH is co-expressed with a gene of interest (GOI). Without SpoTH co-expression, activation of the GOI decreases the expression of a constitutive gene and growth rate by over 80% and 40%, respectively. By contrast, with SpoTH co-expression, the ribosome binding site (RBS) of SpoTH can be tuned to achieve inappreciable change of either the expression of the constitutive gene or growth rate when the GOI is activated to the same level. We finally employ the actuator to keep growth rate constant while expressing dCas9 to a level that would otherwise decrease growth rate by more than 40% due to toxicity. This actuator will be relevant in applications where high cellular protein concentrations are desired without growth defects or global reduction of gene expression. Analysis of Drosophila cardiac hypertrophy by micro-computerized tomography for genetic dissection of heart growth mechanisms Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

NEW AND NOTEWORTHYGenetic analysis of cardiac hypertrophy in Drosophila holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. However, this potential has been hindered by a lack of rapid and effective methods for analysis of heart size in flies. Here we demonstrate that analysis of the Drosophila heart with micro-computerized tomography yields accurate and highly reproducible heart size measurements that can be used to efficiently analyze heart growth and cardiac hypertrophy in Drosophila. Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, show that there are stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.

Author summaryIn this work, we use experimental and modeling approaches to estimate the surface density of the glial glutamate transporters GLAST and GLT-1 in mouse hippocampal astrocytes. The results show that the surface density of glutamate transporters in hippocampal astrocytes is similar in 7-8 week old mice and rats. In mice, GLAST and GLT-1 reach their peak expression at ~ 6 months of age. Their expression, however, varies widely across different sub-cellular compartments. These findings indicate that the identity of the sub-cellular compartment of an astrocyte in contact with a glutamatergic synapses, in addition to the extent of its synaptic coverage, is a main factor to limit glutamate spillover and receptor activation at a distance from the release site. DetecDiv, a generalist deep-learning platform for automated cell division tracking and survival analysis Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and the real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays. Impedance scaling for gold and platinum microelectrodes ObjectiveElectrical measurement of the activity of individual neurons is a primary goal for many invasive neural electrodes. Making these "single unit" measurements requires that we fabricate electrodes small enough so that only a few neurons contribute to the signal, but not so small that the impedance of the electrode creates overwhelming noise or signal attenuation. Thus, neuroelectrode design often must strike a balance between electrode size and electrode impedance, where the impedance is often assumed to scale linearly with electrode area.

Approach and main resultsHere we study how impedance scales with neural electrode area and find that the 1 kHz impedance of Pt electrodes (but not Au electrodes) transitions from scaling with area (r-2) to scaling with perimeter (r-1) when the electrode radius falls below 10 microns. This effect can be explained by the transition from planar to spherical diffusion behavior previously reported for electrochemical microelectrodes. Significance: These results provide important intuition for designing small, single unit recording electrodes. Specifically, for materials where the impedance is dominated by a pseudo-capacitance that is associated with a diffusion limited process, the total impedance will scale with perimeter rather than area when the electrode size becomes comparable with the diffusion layer thickness. For Pt electrodes this transition occurs around 10 um radius electrodes. At even lower frequencies (1 Hz) impedance approaches a constant. This transition to r-1 scaling implies that electrodes with a pseudo-capacitance can be made smaller than one might expect before thermal noise or voltage division limits the ability to acquire high-quality single-unit recordings. 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. Augmenting Recombinant Antibody Production in HEK293E Cells: Optimising Transfection and Culture Parameters BackgroundOptimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations.

MethodsInvestigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements.

ResultsUsing 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 g respectively in a cost-effective manner. With the addition of peptone, {kappa} light chain increased by ~4-fold to 1032 g while whole antibody increased to a lesser extent by ~2.5-fold to 51 g, with benefits potentially for antibodies limited by their light chains in production.

ConclusionsOur optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells.

Statement of SignificanceRecombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~4-fold light chain alone production increase to 1032 g and a 2.5-fold whole antibody production increase to 51 g from 2 mL transfections. Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and less-functional Misfolded protein conformations with decreased functionality can bypass the proteostasis machinery and remain soluble in vivo. This is an unexpected phenomenon as several cellular quality control mechanisms have evolved to rid cells of misfolded proteins. Three questions, then, are: how is it structurally possible for long-lived, soluble, misfolded proteins to bypass the proteostasis machinery and processes? How widespread are these soluble, misfolded states across the proteome? And how long do they persist for? Here, we address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of all proteins exhibit subpopulations of misfolded conformations that are likely to bypass molecular chaperones, avoid aggregation, and not be rapidly degraded. These misfolded states may persist for months or longer for some proteins. Structurally characterizing these misfolded states, we observe they have a large amount of native structure, but also contain localized misfolded regions from non-native changes in entanglement, in which a protein segment threads through a loop formed by another portion of the protein that is not found in the native state. The surface properties of these misfolded states are native like, suggesting they may bypass the proteostasis machinery and its regulatory processes to remain soluble, while their entanglements make these states long-lived kinetic traps, as disentanglement requires unfolding of already folded portions of the protein. In terms of function, we predict that one-third of proteins have subpopulations that misfold into less-functional states that have structurally perturbed functional sites yet remain soluble. Data from limited-proteolysis mass spectrometry experiments, which interrogate the misfolded conformations populated by proteins upon unfolding and refolding, are consistent with the structural changes seen in the entangled states of glycerol-3-phosphate dehydrogenase upon misfolding. These results provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass cellular quality controls, and indicate, unexpectedly, this may be a wide-spread phenomenon in proteomes. Such entanglements are observed in many native structures, suggesting the non-native entanglements we observe are plausible. More broadly, these near-native entangled structures suggest a hypothesis for how synonymous mutations can modulate downstream protein structure and function, with these mutations partitioning nascent proteins between these kinetically trapped states. Flexible control of Pavlovian-instrumental transfer based on expected reward value The Pavlovian-instrumental transfer (PIT) paradigm is widely used to assay the motivational influence of reward-predictive cues, reflected by their ability to invigorate instrumental behavior. Leading theories of incentive learning assume that cues are assigned motivational properties based on the value of the rewards they predict. We outline an alternative account of PIT which recognizes that reward-predictive cues may suppress rather than motivate instrumental behavior under certain conditions. We posit that cues capable of triggering a strong reward expectancy will tend to inhibit instrumental behavior, which is exploratory by nature, in order to facilitate retrieval of the expected reward before it is lost or stolen. According to this view, the motivation to engage in instrumental behavior during a cue should be inversely related to the value of the predicted reward, since there is more to lose by failing to secure a high-value reward than a low-value reward. We tested this hypothesis in rats using a high-conflict PIT protocol which elicits competing instrumental and food-port entry responses. In Experiment 1, cues signaling different reward magnitudes (1, 3, or 9 pellets) differentially influenced these behaviors. Whereas the 1-pellet cue increased instrumental behavior, cues signaling 3 or 9 pellets suppressed instrumental behavior and elicited high levels of food-port activity. Experiment 2 found that reward-predictive cues suppressed instrumental behavior and increased food-port activity in a flexible manner that was disrupted by post-training reward devaluation. We discuss how the PIT task provides a useful tool for studying cognitive control over cue-motivated behavior in rodents. Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells Cells can help each other replicate by communicating with diffusible molecules. In cell cultures, molecules may diffuse within a cell colony or between adjacent or distant colonies. Determining which cell helps which cells replication is challenging. We developed a systematic approach, integrating modeling and experiments, for determining the length-scales of cell-cell communication (from microns to centimeters). With this approach, we discovered that differentiating murine ES cells, scattered across centimeters on a dish, communicate over millimeters to form one macroscopic entity that survives if and only if its centimeter-scale population-density is above a threshold value. Single-cell-level measurements, transcriptomics, and modeling revealed that this "macroscopic quorum sensing" arises from differentiating ES cells secreting and sensing survival-promoting FGF4 that diffuses over millimeters and activates YAP1-induced survival mechanisms. Through the same mechanism, a lone macroscopic, but not microscopic, colony survives differentiation. Our work rigorously establishes that in vitro ES-cell differentiation relies on macroscopic cooperation. Direct Observation of Heterogeneous Formation of Amyloid Spherulites in Real-time by Super-resolution Microscopy The misfolding of proteins and their aggregation in the form of fibrils or amyloid-like spherulites are involved in a spectrum of pathological abnormalities. Our current understanding of protein amyloid aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalisation Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of a microscopically identical aggregates, e.g. amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level. Salmonella Typhimurium outer membrane protein A (OmpA) renders protection against nitrosative stress by promoting SCV stability in murine macrophages Porins are highly conserved bacterial outer membrane proteins involved in the selective transport of charged molecules across the membrane. Despite their significant contributions to the pathogenesis of Gram-negative bacteria, their precise role in salmonellosis remains elusive. In this study, we investigated the role of porins (OmpA, OmpC, OmpD, and OmpF) in Salmonella Typhimurium (STM) pathogenesis. OmpA played a multifaceted role in STM pathogenesis, and a strain deleted for ompA (STM{Delta} ompA) showed enhanced proneness to phagocytosis and compromised proliferation in macrophages. However, in the epithelial cells, despite being invasion deficient, it was hyper-proliferative. The poor colocalization of STM{Delta} ompA with LAMP-1 confirmed impaired stability of SCV membrane around the intracellular bacteria, resulting in its (STM{Delta} ompA) release into the cytosol of macrophages where it is assaulted with reactive nitrogen intermediates (RNI). The cytosolic localization of STM{Delta} ompA was responsible for the downregulation of SPI-2 encoded virulence factor SpiC, which is required to suppress the activity of iNOS. The reduced recruitment of nitrotyrosine on STM in the macrophage cytosol upon ectopically expressing Listeriolysin O (LLO) explicitly supported the pro-bacterial role of OmpA against the host nitrosative stress. Further, we show that the generation of time-dependent redox burst could be responsible for the enhanced sensitivity of STM{Delta} ompA towards nitrosative stress. The absence of OmpA in STM{Delta} ompA resulted in the loss of integrity and enhanced porosity of the bacterial outer membrane, which was attributed to the upregulated expression of ompC, ompD, and ompF. We showed the involvement of OmpF in the entry of excess nitrite in STM{Delta} ompA, thus increasing the susceptibility of the bacteria towards in vitro and in vivo nitrosative stress. In conclusion, we illustrated a mechanism of strategic utilization of OmpA compared to other porins by wildtype Salmonella for combating the nitrosative stress in macrophages.