Subjective understanding of actions and emotions requires interaction of the semantic and action observation networks Previous studies have investigated the neural basis of action and emotion perception conveyed by whole body postures and movements. However, subjective understanding of other peoples bodily actions and emotions, raises different issues that are currently not well understood and have so far seldom been investigated. In this 7T fMRI study, we proceeded beyond conventional univariate/multivariate analyses with predefined categories, and examined the representational geometry of subjective action- and emotion-understanding by mapping individual subjective reports with word embeddings. Dimensionality reduction revealed that the representations for perceived action and emotion were high dimensional, each correlated to but were not reducible to the predefined action and emotion categories. Searchlight representational similarity analysis showed that the representations in the left middle superior temporal sulcus and left dorsal premotor cortex corresponded to the subjective action and emotion understanding of the participants. Furthermore, using task-residual functional connectivity and hierarchical clustering, we found that areas in the action observation network and the semantic/default-mode network were functionally connected to these two seed regions, and showed similar representations. Our study provides direct evidence that both networks were concurrently involved in subjective action and emotion understanding. Our approach shows how subjective understanding of action and emotion stimuli can be reliably studied and complement insights based on from predefined stimulus categories.
Controllability reveals defining features of information seeking In uncertain environments, seeking information about alternative choice options is essential for adaptive learning and decision-making. However, information seeking is usually confounded with changes-of-mind about the reliability of the preferred option. Here, we exploited the fact that information seeking requires control over which option to sample to isolate its behavioral and neurophysiological signatures. We found that changes-of-mind occurring with control require more evidence against the current option, are associated with reduced confidence, but are nevertheless more likely to be confirmed on the next decision. Multimodal neurophysiological recordings showed that these changes-of-mind are preceded by stronger activation of the dorsal attention network in magnetoencephalography (MEG), and followed by increased pupil-linked arousal during the presentation of decision outcomes. Together, these findings indicate that information seeking increases the saliency of evidence perceived as the direct consequence of ones own actions.

HighlightsO_LIExploration-exploitation dilemmas confound information seeking with changes-of-mind.
C_LIO_LIUnlike changes-of-mind, information seeking requires control over information sampling.
C_LIO_LIInformation seeking is associated with reduced confidence and active hypothesis testing.
C_LIO_LIStronger neurophysiological correlates of attention and arousal during information seeking.
C_LI
Protective host-dependent antagonism among 	Pseudomonas in the Arabidopsis phyllosphere The plant microbiome is a rich biotic environment, comprising numerous taxa. The community structure of these colonizers is constrained by multiple factors, including host-microbe and microbe-microbe interactions, as well as the interplay between the two. While much can be learned from pairwise relationships between individual hosts and microbes, or individual microbes with themselves, the ensemble of interrelations between the host and microbial consortia may lead to different outcomes that are not easily predicted from the individual interactions. Their study can thus provide new insights into the complex relationship between plants and microbes. Of particular importance is how strain-specific such plant-microbe-microbe interactions are, and how they eventually affect plant health. Here, we test strain-level interactions in the phyllosphere between groups of co-existing commensal and pathogenic Pseudomonas among each other and with A. thaliana, by employing synthetic communities of genome-barcoded isolates. We found that commensal Pseudomonas prompted a host response leading to a selective inhibition of a specific pathogenic lineage, resulting in plant protection. The extent of plant protection, however, was dependent on plant genotype, indicating that these effects are host-mediated. There were similar genotype-specific effects on the microbe side, as we could pinpoint an individual Pseudomonas isolate as the predominant cause for this differential interaction. Collectively, our work highlights how within-species genetic differences on both the host and microbe side can have profound effects on host-microbe-microbe dynamics. The paradigm that we have established provides a platform for the study of host-dependent microbe-microbe competition and cooperation in the A. thaliana-Pseudomonas system. SyMBac: Synthetic Micrographs for Training Deep Neural Networks for Accurate Segmentation of Bacterial Cells We present a novel method of bacterial image segmentation using machine learning based on Synthetic Micrographs of Bacteria (SyMBac). SyMBac allows for rapid, automatic creation of arbitrary amounts of training data that combines detailed models of cell growth, physical interactions, and microscope optics to create synthetic images which closely resemble real micrographs, with access to the ground truth positions of cells. This approach provides the following major advantages: (1) Synthetic training data can be generated virtually instantly, and on demand; (2) it is accompanied by perfect ground truth information, which also means large sets of training data can be generated without the need for curation; (3) Biological ground truth and imaging effects can be tuned independently, enabling easy adaptation to different biological conditions, imaging platforms, and imaging modalities. Machine-learning models trained on SyMBac data generate more accurate and precise cell masks than those trained on human annotated data. This enables precise analysis of cell size regulation along changing conditions in a growing bacterial cell culture, revealing novel insights about the physiological dynamics of individual bacterial cells during entry and exit from dormancy.

Author SummaryMicroscopy is integral to microbiology, but accurate processing of microscopy data has been a major obstacle towards quantitative analysis of microbial physiology. The small size of bacterial cells and the comparable size of the microscopes point-spread function has made it virtually impossible to accurately determine the size, shape, and intensity of individual cells from their images. The difficulties of accurately identifying the boundaries of a cell from their image using computational thresholding or human annotators also limits the performance of recently developed machine-learning based image segmentation approaches, since these toolsets rely on the quality and quantity of the ground truth in the training data. In response to all these problems, we have developed SyMBac: Synthetic Micrographs of Bacteria. SyMBac is a tool which rapidly generates arbitrary amounts of synthetic training data of bacteria growing in microfluidic devices. Synthetic images generated by SyMBAc are accompanied by perfect ground truth information, which means large sets of training data can be generated without the need for curation. Our benchmarking results show that deep-learning algorithms trained with SyMBac are far superior, in terms of accuracy and precision of segmentation, compared to the same models trained on human annotated or computationally segmented data. This will significantly advance the field of quantitative microbiology. Modeling microRNA-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 rat model of pregnancy in the male parabiont Male pregnancy is a unique phenomenon in syngnathidae which refers to embryo or fetus incubation by males. However, whether male mammalian animals have the potential to conceive and maintain pregnancy remains unclear. Here, we constructed a rat model of pregnancy in the male parabiont using a four-step strategy. First, a heterosexual parabiotic pair was produced by surgically joining a castrated male rat and a female rat. After 8 weeks, uterus transplantation was performed on the male parabiont. After recovery, blastocyst-stage embryos were transplanted to the grafted uterus of the male parabiont and the native uterus of the female parabiont. Caesarean section was performed at embryonic day 21.5. The success rate of the model was 3.68%, and pups were only delivered from the male parabiont under pregnant blood exposure. Our results reveal the potential for rat embryonic development in male parabionts, which may have a profound impact on reproductive biology.

One-sentence summaryA rat model of pregnancy was constructed in the male parabiont using a four-step strategy.



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org.highwire.dtl.DTLVardef@1f0694eorg.highwire.dtl.DTLVardef@91997forg.highwire.dtl.DTLVardef@f47086org.highwire.dtl.DTLVardef@7a33f_HPS_FORMAT_FIGEXP  M_FIG C_FIG Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Integrative transcriptomics reveals ectopic lipid homeostasis mechanisms in non-endocrine cells of the teleost pituitary Directing both organismal homeostasis and physiological adaptation, the pituitary is a key endocrine gland in all vertebrates. It communicates the needs of the organism to different organs by secreting hormones into the bloodstream. Here, we have used the model fish medaka to investigate the developmental dynamics in the pituitary using a comprehensive RNA-seq time series. By linking developmental expression trends to single-cell RNA-seq profiles, we show how the transcriptional activities of cell types change during sexual maturation. One of the most prominent changes is the decline of the non-endocrine folliculo-stellate cell populations, and especially of rare cells expressing genes encoding secreted lipid transport proteins. As these genes are typically associated with the liver, this reveals the existence of unexpected connections between endocrine communication, lipid homeostasis, and sexual maturation. Common coupling map advances GPCR-G protein selectivity Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein  couplome. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on GPCR-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling towards the exploitation of G protein signaling bias in design of safer drugs. Royal knifefish generate powerful suction feeding through large neurocranial elevation and high epaxial muscle power Suction feeding in ray-finned fishes involves powerful buccal cavity expansion to accelerate water and food into the mouth. Previous XROMM studies in largemouth bass (Micropterus salmoides), bluegill sunfish (Lepomis macrochirus), and channel catfish (Ictalurus punctatus) have shown that more than 90% of suction power in high performance strikes comes from the axial musculature. Thus, the shape of the axial muscles and skeleton may impact suction feeding mechanics. Royal knifefish (Chitala blanci) have an unusual postcranial morphology, with a ventrally flexed vertebral column and relatively large mass of epaxial muscle. Based on their body shape, we hypothesized that royal knifefish would generate high power strikes by utilizing large neurocranial elevation, vertebral column extension, and epaxial shortening. As predicted, C. blanci generated high suction expansion power compared to the other three species studied to date (up to 160 W), which was achieved by increasing both the rate of volume change and the intraoral subambient pressure. The large epaxial muscle (25% of body mass) shortened at high velocities to produce large neurocranial elevation and vertebral extension (up to 41 deg, combined), as well as high muscle mass-specific power (up to 800 W kg-1). For the highest power strikes, axial muscles generated 95% of the power, and 64% of the axial muscle mass consisted of the epaxial muscles. The epaxial-dominated suction expansion of royal knifefish supports our hypothesis that postcranial morphology may be a strong predictor of suction feeding biomechanics.

SUMMARY STATEMENTRoyal knifefish rely on their distinct postcranial morphology--with a curved vertebral column and large dorsal body muscles--to produce large neurocranial elevation and powerful suction feeding. bMSI-CAST: a systematic method for next generation sequencing-based microsatellite instability detection in plasma cell-free DNA Microsatellite instability (MSI) is a well-established prognostic and predictive biomarker in certain types of cancers. MSI detection using tumour tissue is often limited by the availability of specimens. Next generation sequencing (NGS)-based MSI detection in plasma cell-free DNA (cfDNA) is challenged by a much lower signal-to-noise ratio. We developed a highly accurate cfDNA MSI detection method called bMSI-CAST (blood MSI Caller Adjusted with Sequence duplicaTes), with improvement on three features including a set of locus selection principles ensuring loci with high robustness and compatibility across sequencing platforms, an MSI-specific duplicate removal strategy, and a calling algorithm that dynamically matches baselines with a broad range of duplication levels. Analytical validation via MSI-high (MSI-H) cell gDNA showed an LOD of 0.15%. Furthermore, in an analysis of 95 evaluable cfDNA samples from patients with gastrointestinal cancers, bMSI-CAST exhibited a positive predictive agreement (PPA) of 92.9% (39/42) and negative predictive agreement (NPA) of 100% (53/53) with tissue MSI-PCR. In conclusion, bMSI-CAST provides novel and advanced solutions to key aspects fundamental to cfDNA MSI calling but not sufficiently addressed by existing methods, and it is a validated method ready to be applied to aid clinical decisions for cancer patients. Glycolysis/gluconeogenesis specialization in microbes is driven by biochemical constraints of flux sensing Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic re-organization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida. We combined growth dynamics and dynamic changes of intracellular metabolite levels with a coarse-grained model that integrates fluxes, regulation, protein synthesis and growth and uncovered fundamental limitations of the regulatory network: after nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade-offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.

Graphical synopsis O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Standfirst textMicrobes face a series of fundamental trade-offs that limit their ability to optimize simultaneously for both glycolytic and gluconeogenic growth.

Bullet pointsO_LILag times between glycolysis and gluconeogenesis show asymmetry in many microbes: A long lag in one direction, but a short lag in the other.
C_LIO_LILong lag times are caused by an inability to sense fluxes after nutrient shifts.
C_LIO_LIWith existing regulation, lag time asymmetry can only be overcome by reducing either growth rate or increasing futile cycling in metabolism.
C_LI An End-to-End Deep Learning Speech Coding and Denoising Strategy for Cochlear Implants Cochlear implant (CI) users struggle to understand speech in noisy conditions. To address this problem, we propose a deep learning speech denoising sound coding strategy that estimates the CI electric stimulation patterns out of the raw audio data captured by the micro-phone, performing end-to-end CI processing. To estimate the relative denoising performance differences between various approaches, we compared this technique to a classic Wiener filter and to a convTasNet. Speech enhancement performance was assessed by means of signal-to-noise-ratio improvement and the short-time objective speech intelligibility measure. Additionally, 5 CI users were evaluated for speech intelligibility in noise to assess the potential benefits of each algorithm. Our results show that the proposed method is capable of replacing a CI sound coding strategy while preserving its general use for every listener and performing speech enhancement in noisy environments, without sacrificing algorithmic latency. Hydrophobic mismatch is a key factor in protein transport on the Tat pathway The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes. 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. Sex-specific effects of an IgE polymorphism on immunity, susceptibility to infection and reproduction in a wild rodent The genotype of an individual is an important predictor of their immune function, and subsequently, their ability to control or avoid infection and ultimately contribute offspring to the next generation. However, the same genotype, subjected to different environments, can also result in different outcomes. The sexes represent two such different environments. Sexual dimorphism is widespread across the animal kingdom. Despite this, very little is known about the importance of sex for the expression of genotype in the context of health and disease, particularly in natural populations. We identified a synonymous polymorphism in the high-affinity Immunoglobulin E (IgE) receptor (GC and non-GC haplotypes) that has sex-specific effects on immune gene expression, susceptibility to infection and reproductive success of individuals in a natural population of field voles (Microtus agrestis). We found that the effect of the GC haplotype on the expression of genes affecting inflammation displayed a significant interaction with sex. While males with the GC haplotype had upregulated pro-inflammatory genes, in particular the pro-inflammatory cytokine Il33, females had upregulated anti-inflammatory genes, in particular the anti-inflammatory cytokine inhibitor Socs3. Furthermore we found that the effect of the GC haplotype on the probability of infection with a common microparasite, Babesia microti, displayed a significant interaction with sex. While males with the GC haplotype did not differ significantly in their susceptibility to infection, females with the GC haplotype were more likely to be infected. Finally, we found that the effect of the GC haplotype on reproductive success also displayed a significant interaction with sex. While males with the GC haplotype had a lower reproductive success, females with the GC haplotype did not differ in the number of offspring they produced. To our knowledge, this is the first time that a polymorphism with sex-specific effects across all three levels (immune gene expression, susceptibility to infection and reproductive success) has been documented in a natural population. The pathogen-encoded signaling receptor Tir exploits host-like intrinsic disorder to assist infection The translocated intimin receptor (Tir) is a central effector of Attaching and Effacing (A/E) pathogens responsible for worldwide foodborne disease cases. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes to assist infection by targeting multiple host proteins. We sought to understand the basis for Tir binding diversity in signaling. Here, we establish that Tir is a disordered protein with host-like binding motifs. A trait we find prevalent in several other effectors secreted by A/E bacteria. We disclose that Tir has a disordered C-terminal intracellular tail (C-Tir) with non-random structural preferences at phosphorylation sites, including host-like tyrosine-based motifs, with versatile lipid- and SH2 domain binding capability pre-phosphorylation. We show that multi-site tyrosine phosphorylation enables C-Tir to engage SH2 domains in a multivalent manner, consistent with Tirs scaffold/hub role for host proteins. Last, we uncover Tirs ability to dimerizes via its partially disordered N-terminal intracellular domain. Collectively, our findings provide an updated picture of Tirs intracellular side, highlighting its ability to mimic host disordered membrane receptors versatility as a molecular strategy for host evasion.

SummaryTir is a cell-surface receptor secreted by life-threatening pathogens. Upon delivery into host cells, Tir inserts the host plasma membrane providing a means for these extracellular pathogens to control host intracellular processes. To prevent pathogens from relying on Tir, it is essential to understand its intracellular mechanics. This paper provides a coherent picture of the intracellular side of Tir, highlighting its ability to copycat the interactions of disordered intracellular domains of host immune receptors. This copycatting allows the bacterial pathogens to modulate critical host processes, allowing infection to spread further without triggering the immune system response. This work proposes that other bacterial secreted pathogenic proteins exploit intrinsic disorder to hijack human cells, suggesting a widespread host subversion mechanism. Anti- SARS-CoV-2 Receptor Binding Domain Antibody Evolution after mRNA Vaccination Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B-cell responses that continue to evolve for at least one year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern1. As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested1, 2. Here, we examine memory B cell evolution 5 months after vaccination with either Moderna (mRNA-1273) or Pfizer- BioNTech (BNT162b2) mRNA vaccines in a cohort of SARS-CoV-2 naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge 5 months after vaccination of naive individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with breadth equivalent to those obtained by vaccinating convalescent individuals. 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. Prostate megalin is regulated by serum hormone levels and imports globulin-bound testosterone Vitamin D deficiency associates with an increased risk of prostate cancer (PCa) mortality and is hypothesized to contribute to PCa aggressiveness and disparities in African Americans. We previously reported a relationship between African-ancestry, circulating and intraprostatic vitamin D metabolites and prostatic expression of megalin, an endocytic receptor that internalizes globulin-bound hormones. Here, we show prostatic 5-dihydrotestosterone (DHT) levels are higher in African American men and inversely correlate with serum 25-hydroxyvitamin D (25D) status. We further demonstrate that megalin imports sex hormone-binding globulin-bound testosterone in prostate cells and prostatic loss of Lrp2 (megalin) results in reduced prostate androgen levels. Megalin expression was suppressed by 25D and DHT in cell lines, patient-derived prostate epithelial cells, and prostate tissue explants, supporting a negative feedback loop. Megalin levels are reduced in localized PCa by Gleason Grade and in patients with future disease recurrence. Our findings highlight the impact of vitamin D deficiency on prostate androgen levels, which are known drivers of PCa, and reveal a potential mechanistic link between vitamin D and the PCa disparities observed in African Americans. A class of identifiable phylogenetic birth-death models In a striking result, Louca and Pennell (2020) recently proved that a large class of phylogenetic birth-death models are statistically unidentifiable from lineage-through-time (LTT) data: any pair of sufficiently smooth birth and death rate functions is "congruent" to an infinite collection of other rate functions, all of which have the same likelihood for any LTT vector of any dimension. As Louca and Pennell argue, this fact has distressing implications for the thousands of studies that have utilized birth-death models to study evolution.

In this paper, we qualify their finding by proving that an alternative and widely used class of birth-death models is indeed identifiable. Specifically, we show that piecewise constant birth-death models can, in principle, be consistently estimated and distinguished from one another, given a sufficiently large extant time tree and some knowledge of the present-day population. Subject to mild regularity conditions, we further show that any unidentifiable birth-death model class can be arbitrarily closely approximated by a class of identifiable models. The sampling requirements needed for our results to hold are explicit, and are expected to be satisfied in many contexts such as the phylodynamic analysis of a global pandemic. Load adaptation of endocytic actin networks Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load, as has been observed in lamellipodia, has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feedback on assembly. By manipulating membrane tension and Arp2/3 complex activity we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions. A rationalized definition of tumor suppressor microRNAsexcludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors. Risky choice: probability weighting explains Independence Axiom violations in monkeys Expected Utility Theory (EUT) provides axioms for maximizing utility in risky choice. The Independence Axiom (IA) is its most demanding axiom: preferences between two options should not change when altering both options equally by mixing them with a common gamble. We tested common consequence (CC) and common ratio (CR) violations of the IA over several months in thousands of stochastic choices using a large variety of binary option sets. Three monkeys showed consistently few outright Preference Reversals (8%) but substantial graded Preference Changes (46%) between the initial preferred gamble and the corresponding altered gamble. Linear Discriminant Analysis (LDA) indicated that gamble probabilities predicted most Preference Changes in CC (72%) and CR (88%) tests. The Akaike Information Criterion indicated that probability weighting within Cumulative Prospect Theory (CPT) explained choices better than models using Expected Value (EV) or EUT. Fitting by utility and probability weighting functions of CPT resulted in nonlinear and non-parallel indifference curves (IC) in the Marschak-Machina triangle and suggested IA non-compliance of models using EV or EUT. Indeed, CPT models predicted Preference Changes better than EV and EUT models. Indifference points in out-of-sample tests were closer to CPT-estimated ICs than EV and EUT ICs. Finally, while the few outright Preference Reversals may reflect the long experience of our monkeys, their more graded Preference Changes corresponded to those reported for humans. In benefitting from the wide testing possibilities in monkeys, our stringent axiomatic tests contribute critical information about risky decision-making and serve as basis for investigating neuronal decision mechanisms. 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.