Daily caffeinated soda intake in children is associated with neurocognitive vulnerabilities of substance misuse and predicts higher risk of alcohol sipping in 12 months Caffeinated soda contains two addictive substances, sugar and caffeine, and is the most preferred means of caffeine consumption among children. However, it remains unexplored if habitual caffeinated soda intake in childhood is associated with a higher risk of alcohol misuse in the future. Here, we investigated the neurocognitive correlates of daily consumption of caffeinated soda and examined whether daily caffeinated soda intake is associated with a higher risk of alcohol initiation in children. By applying machine learning and hierarchical regression to a large dataset from the Adolescent Brain Cognitive Development (ABCD) Study, we investigated the relationship between daily caffeinated soda intake and the well-known risk factors of substance misuse, including impaired working memory, high impulsivity, and altered reward processing. We next examined whether daily caffeinated soda intake could predict greater alcohol sipping in 12 months. The results showed that daily consumption of caffeinated soda was associated with impaired working memory, higher impulsivity, and altered reward processing, with increased amygdala activation during the emotional working memory task and hypoactivity in the nucleus accumbens and posterior cingulate cortex during reward processing tasks. Daily drinkers reported higher rates of alcohol sipping compared to non-drinkers after 12 months, and daily intake of caffeinated soda mediated the relationship between the neurocognitive risk factors and future alcohol sipping. These results suggest that daily caffeinated soda intake in childhood (1) is associated with impaired neurocognitive functioning, and (2) may work as a predictor for alcohol initiation, which has significant implications for public health recommendations.

Significance StatementIs daily caffeinated soda intake associated with higher risks of substance misuse? If so, which specific risk factors of substance misuse are involved? Moreover, can daily caffeinated soda intake predict future alcohol initiation? These are the questions that many parents are asking but surprisingly there is no clear guideline, and its potential vulnerabilities of substance misuse remain largely unknown. Here, by applying machine learning and hierarchical regression approaches, we demonstrate that daily caffeinated soda intake is associated with neurocognitive risk factors of substance misuse including impaired working memory, higher impulsivity, and altered reward processing. We also present that daily caffeinated soda consumption could predict higher alcohol sipping rates in 12 months. These results have significant implications for public health recommendations.
A dopamine-dependent decrease in dorsomedial striatum direct pathway neuronal activity is required for learned motor coordination It has been suggested that the dorsomedial striatum (DMS) facilitates the early stages of motor learning for goal-directed actions, whereas at later stages, control is transferred to the dorsolateral striatum (DLS), which enables the motor actions to become a skill or habit. It is unknown whether these striatal regions are simultaneously active while expertise is acquired during skill learning. To address this question, we developed the "treadmill training task" to track changes in mouse locomotor coordination during practice at running that simultaneously provides a means to measure local neuronal activity using photometry. We analyzed body position and paw movement to evaluate changes in motor coordination over practice sessions on the treadmill using DeepLabCut and custom-built code. By correlating improvements in motor coordination during training with simultaneous neuronal calcium activity in the striatum, we found that DMS direct pathway neurons exhibited decreased activity as the mouse gains proficiency at running. In contrast, direct pathway activity in the DLS was similar throughout training and did not correlate with learned skill proficiency. Pharmacological blockade of D1 dopamine receptors in these subregions during task performance confirmed that dopamine neurotransmission in the DMS direct pathway activity is necessary for efficient motor coordination learning, while dopamine signalling in the DLS is important for both coordination learning and maintenance of the acquired skill. These results provide new tools to measure changes in fine motor skills during simultaneous recordings of brain activity, revealing fundamental features of the neuronal substrates of motor learning.
POPULATION GENOMIC ANALYSIS OF CRYPTOCOCCUS BRAZILIAN ISOLATES REVEALS AN AFRICAN TYPE SUBCLADE DISTRIBUTION The genomes of a large number of Cryptococcus neoformans isolates have been sequenced and analyzed in recent years. These genomes have been used to understand the global population structure of this opportunistic pathogen. However, only a small number of South American isolates have been considered in these studies, and the population structure of C. neoformans in this part of the world remains elusive. Here, we analyzed the genomic sequences of 53 Brazilian Cryptococcus isolates and deciphered the C. neoformans population structure in this country. Our data reveal an African-like structure that suggested repeated intercontinental transports from Africa to South America. We also identified a mutator phenotype in one VNBII Brazilian isolate, exemplifying how fast-evolving isolates can shape the Cryptococcus population structure. Finally, phenotypic analyses revealed wide diversity but not lineage specificity in the expression of classical virulence traits within the set of isolates. Classification, Using Field Potential Images, of Pacemaker Micro-coordination in the Mouse Intestine The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals. 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. Mutualism-enhancing mutations dominate early adaptation in a microbial community Species interactions drive species evolution while evolution shapes these interactions. The resulting eco-evolutionary dynamics, their outcomes and their repeatability depend on how adaptive mutations available to community members affect fitness and ecologically relevant traits. However, the diversity of adaptive mutations available to community members is not well characterized, and we do not know how this diversity is affected by the ecological milieu. Here we use barcode lineage tracking to address this gap in a competitive mutualism between the yeast Saccharomyces cerevisiae and the alga Chlamydomonas reinhardtii. We find that yeast has access to many adaptive mutations with diverse ecological consequences, in particular, those that increase and reduce the yields of both species. The presence of alga does not change which mutations are adaptive in yeast (i.e., there is no fitness trade-off for yeast between growing alone or with alga), but rather shifts selection to favor yeast mutants that increase the yields of both species and make the mutualism stronger. Thus, in the presence of alga, we find that yeast repeatably evolves to become more cooperative, even though cooperativity is not directly favored by natural selection in our system. Our results demonstrate that ecological interactions not only alter the trajectory of evolution but also dictate its repeatability; in particular, weak mutualisms can repeatably evolve to become stronger. A memetic algorithm enables efficient local and global all-atom protein-protein docking with backbone and sidechain flexibility Protein complex formation is encoded by specific interactions at the atomic scale, but the computational cost of modeling proteins at this level often requires the use of simplified energy models and limited conformational flexibility. In particular, the use of all-atom energy functions, backbone and sidechain flexibility results in rugged energy landscapes that are difficult to explore. In this study we develop a protein-protein docking algorithm, EvoDOCK, that combine the strength of a differential evolution algorithm for efficient exploration of the global search space with the benefits of a local optimization method to refine detailed atomic interactions. EvoDOCK enabled accurate and fast local and global protein-protein docking using an all-atom energy function with side-chain flexibility. Comparison with a standard method built on Monte Carlo optimization demonstrated improved accuracy and with increases in computational speed of up to 35 times. The evolutionary algorithm also enabled efficient atomistic docking with backbone flexibility. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. Macroscopic control of synchronous electrical signaling with chemically-excited gene expression Excitable cells can convert electrical signals into chemical outputs to facilitate the active transport of information across larger distances. This electrical-to-chemical conversion requires a tightly regulated expression of ion channels. Alterations of ion channel expression provide landmarks of numerous pathological diseases, such as cardiac arrhythmia, epilepsy, or cancer. Although the activity of ion channels can be locally regulated by external light or chemical stimulus, it remains challenging to coordinate the expression of ion channels on extended spatial-temporal scales in a non-invasive manner. Here, we have engineered yeast S. cerevisiae to read and convert local chemical concentrations into a dynamic electrical field distributed across cell populations. The core mechanism encodes a chemically-excitable dual-feedback gene circuit that precisely tunes the expression domain of potassium channels, globally coordinating cyclic firing of the plasma membrane potential (PMP). We demonstrate that this mechanism leverages an engineered constitutively open bacterial potassium channel KcsA to directly couple chemical stimuli with ion flux through gene expression and it can interface with the host ion channels through the pulsatile production of toxins. Our study provides a robust synthetic transcriptional toolbox underlying the conversion of local chemical environments into spatiotemporally organized electrical impulses for various cellular engineering, synthetic biology, and potential therapeutic applications. Regulation of blood-brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine N-oxide BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied.

ResultsHere we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner.

ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function. Integrated analysis of cervical squamous cell carcinoma cohorts from three continents reveals conserved subtypes of prognostic significance. Human papillomavirus (HPV)-associated cervical cancer represents one of the leading causes of cancer death worldwide. Although low-middle income countries are disproportionately affected, our knowledge of the disease predominantly originates from populations in high-income countries. Using the largest multi-omic analysis of cervical squamous cell carcinoma (CSCC) to date, totalling 643 tumours and representing patient populations from the USA, Europe and Sub-Saharan Africa, we identify two CSCC subtypes (C1 and C2) with differing prognosis. C1 tumours are largely HPV16-driven, display increased cytotoxic T-lymphocyte infiltration and frequently harbour PIK3CA and EP300 mutations. C2 tumours are associated with shorter overall survival, are frequently driven by HPVs from the HPV18-containing alpha-7 clade, harbour alterations in the Hippo signalling pathway and increased expression of immune checkpoint genes, B7-H3 (also known as CD276) and NT5E (also known as CD73) and PD-L2 (also known as PDCD1LG2). In conclusion, we identify two novel, therapy-relevant CSCC subtypes that share the same defining characteristics across three geographically diverse cohorts. 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. The Gut Microbiome of Exudivorous Marmosets in the Wild and Captivity Mammalian captive dietary specialists like folivores are prone to gastrointestinal distress and primate dietary specialists suffer the greatest gut microbiome diversity losses in captivity compared to the wild. Marmosets represent another group of dietary specialists, exudivores that eat plant exudates, but whose microbiome remains relatively less studied. The common occurrence of gastrointestinal distress in captive marmosets prompted us to study the Callithrix gut microbiome composition and predictive function through bacterial 16S ribosomal RNA V4 region sequencing. We sampled 59 wild and non-wild Callithrix across four species and their hybrids. Host environment had a stronger effect on the gut microbiome than host taxon. Wild Callithrix gut microbiomes were enriched for Bifidobacterium, which process host-indigestible carbohydrates. Captive marmoset guts were enriched for Enterobacteriaceae, a family containing pathogenic bacteria. While gut microbiome function was similar across marmosets, Enterobacteriaceae seem to carry out most functional activities in captive host guts. More diverse bacterial taxa seem to perform gut functions in wild marmosets, with Bifidobacterium being important for carbohydrate metabolism. Non-wild marmosets showed gut microbiome composition aspects seen in human gastrointestinal diseases. Thus, captivity may perturb the exudivore gut microbiome, which raises implications for captive exudivore welfare and calls for husbandry modifications. Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. Micro topographical instruction of bacterial attachment, biofilm formation and in vivo host response Bio-instructive materials that prevent bacterial biofilm formation and drive an appropriate host immune response have the potential to significantly reduce the burden of medical device-associated infections. As bacterial surface attachment is sensitive to surface topography, we experimentally surveyed 2,176 combinatorially generated shapes using an unbiased high-throughput micro topographical polystyrene polymer chip screen. This identified topographies that reduced colonization in vitro by up to 15-fold compared with a flat surface for both motile and non-motile bacterial pathogens. Equivalent reductions were achieved on polyurethane, a polymer commonly used in medical devices. Using machine learning methods, a set of design rules based on generalisable topo-descriptors was established for predicting bacteria-resistant micro topographies. In a murine foreign body infection model, anti-attachment topographies were shown to be refractory to P. aeruginosa colonization and serendipitously, the fibrotic response to the implant was reduced, offering control of foreign body response using simple topographical patterning of non-eluting implants. 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. Quantifying isolation-by-resistance and connectivity in dendritic ecological networks Statistical methods that quantify individual movements have limited applicability within dendritic ecological networks (DENs), to include riverscapes. We promote an approach herein by deriving an analytical framework (=DO_SCPLOWISTC_SCPLOWNO_SCPLOWETC_SCPLOW and RO_SCPLOWESISTC_SCPLOWNO_SCPLOWETC_SCPLOW) that more appropriately quantifies differentiation by meshing individual DEN segments with fitted distances and annotated environmental features. We first explored the spatial arrangement of diversity in a river network by fitting pairwise distances against graph edges. We then derived effective resistance models (e.g., as a composite of an array of possible environmental covariates) by using a genetic algorithm for heuristic model selection, invoking circuit theory (CO_SCPLOWIRCUITC_SCPLOWSO_SCPLOWCAPEC_SCPLOW) to form a graph-based complement to RO_SCPLOWESISTANCEC_SCPLOWGA (a landscape ecology package). Although numerous pairwise distance metrics can be employed, we utilized genetic distances encompassing 13,218 ddRAD loci from N=762 Speckled Dace (Cyprinidae: Rhinichthys osculus) sampled at 78 Colorado River localities ({micro}=9.77/site). When distances for each locus were fitted into the network, we found intraspecific divergences due to stream reaches were greater than expected when distances alone. In fitting model-averaged  effective resistance models to the same network, we showed the manner by which a host of anthropogenic and environmental variables contribute to patterns of connectivity underlying riverscape genetic differentiation. Our framework represents a contemporary approach to deriving metapopulation/ metacommunity structure within DENs, in that it allows: (a) The extension of projections into unsampled temporal/spatial components; (b) Comparisons to be made among species and/or drainages, both of which benefit multi-species management; and (c) the quantification of locus-specific patterns, from which adaptive hypotheses could then be derived. O_QD"The intimate relation of hydrological conditions to fish life is proverbial; to be hopelessly or dangerously maladjusted to ones environment is to be a  fish out of water" (Hubbs 1941).

C_QD SARS-CoV-2 Beta Variant Substitutions Alter Spike Glycoprotein Receptor Binding Domain Structure and Stability The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic has visited a terrible cost on the world in the forms of disease, death, and economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 have seemingly brought within reach the end of the pandemic. However, the virus has acquired mutations; and emerging variants of concern (VOC) are more infectious and reduce the efficacy of existing vaccines. While promising efforts to combat these variants are underway, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain of three amino-acid substitutions found in several variants of concern, including alpha (B.1.1.7), beta (B.1.351), and gamma (P.1). We found that these substitutions alter the RBD structure, stability, and ability to bind to ACE2, in such a way as to possibly have opposing and compensatory effects. These findings provide new insights into how these VOC may have been selected for infectivity while maintaining the structure and stability of the receptor binding domain. Fatal COVID-19 ARDS associated with incomplete AEC1 differentiation from the transitional state without senescence or 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. Aldh2 is a lineage-specific metabolic gatekeeper in melanocyte stem cells Melanocyte stem cells (McSCs) in zebrafish serve as an on-demand source of melanocytes during growth and regeneration, but metabolic programs associated with their activation and regenerative processes are not well known. Here, using live imaging coupled with scRNA-sequencing, we discovered that quiescent McSCs during regeneration activate a dormant embryonic neural crest transcriptional program followed by an aldehyde dehydrogenase (Aldh) 2 metabolic switch to generate progeny. Unexpectedly, while ALDH2 is well known for its aldehyde clearing mechanisms we find that in regenerating McSCs, Aldh2 activity is required to generate formate - the one-carbon (1C) building block for nucleotide biosynthesis - through formaldehyde metabolism. Consequently, we find that disrupting the 1C cycle with low-doses of methotrexate caused melanocyte regeneration defects. In the absence of Aldh2, we find that purines (but not pyrimidines) are the metabolic end product sufficient for activated McSCs to generate progeny. Together, our work reveals McSCs undergo a two-step cell state transition during regeneration, and that the reaction products of Aldh2 enzymes have tissue-specific stem cell functions that meet metabolic demands in regeneration.

SUMMARY STATEMENTIn melanocyte regeneration, quiescent McSCs respond by re-expressing a neural crest identity, followed by an Aldh2-dependent metabolic switch to generate progeny. Epithelial and neutrophil interactions and coordinated response to Shigella in a human intestinal enteroid-neutrophil co-culture model Polymorphonuclear neutrophils (PMN) are recruited to the gastrointestinal mucosa in response to inflammation, injury, and infection. Herein, we report the development and the characterization of an ex vivo tissue co-culture model consisting of human primary intestinal enteroid monolayers and PMN, and a mechanistic interrogation of PMN-epithelial cell interaction and response to Shigella, a primary cause of childhood dysentery. Cellular adaptation and tissue integration, barrier function, PMN phenotypic and functional attributes, and innate immune responses were examined. PMN within the enteroid monolayers acquired a distinct activated/migratory phenotype that was influenced by direct epithelial cell contact as well as by molecular signals. Seeded on the basal side of the intestinal monolayer, PMN intercalated within the epithelial cells and moved paracellularly toward the apical side. Co-cultured PMN also increased basal secretion of IL-8. Shigella added to the apical surface of the monolayers evoked additional PMN phenotypic adaptations, including increased expression of cell surface markers associated with chemotaxis and cell degranulation (CD47, CD66b, and CD88). Apical Shigella infection triggered rapid transmigration of PMN to the luminal side, NET formation as well as bacterial phagocytosis and killing. Shigella infection modulated cytokine production in the co-culture; apical MCP-1, TNF-, and basolateral IL-8 production were downregulated, while basolateral IL-6 secretion was increased. We demonstrated, for the first time, PMN phenotypic adaptation, mobilization, and coordinated epithelial cell-PMN innate response upon Shigella infection in the human intestinal environment. The enteroid monolayer-PMN co-culture represents a technical innovation for mechanistic interrogation of gastrointestinal physiology, host-microbe interaction, innate immunity, and evaluation of preventive/therapeutic tools.

ImportanceStudies of mucosal immunity and microbial host cell interaction have traditionally relied on animal models and in vitro tissue culture using immortalized cancer cell lines, which render non-physiological and often unreliable results. Herein we report the development and characterization of an ex vivo enteroid-PMN co-culture consisting of normal human intestinal epithelium and a mechanistic interrogation of PMN and epithelial cell interaction and function in the context of Shigella infection. We demonstrated tissue-driven phenotypic and functional adaptation of PMN and a coordinated epithelial cell and PMN response to Shigella, a primary cause of dysentery in young children in the developing world. Mechanical loading of cranial joints minimizes the craniofacial phenotype in Crouzon syndrome Children with syndromic forms of craniosynostosis undergo a plethora of surgical interventions to resolve the clinical features caused by the premature fusion of cranial sutures. While surgical correction is reliable, the need for repeated rounds of invasive treatment puts a heavy burden on the child and their family.

This study explores a non-surgical alternative using mechanical loading of the cranial joints to prevent or delay craniofacial phenotypes associated with Crouzon syndrome.

We treated Crouzon syndrome mice before the onset of craniosynostosis by cyclical mechanical loading of cranial joints using a custom designed set-up.

Cranial loading applied to the frontal bone partially restores normal skull morphology, significantly reducing the typical brachycephalic appearance. This is underpinned by the delayed closure of the coronal suture and of the intersphenoidal synchondrosis.

This study provides a novel treatment alternative for syndromic craniosynostosis which has the potential to be an important step towards replacing, reducing or refining the surgical treatment of all craniosynostosis patients. Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germ line specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESCs self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification. A terrain treadmill to study animal locomotion through large obstacles A major challenge to understanding locomotion in complex 3-D terrain with large obstacles is to create tools for controlled, systematic lab experiments. Existing terrain arenas only allow observations at small spatiotemporal scales ([~]10 body length, [~]10 stride cycles). Here, we create a terrain treadmill to enable high-resolution observations of animal locomotion through large obstacles over large spatiotemporal scales. An animal moves through modular obstacles on an inner sphere, while a rigidly-attached, concentric, transparent outer sphere rotated with the opposite velocity via closed-loop feedback to keep the animal on top. During sustained locomotion, a discoid cockroach moved through pillar obstacles for 25 minutes ({approx} 2500 strides) over 67 m ({approx} 1500 body lengths), and was contained within a radius of 4 cm (0.9 body length) for 83% of the duration, even at speeds of up to 10 body length/s. The treadmill enabled observation of diverse locomotor behaviors and quantification of animal-obstacle interaction.

Summary StatementA device keeps a small animal on top of a sphere while it traverses obstacles, creating a "terrain treadmill" to study locomotion over a long time and distance at high-resolution. The DC1 domain protein BINUCLEATE POLLEN is required for pollen development in Arabidopsis thaliana Development of the male gametophyte is a tightly regulated process that requires precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying the defective BNP allele failed to complete mitosis II and are impaired in pollen germination. By yeast two hybrid screening and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors we found NAC family transcriptional regulators Vascular plant One-Zinc finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP, as in their absence in pollen from bnp/BNP plants, VOZ1 nuclear localization resulted impaired. As voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have a role of scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators. 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.