The isoforms of pyruvate kinase act as nutrient sensors for the β-cell KATP channel Insulin release from pancreatic {beta}-cells is triggered by the metabolism-dependent closure of ATP-sensitive K+ (KATP) channels. Here, we used isoform-specific deletion of pyruvate kinase (PK) to identify the unique roles of PKm1 and PKm2 in the {beta}-cell nutrient response. In the absence of PK activity, cytosolic ATP/ADP was insufficient to close KATP channels. Both PK isoforms were found on the plasma membrane, where they endow KATP channels with nutrient sensitivity and specificity. Although PKm1 accounts for >90% of cellular activity, PKm2 controls glucosedependent KATP closure. Amino acids, which generate mitochondrial phosphoenolpyruvate (PEP) without allosterically activating PKm2, depend on PKm1 to raise ATP/ADP and close KATP channels. Shifting the {beta}-cell from PKm2 to PKm1 correspondingly increased amino acid sensitivity and boosted secretory output. In addition to these membrane depolarizing "on-switches," {beta}-cell deletion of PCK2 exposed a mitochondrial PEP-dependent "off-switch" that facilitates Ca2+ extrusion. Restricting PCK2 prolonged Ca2+ influx and increased insulin secretion. In summary, it is not the KATP channel alone, but the PK-KATP microcompartment that confers nutrient sensitivity to the {beta}-cell plasma membrane. The differential response of the PK isoforms to the glycolytic and mitochondrial sources of PEP influences {beta}-cell nutrient preference, and controls the oscillatory cycle regulating insulin secretion.
TCAB1 prevents nucleolar accumulation of the telomerase RNA to promote telomerase assembly Localization of a wide variety of RNAs to non-membrane bound cellular compartments such as nucleoli, Cajal bodies, and stress-granules is critical for their function and stability. The molecular mechanisms that underly the recruitment and exclusion of specific RNAs from these phase-separated organelles is poorly understood. Telomerase is a ribonucleoprotein (RNP), that is composed of the reverse transcriptase protein TERT, the telomerase RNA (TR), and several auxiliary proteins that associate with TR, including TCAB1. Here we show that, that in the absence of TCAB1, TR is sequestered in the nucleolus, while TERT localizes to the nucleoplasm and is excluded from the nucleolus, which prevents telomerase assembly. Thus, nuclear compartmentalization by the non-membrane bound nucleolus counteracts telomerase assembly and TCAB1 is required to exclude the telomerase RNA from the nucleolus. Our work provides general insight into the mechanism and functional consequences of RNA recruitment to organelles formed by phase-separation and proposes a new model explaining the critical role of TCAB1 in telomerase function.
Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. 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. A cell type-specific multiomics uncovers a guard cell-specific RAF15-SnRK2.6/OST1 kinase cascade Multicellular organisms such as plants contain different cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances understanding of organization and function at the organismal level. Here we report a highly-sensitive and efficient cell type-specific multiomics pipeline, combining simplified flow cytometry-based cell sorting for fluorescent protoplasts and optimized nanoscale proteomics and metabolomics methods, which allow in-depth analysis of the proteomes and metabolomes of a particular cell type. Using this method, we quantitatively compared the proteomes and metabolomes of guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond rapidly to various environmental stimuli. We uncovered a guard cell-specific kinase cascade whereby RAF15 and OST1 mediate ABA-induced stomatal closure. This pipeline can be applied to various cell types in plant or non-plant systems to learn how cells function in highly organized multicellular organisms. Placental uptake and metabolism of 25(OH)Vitamin D determines its activity within the fetoplacental unit Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy. If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context. Oct1 recruits the histone lysine demethylase Utx to promote lineage specification The pathways used by cells to transition between undifferentiated, pluripotent state and tissue-specific states are incompletely understood. Here we show that the widely-expressed transcription factor Oct1/Pou2f1 activates silent, developmental lineage-appropriate genes to "canalize" developmental progression. Using Oct1 inducible knockout embryonic stem cells, we show that that Oct1 deficiency impairs mesodermal and terminal muscle differentiation in a manner that can be rescued by Oct1 retroviral expression. Using bulk RNA-seq, we show that mesoderm-specific genes are not correctly induced early in the differentiation timecourse. Single-cell gene expression profiling reveals that Oct1-deficient cells lose coherence in temporal induction of lineage programs, and show inappropriate developmental lineage branching resulting in poorly differentiated cells state with epithelial characteristics and hallmarks of oxidative stress. In embryonic stem cells, Oct1 co-binds with Oct4 to genes critical for mesoderm induction. The Utx/Kdm6a histone lysine demethylase also binds to many of these genes, and using a prototypic Pax3 gene we show that Oct1 recruits Utx to remove inhibitory H3K27me3 marks and activate expression. The specificity of the ubiquitous Oct1 protein for mesodermal genes can be explained by cooperative interactions with lineage-driving Smad transcription factors, as we show that Smad and Oct binding sites frequently coexist mesoderm-specific genes, that Oct1 and Smad3 interact, and that the sites and factors act cooperatively at the Myog enhancer. Overall, these results identify Oct1 as a key mediator of the induction of mesoderm lineage-specific genes. Cascade screening following a polygenic risk score test: what is the risk of a relative conditional on a high score of a proband? Polygenic risk scores (PRSs) for predicting disease risk have become increasingly accurate, leading to rising popularity of PRS tests. Consider an individual whose PRS test has placed him/her at the top q-quantile of genetic risk. Recently, Reid et al. (Circ Genom Precis Med. 2021;14:e003262) have investigated whether such a finding should motivate cascade screening in the probands siblings. Specifically, using data from the UK biobank, Reid et al. computed the empirical probability of a sibling of the proband to also have a PRS at the top q-quantile. In this short note, I use the liability threshold model to compute this probability analytically (for either a sibling of the proband or for a more distant relative), showing excellent agreement with the empirical results of Reid et al., including that this probability is disease-independent. Further, I compute the probability of the relative of the proband to be affected, as a function of the quantile threshold q, the proportion of variance explained by the score, and the disease prevalence. Evolutionary dynamics of sex-biased genes expressed in cricket brains and gonads Sex-biased gene expression, particularly sex-biased expression in the gonad, has been linked to rates of protein sequence evolution (nonsynonymous to synonymous substitutions, dN/dS) in animals. However, in insects, sex-biased expression studies remain centered on a few holometabolous species, and moreover, other major tissue types such as the brain remain underexplored. Here, we studied sex-biased gene expression and protein evolution in a hemimetabolous insect, the cricket Gryllus bimaculatus. We generated novel male and female RNA-seq data for two sexual tissue types, the gonad and somatic reproductive system, and for two core components of the nervous system, the brain and ventral nerve cord. From a genome-wide analysis, we report several core findings. Firstly, testis-biased genes had accelerated evolution, as compared to ovary-biased and unbiased genes, which was associated with positive selection events. Secondly, while sex-biased brain genes were much less common than for the gonad, they exhibited a striking tendency for rapid protein evolution, an effect that was stronger for the female than male brain. Further, some sex-biased brain genes were linked to sexual functions and mating behaviors, which we suggest may have accelerated their evolution via sexual selection. Thirdly, a tendency for narrow cross-tissue expression breadth, suggesting low pleiotropy, was observed for sex-biased brain genes, suggesting relaxed purifying selection, which we speculate may allow enhanced freedom to evolve adaptive protein functional changes. The findings of rapid evolution of testis-biased genes and male and female-biased brain genes are discussed with respect to pleiotropy, positive selection, and the mating biology of this cricket. Methylation pattern of polymorphically imprinted nc886 is not conserved across mammalia BackgroundIn humans, the nc886 locus is a polymorphically imprinted metastable epiallele. Periconceptional conditions have an effect on the methylation status of nc886, and further, this methylation status is associated with health outcomes in later life, in line with the Developmental Origins of Health and Disease (DOHaD) hypothesis. Animal models would offer opportunities to study the associations between periconceptional conditions, nc886 methylation status and metabolic phenotypes further. Thus, we set out to investigate the methylation pattern of the nc886 locus in non-human mammals.

DataWe obtained DNA methylation data from the data repository GEO for mammals, whose nc886 gene included all three major parts of nc886 and had sequency similarity of over 80% with the human nc886. Our final sample set consisted of DNA methylation data from humans, chimpanzees, bonobos, gorillas, orangutangs, baboons, macaques, vervets, marmosets and guinea pigs.

ResultsIn human data sets the methylation pattern of nc886 locus followed the expected bimodal distribution, indicative of polymorphic imprinting. In great apes, we identified a unimodal DNA methylation pattern with 50% methylation level in all individuals and in all subspecies. In Old World monkeys, the between individual variation was greater and methylation on average was close to 60%. In guinea pigs the region around the nc886 homologue was non-methylated. Results obtained from the sequence comparison of the CTCF binding sites flanking the nc886 gene support the results on the DNA methylation data.

ConclusionsOur results indicate that unlike in humans, nc886 is not a polymorphically imprinted metastable epiallele in non-human primates or in guinea pigs, thus implying that animal models are not applicable for nc886 research. The obtained data suggests that the nc886 region may be classically imprinted in great apes, and potentially also in Old World monkeys, but not in guinea pigs. The multi-level phenotypic impact of synonymous substitutions: heterologous gene expression in human cells. Redundancy in the genetic code allows for differences in transcription and/or translation efficiency between sequences carrying synonymous polymorphisms, potentially leading to phenotypic changes. It is commonly admitted that the evolution codon usage bias ("CUB", the over-representation of certain codons in a genome, a gene or in positions along a gene) are driven by a combination of neutral and selective processes, but their relative contribution is a matter of debate, especially in mammals. Particularly, integrative studies quantifying the phenotypic impact of CUB at different molecular and cellular levels are lacking. Here we report a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells.

Six synonymous versions of the shble antibiotic resistance gene were generated, fused to a fluorescent reporter, and independently expressed in HEK293 cells. Multiscale phenotype was assessed by means of: i) mRNA-to-DNA and protein-to-mRNA ratios for each shble version; ii) cellular fluorescence, using flow cytometry, as a proxy for single-cell level expression; and iii) real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cell fitness.

We show that differences in CUB strongly impact the molecular and the cellular phenotype: i) they result in large differences in mRNA and in protein levels, as well in mRNA-to-protein ratio; ii) they introduce splicing events not predicted by current algorithms; iii) they lead to reproducible phenotypic heterogeneity; iv) they lead to a trade-off between the benefit of antibiotic resistance and the burden of heterologous expression.

We interpret that CUB modulate mRNA availability and suitability for translation in human cells, leading to differences in protein levels and eventually eliciting phenotypic differences.

AUTHOR SUMMARYThe genetic code is redundant, with several codons encoding for the same amino acid. These synonymous codons are not used with equal frequencies. Instead, codon usage bias (CUB) varies between species, genes, and even positions along a gene. At each of these levels, CUB are shaped by the overall balance between mutational biases and selection forces. To shed light on the molecular mechanisms underlying molecular and organism phenotypes, integrative studies quantifying the phenotypic impact of CUB at different levels of biological integration are necessary. Here, we monitored the multiscale changes induced by six synonymous versions of an antibiotic resistance gene independently expressed in a human cell line. We show that: 1. both mRNA levels, protein levels, and their ratios are affected by CUB; 2. potential effects on mRNA (splicing and availability for translation) seem to constitute the main level of action; 3. cell fitness is severely impacted by a trade-off between the burden of heterologous expression and the benefits of antibiotic resistance. This integrative study provides new insights on translation regulation and the associated phenotypic impact in human cells, associated to CU. The "LINC" in between Delta40p53-miRNA axis in the regulation of cellular processes Recently, we showed that {Delta}40p53, the translational isoform of p53, can inhibit cell growth independently of p53 by regulating microRNAs. Here, we explored the role of {Delta}40p53 in regulating the lncRNA-miRNA axis. Based on early research from our laboratory, we selected LINC00176 for further exploration. LINC00176 levels were affected by the overexpression and knockdown of {Delta}40p53. Further, under DNA damage, ER stress, and glucose deprivation, LINC00176 was upregulated in HCT116 p53-/- cells (harboring only {Delta}40p53) compared to HCT116 p53+/+ cells. Additionally, ChIP and RNA stability assays revealed that {Delta}40p53 transactivates LINC00176 transcriptionally and stabilizes it post-transcriptionally. We ectopically overexpressed and knocked down LINC00176 in HCT116 p53-/- cells, which affected proliferation, cell viability, and the expression of epithelial markers. Finally, RNA pulldown revealed that LINC00176 sequesters several putative miRNA targets. These results provide important insights into the pivotal role of {Delta}40p53 in regulating the lncRNA-miRNA axis.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/489891v2_ufig1.gif" ALT="Figure 1">
View larger version (31K):
org.highwire.dtl.DTLVardef@1035f85org.highwire.dtl.DTLVardef@74185borg.highwire.dtl.DTLVardef@1eb3704org.highwire.dtl.DTLVardef@168ebab_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Model depicting the regulation of {Delta}40p53-LINC00176-miRNA axis

(A) In absence of {Delta}40p53, LINC00176 levels do not increase which leads to increased pool of free miRNAs which probably binds to several mRNA targets preventing their function, ultimately resulting in increased cell proliferation. (B) However, when {Delta}40p53 is present it upregulates LINC00176 levels both at the level of transcription and post-transcription, which sequesters oncogenic miRNAs preventing it to bind to their respective mRNA targets, which ultimately results in decreased cell proliferation.

C_FIG Robust and annotation-free analysis of alternative splicing across diverse cell types in mice Although alternative splicing is a fundamental and pervasive aspect of gene expression in higher eukaryotes, it is often omitted from single-cell studies due to quantification challenges inherent to commonly used short-read sequencing technologies. Here, we undertake the analysis of alternative splicing across numerous diverse murine cell types from two large-scale single-cell datasets--the Tabula Muris and BRAIN Initiative Cell Census Network--while accounting for understudied technical artifacts and unannotated isoforms. We find strong and general cell-type-specific alternative splicing, complementary to total gene expression but of similar discriminatory value, and identify a large volume of novel isoforms. We specifically highlight splicing variation across different cell types in primary motor cortex neurons, bone marrow B cells, and various epithelial cells; and show that the implicated transcripts include many genes which do not display total expression differences. To elucidate the regulation of alternative splicing, we build a custom predictive model based on splicing factor activity, recovering several known interactions while generating new hypotheses, including potential regulatory roles for novel alternative splicing events in critical genes including Khdrbs3 and Rbfox1. We make our results available using public interactive browsers to spur further exploration by the community. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI Heterologous vaccination regimens with self-amplifying RNA and Adenoviral COVID vaccines induce robust immune responses in mice Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two dose heterologous vaccination regimens than single dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies. Including imprecisely georeferenced specimens improves accuracy of species distribution models and estimates of niche breadth: Don't let the perfect be the enemy of the good Museum and herbarium specimen records are frequently used to assess species conservation status and responses to climate change. Typically, records of occurrence with imprecise geolocality information are discarded because they cannot be matched confidently to environmental conditions, and are thus expected to increase uncertainty in downstream analyses. However, using only those records that can be precisely georeferenced risks undersampling of species environmental and geographic distributions. Using simulated and real species, we compared the effect of discarding versus retaining imprecise records on the accuracy of ecological niche models (ENMs), and estimates of niche breadth and extent of occurrence. Imprecise records were assigned to locations or climate values using a conservative approach. For simulated species, including imprecisely-georeferenced records alongside precisely-georeferenced records improved the accuracy of ENMs projected to the present and the future. Improvements were especially notable for species with <~20 precise occurrences, but accuracy continued to improve and eventually matched the accuracy of ENMs using all records with no georeferencing error. Using only precise records underestimated loss in suitable habitat and overestimated the amount of suitable habitat in both the present and future. Including imprecise records also improved univariate and multivariate estimates of niche breadth, and of species extent of occurrence. An analysis of 44 species in the genus Asclepias (milkweeds) revealed similarly large differences between cases using only precise records and those using precise plus imprecise records. Although the inclusion of imprecisely-georeferenced occurrence records may not always be appropriate, careful consideration must be made regarding the trade-offs between fidelity of the match between occurrences and the environment, versus sample size and how well precisely-geolocated records sample species environmental and geographic distributions. The preponderance of imprecisely georeferences specimens in museums and herbaria could be gainfully employed to address known shortfalls in sampling of species distributions and climatic niche tolerances. Cell Intrinsic Control of Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells by pH Many biological processes are controlled by cell death and proliferation. The present study found that intracellular pH positively correlated with proliferation but negatively with apoptosis. Acid treatments lowered intracellular pH and preferentially depleted highly proliferating lymphocytes, whereas alkaline treatments increased intracellular pH and enhanced lymphocyte proliferation. Likewise, acid treatments induced apoptosis and alkaline treatments maintained proliferation of tumor cells. These findings support a unified theory of regulation of cell death and proliferation where an intracellular pH balance controls both events, and the mitochondria act as the pH-stats of the cell. Accordingly, the Warburg effect is explained as necessary for proliferating cells to have a high intracellular pH environment to accelerate proliferation and avoid low pH-induced death. Circular RNA's competing endogenous gene pair as motif in serous ovarian cancer The non-coding RNA (ncRNA) regulation apprears to be associated to the diagnosis and targeted therapy of complex diseases. Motifs of non-coding RNAs and genes in the competing endogenous RNA (ceRNA) network would probably contribute to the accurate prediction of serous ovarian carcinoma (SOC). Hence, we profiled a whole transcriptome of eight human SOCs and eight controls and constructed a ceRNA network including mRNAs, long ncRNAs, and circular RNAs (circRNAs). We identified the mRNA-ncRNA-mRNA motifs in the ceRNA network named the non-coding RNAs competing endogenous gene pairs (ceGPs), through the denoised individualized pair analysis of gene expression (deiPAGE) proposed in this study. 18 cricRNAs ceGPs (cceGPs) were identified from multiple cohorts and were fused as an indicator (SOC index) for SOC discrimination, which carried a high predictive capacity in independent cohorts. It was found that the index was negatively correlated with the CD8+/CD4+ ratio in tumour-infiltration, reflecting the migration and growth of tumour cells in ovarian cancer progression. Frog nest foams exhibit pharmaceutical foam-like properties Foams have frequently been used as systems for the delivery of cosmetic and therapeutic molecules; however, there is high variability in the foamability and long-term stability of synthetic foams. The development of pharmaceutical foams that exhibit desirable foaming properties, delivering appropriate amounts of the active pharmaceutical ingredient (API) and that have excellent biocompatibility is of great interest. The production of stable foams is rare in the natural world; however, certain species of frogs have adopted foam production as a means of providing a protective environment for their eggs and larvae from a predators and parasites, to prevent desiccation, to control gaseous exchange, temperature extremes, and to reduce UV damage. These foams show great stability (up to 10 days in tropical environments) and are highly biocompatible due to the sensitive nature of amphibian skin. This work demonstrates for the first time, that nests of the Tungara frog (Engystomops pustulosus) is stable ex situ with useful physiochemical and biocompatible properties and is capable of encapsulating a range of compounds, including antibiotics. These protein foam mixtures share some properties with pharmaceutical foams and may find utility in a range of pharmaceutical applications such as a topical drug delivery systems (DDS). 5-HT2C agonists and antagonists block different components of behavioral responses to potential, distal, and proximal threat in zebrafish Serotonin (5-HT) receptors have been implicated in responses to aversive stimuli in mammals and fish, but its precise role is still unknown. Moreover, since at least seven families of 5-HT receptors exist in vertebrates, the role of specific receptors is still debated. Aversive stimuli can be classified as indicators of proximal, distal, or potential threat, initiating responses that are appropriate for each of these threat levels. Responses to potential threat usually involve cautious exploration and increased alertness, while responses to distal and proximal threat involve a fight-flight-freeze reaction. We exposed adult zebrafish to a conspecific alarm substance (CAS) and observed behavior during (distal threat) and after (potential threat) exposure, and treated with the 5-HT2C receptor agonists MK-212 or WAY-161503 or with the antagonist RS-102221. The agonists blocked CAS-elicited defensive behavior (distal threat), but not post-exposure increases in defensive behavior (potential threat), suggesting inhibition of responses to distal threat. MK-212 blocked changes in freezing elicited by acute restraint stress, a model of proximal threat, while RS-102221 blocked changes in geotaxis elicited this stressor. We also found that RS-102221, a 5-HT2C receptor antagonist, produced small effect on behavior during and after exposure to CAS.

Preprint: https://www.biorxiv.org/content/10.1101/2020.10.04.324202; Data and scripts: https://github.com/lanec-unifesspa/5-HT-CAS/tree/master/data/5HT2C Colicin E1 opens its hinge to plug TolC The double membrane architecture of Gram-negative bacteria forms a barrier that is effectively impermeable to extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, Escherichia coli that hijacks the outer membrane proteins TolC and BtuB to enter the cell. Here we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryoEM, the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore--an architectural rearrangement from colicin E1s unbound conformation. This binding is stable in live E. coli cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plugs the channel, inhibiting its native efflux function as an antibiotic efflux pump and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other outer membrane protein functions. Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small biaryl Fragments toward Discovery of biased D1 agonists Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands. Sarcoplasmic reticulum calcium handling in young DBA/2J vs C57BL/10 mdx models of Duchenne muscular dystrophy The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice. Substrate traits shape the structure of microbial community engaged in metabolic division of labor Metabolic division of labor (MDOL) is widespread in nature, whereby a complex metabolic pathway is shared between different strains within a community for mutual benefit. However, little is known about how the mutual interactions in the microbial community engaged in MDOL are regulated. We hypothesized that when degradation of an organic compound is carried out via MDOL, the substrate traits (i.e., concentration and its toxicity) modulate the benefit allocation between the two microbial populations, thus affecting the structure of this community. We tested this hypothesis by combining mathematical modelling with experiments using engineered synthetic microbial consortia. Numerous modelling analyses suggested that the proportion of the population executing the first metabolic step can be simply estimated by Monod-like formulas governed by substrate traits. The model and the proposed formula quantitatively predicted the structure of our synthetic consortia composed of two strains degrading salicylate through MDOL. Individual-based modelling and colony pattern formation assays further indicated that our rule is also applicable to estimating community structure in spatially structured environments. Our results demonstrate that the structure of the microbial communities can be quantitatively predicted from simple environmental factors, such as substrate concentration and its toxicity, which provides novel perspectives on understanding the assembly of natural communities, as well as insights into how to manage artificial microbial systems. Shedding Light on the Circadian Clock of the Threespine Stickleback The circadian clock is an internal timekeeping system shared by most organisms, and knowledge about its functional importance and evolution in natural environments is still needed. Here, we investigated the circadian clock of wild-caught threespine sticklebacks (Gasterosteus aculeatus) at the behavioural and molecular levels. While their behaviour, ecology, and evolution are well studied, information on their circadian rhythms are scarce. We quantified the daily locomotor activity rhythm under a light-dark cycle (LD) and under constant darkness (DD). Under LD, all fish exhibited significant daily rhythmicity, while under DD, only 18% of individuals remained rhythmic. This interindividual variation suggests that the circadian clock controls activity only in certain individuals. Moreover, under LD, some fish were almost exclusively nocturnal, while others were active around the clock. Furthermore, the most nocturnal fish were also the least active. These results suggest that light masks activity (i.e. suppresses activity without entraining the internal clock) more strongly in some individuals than others. Finally, we quantified the expression of five clock genes in the brain of sticklebacks under DD using qPCR. We did not detect circadian rhythmicity, which could either indicate that the clock molecular oscillator is highly light-dependent, or that there was an oscillation but that we were unable to detect it. Overall, our study suggests that a strong circadian control on behavioural rhythms may not necessarily be advantageous in a natural population of sticklebacks and that the daily phase of activity varies greatly between individuals because of a differential masking effect of light.

Summary statementWe found that in wild-caught threespine sticklebacks, the circadian clock does not control locomotor activity in most, but not all, individuals. Sticklebacks are mostly nocturnal, although interindividual variation exists.