Holobiont Evolution: Population Theory for the Hologenome This article develops mathematical theory for the population dynamics of microbiomes with their hosts and theory for microbiome evolution caused by holobiont selection.

A holobiont is the union of a host with its microbiome. Holobiont selection is natural selection on the combined host and microbiome as a unit. The hologenome is the union of genes in the microbiome with genes in the host nucleus. The deep integration within many holobionts of microbiomes with their hosts extends to development, metabolism, physiology and behavior. The kind of eco-evolutionary processes producing this integration is unclear.

For a microbe to join a hosts microbiome, its population-dynamic parameters must sufficiently align with the hosts population-dynamic parameters. Accordingly, a microbe might be excluded from the microbiome, might enter the microbiome in a way leading to its subsequent gradual shedding from the holobiont, might join the microbiome and coexist with the host, or might, for a deleterious microbe, enter the microbiome culminating in the extinction of both it and the host.

A horizontally transmitted microbiome constitutes a genetic system that features "collective inheritance" capable of underwriting Darwinian descent with modification. The microbial source pool in the environment is the counterpart for the microbiome of the gamete pool for the nuclear genome. Poisson sampling of the microbial source pool is the counterpart of binomial sampling of the gamete pool. However, holobiont selection on the microbiome does not lead to a counterpart of the Hardy-Weinberg Law, a concept of selective neutrality, or directional selection that produces fixation of the microbial gene with the highest fitness. The effectiveness of holobiont selection on microbial genes is limited compared to the effectiveness of natural selection on nuclear genes. Nonetheless, holobiont selection can affect the composition of the microbiome by permitting the coexistence of a competitively inferior microbial strain with a superior strain in a colonization-extinction equilibrium and by permitting a polymorphism between altruistic and selfish microbial strains.

An altruistic microbe might strike an optimal balance between lowering within-host fitness to increase holobiont fitness. However, altruistic microbes are replaced by otherwise identical microbes that contribute nothing to holobiont fitness. This replacement can be reversed by hosts that discriminate against colonization by selfish microbes--likely brought about by hosts making antibodies against selfish microbes. This discrimination leads to a process of microbial species sorting orchestrated by the host immune system. Holobiont assembly with host-orchestrated species sorting (HOSS) followed by microbial competition, rather than coevolution or multi-level selection, is the likely cause of host-microbiome integration.
The Pogonomyrmex californicus social niche polymorphism is a polygenic trait involving a young supergene Social insects vary considerably in their social organization both between and within species. In the California harvester ant, Pogonomyrmex californicus (Buckley 1867), colonies are commonly founded and headed by a single queen (haplometrosis, primary monogyny). However, in some populations in California (USA), unrelated queens cooperate not only during founding (pleometrosis) but throughout the life of the colony (primary polygyny). The genetic architecture and evolutionary dynamics of this complex social niche polymorphism (haplometrosis vs pleometrosis) have remained unknown. Here, we provide a first analysis of its genomic basis and evolutionary history. We discover a recently evolved (< 200 k years), 8 Mb non-recombining region segregating with the observed social niche polymorphism, showing characteristics of a supergene comparable to those underlying social polymorphisms in other ant species. However, we also find remarkable differences to the other so far described social supergenes. Particularly, four additional genomic regions not in linkage with the supergene show signatures of a selective sweep in the pleometrotic population. Within these regions, we find for example genes crucial for epigenetic regulation via histone modification (chameau) and DNA methylation (dnmt1). Our results suggest that social morph in this species is a polygenic trait including an incipient supergene that evolved less than 200 000 years ago.
Discovery of an Antarctic ascidian-associated uncultivated Verrucomicrobia with antimelanoma palmerolide biosynthetic potential The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially-produced hybrid non-ribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74 Kbp candidate BGC encoding the multi-modular enzymatic machinery (hybrid Type I-trans-AT polyketide synthase-non-ribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide-gene targets and a single 16S rRNA gene variant (R=0.83 - 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a near-complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as Candidatus Synoicihabitans palmerolidicus. The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium.

ImportancePalmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply-branching genus, Candidatus Synoicihabitans palmerolidicus, in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. S. palmerolidicus 4.29 Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways enabling its ascidian-associated lifestyle. The palmerolide-producers genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Effects of substrate on shrimp growth, water quality and bacterial community in the biofloc system nursing Penaeus vannamei postlarvae This study aimed to investigate the effects of substrate on water quality, shrimp growth and bacterial community in the biofloc system with a salinity of 5{per thousand}. Two treatments, biofloc system with (sB) or without (nB) addition of elastic solid packing filler (nylon) as substrate, were set up. Penaeus vannamei postlarvae (PL, [~] stage 15) were stocked at a density of 4000 PL m-3 in each treatment with triplicates for a 28-days culture experiment, taking glucose as carbon source (C:N 15:1). Results showed that the survival rate (96.3{+/-}3.6%), FCR (0.76{+/-}0.06) and productivity (1.54{+/-}0.12 kg m-3) in sB treatment were significantly better than those in nB treatment (81.0{+/-}7.1%, 0.98{+/-}0.08 and 1.14{+/-}0.09 kg m-3, P<0.05). All water parameters were in the recommended ranges. Substrate showed significant effect on TAN, TSS, turbidity, biofloc volume, pH and carbonate alkalinity (P < 0.05). Actinobacteria (4.0-22.7%), Bacteroidetes (10.4-33.5%), Firmicutes (0.2-11.2%), Planctomycetes (4.0-14.9%) and Proteobacteria (29.4-59.0%) were the most dominant phyla for both treatments. However, the bacterial community in sB treatment showed to be significantly different from that in nB treatment (Jaccard distance 0.94{+/-}0.01, P=0.001). Substrate showed significant effects on Shannon, Heip, Pielou and Simpson index, as well as relative abundances of Actinobacteria, Bacteroidetes and Planctomycetes (P < 0.05). The results suggested that addition of substrate affected the shrimp growth, water quality and bacterial community in the biofloc system nursing P. vannamei PL with a 5{per thousand} salinity. Allopurinol Blocks The Formation And Progression Of Aortic Aneurysm In A Mouse Model Of Marfan Syndrome BackgroundThe pathogenesis and progression of aortic aneurysm in Marfan syndrome (MFS) involves dysregulated TGF-{beta} and nitric oxide signaling, altered hemodynamics, and biomechanical forces. Increasing evidence indicates that redox stress participates in MFS aortopathy, though its contribution is not well established. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mice aortic samples. Here we address the contribution of xanthine oxidoreductase (XOR), which is transcribed as dehydrogenase (XDH) and then post-translationally modified to oxidase (XO), catabolizing mainly purines into uric acid and ROS.

Methods and ResultsWe report that in MFS patients, XOR protein levels increased in aortic samples. In MFS mice (Fbn1C1041G/+), XOR mRNA transcripts augmented in the aorta of young but not older MFS mice. Moreover, the enzymatic activity of XO form increased with respect to XDH. The administration of the XOR inhibitor allopurinol (ALO) blocked the progression of aortic root aneurysm in MFS mice. ALO was also protective when administrated before the appearance of aneurysm. MFS-induced elastic fiber fragmentation, fibrotic remodeling, nuclear translocation of pNRF2, and increased 3-nitrotyrosine levels in the aortic tunica media as well as endothelial dysfunction were all prevented by allopurinol. The large overproduction of H2O2 in MFS aorta did not occur in allopurinol-treated mice whose uric acid plasma levels remained unaltered.

ConclusionsThis study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and supports a clinical trial for ALO in the pharmacological treatment of MFS aortopathy.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/464182v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@eab02aorg.highwire.dtl.DTLVardef@1d3b39eorg.highwire.dtl.DTLVardef@1b54d0forg.highwire.dtl.DTLVardef@1d61ad0_HPS_FORMAT_FIGEXP  M_FIG C_FIG The effect of recent habitat change on genetic diversity at putatively adaptive and neutral loci in Primula veris in semi-natural grasslands Recent habitat change in semi-natural grasslands due to a lack of management has been shown to affect the genetic diversity of grassland plants. However, it is unknown how a change in local environment affects genetic diversity at adaptive loci. We applied RADseq (restriction-site associated DNA sequencing) to extract > 3,000 SNPs across 568 individuals from 32 Estonian populations of Primula veris, a plant species common to semi-natural grasslands. We evaluated the effect of recent grassland overgrowth following management abandonment on the genetic diversity at putatively neutral and adaptive loci, which we distinguished by applying three methods, i.e., linear and categorical environmental association analyses, and an FST outlier test. For validation, we randomised the genotype to sample assignments. Effects of recent habitat change on genetic diversity differed between neutral and adaptive SNP sets. Genetic diversity assessed at putatively neutral loci was similar in open and overgrown habitats but showed a significant difference between these habitat types at putatively adaptive loci: overgrown (i.e. newly established) habitats exhibited higher genetic diversity at putatively adaptive loci than open (i.e. old) habitats, likely due to the exertion of novel selection pressures imposed by new habitat conditions. This increase in genetic diversity at putatively adaptive loci in the new environment points to currently ongoing selection processes where genetic adaptation to the old habitat is potentially lost through altered allele frequencies. Our study suggests that a recent change in local habitat conditions may not be reflected in neutral loci whereas putatively adaptive loci can inform about potential selection processes. Genome-wide association study of musical beat synchronization demonstrates high polygenicity Moving in synchrony to the beat is a fundamental component of musicality. Here, we conducted a genome-wide association study (GWAS) to identify common genetic variants associated with beat synchronization in 606,825 individuals. Beat synchronization exhibited a highly polygenic architecture, with sixty-nine loci reaching genome-wide significance (p<5x10-8) and SNP-based heritability (on the liability scale) of 13%-16%. Heritability was enriched for genes expressed in brain tissues, and for fetal and adult brain-specific gene regulatory elements, underscoring the role of central nervous system-expressed genes linked to the genetic basis of the trait. We performed validations of the self-report phenotype (through internet-based experiments) and of the GWAS (polygenic scores for beat synchronization were associated with patients algorithmically classified as musicians in medical records of a separate biobank). Genetic correlations with breathing function, motor function, processing speed, and chronotype suggest shared genetic architecture with beat synchronization and provide avenues for new phenotypic and genetic explorations. T606-phosphorylation deprives the function of Kaiso as a transcription factor and cancer repressor It is well known that Kaiso protein encoded by ZBTB33 gene is a transcription repressor and that Kaiso-P120ctn interaction increases the shift of Kaiso from the nucleus into the cytoplasm. However, the regulatory mechanisms of Kaiso compartmentalization are far from clear. Here, we reported that AKT1 could phosphorylate 606-threonine residue (T606) within the RSSTIP motif of Kaiso in the cytoplasm. The T606-phosphorylated Kaiso (pT606-Kaiso) could directly bind to 14-3-3 family proteins and the depletion of T606 phosphorylation by T606A mutation abolished most of the Kaiso-14-3-3 binding. In addition, the Kaiso-P120ctn interaction was essential for the pT606-Kaiso accumulation in the cytoplasm. Notably, enforced 14-3-3{sigma} (SFN) overexpression could increase the pT606-Kaiso accumulation in the cytoplasm and de-repress the transcription of Kaiso target gene CDH1. Decreased amounts of both pT606-Kaiso and CDH1 proteins were frequently observed in human gastric cancer tissues relative to paired normal controls. The mRNA levels of 14-3-3{sigma} and Kaiso target gene CDH1 were positively and significantly correlated with each other in bioinformatics analyses using publicly available RNA-seq datasets for human normal tissues (n=11688, r=0.60, p<0.001) in the GTEx project and for cancer cell lines (n=1156, r=0.41, p<0.001) in the CCLE project. Furthermore, Kaiso T606A mutant (unable to be phosphorylated) significantly increased the migration and invasion of cancer cells in vitro as well as boosted the growth of these cells in vivo. In conclusion, Kaiso could be phosphorylated by AKT1 at the T606 and the pT606-Kaiso accumulates in the cytoplasm through binding to 14-3-3/P120ctn that de-represses the expression of Kaiso target gene CDH1 in normal tissues. Decreased Kaiso phosphorylation may contribute to the development of gastrointestinal cancer. The status of Kaiso phosphorylation is a determinant factor for the role of Kaiso in the development of cancer. A theoretical model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers. The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (Neuropathy, Ataxia and Retinitis Pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-c-oxidase preventing yeast growth on oxidative substrates. The over-expression of the mitochondrial oxodicarboxylate carrier (Odc1p) is able to rescue the growth on oxidative substrate by increasing the substrate-level phosphorylation of ADP coupled to conversion of -ketoglutarate (AKG) into succinate with an increase in Complex IV activity. Previous studies showed that equivalent point mutations in ATP synthase behave similarly and can be rescued by Odc1p overexpression and/or uncoupler of OXPHOS from ATP synthesis. In order to better understand the mechanism of ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as respiratory substrate. We describe the different possible metabolite output and the ATP/O ratio values as a function of ATP synthase inhibition. INPP4B drives lysosome biogenesis to restrict leukemic stem cell differentiation and promote leukemogenesis Signaling pathways that control vital features of leukemic stem cells including multipotency, self-renewal, clonal expansion and quiescence remain unclear. Emerging studies illustrate critical roles for lysosomes in hematopoietic and leukemic stem cell fate. By investigating consequences of INPP4B alterations in AML, we have discovered its role in driving leukemic  stemness. We observed that INPP4B is highly expressed leukemic stem cell populations and Inpp4b-deficeint leukemias demonstrate increased disease latency, reduced leukemia initiating potential which is associated with a differentiated leukemic phenotype. Molecular analyses show that Inpp4b-deficient leukemias have compromised lysosomal gene expression, lysosomal content, and lysosomal activity. Our discovery of a novel pathway linking INPP4B, lysosomal biogenesis and leukemic stemness, provides a mechanism to explain the association of high INPP4B expression with poor AML prognosis, and highlights novel patient stratification strategies and LSC-specific leukemic therapies.

Key PointsOur findings highlight a novel pathway linking INPP4B, lysosomal function and leukemic stemness that explains the prognostic role of INPP4B in AML.
Our data reveal the utility of INPP4B as a biomarker of aggressive AML and provide a rationale to explore INPP4B and its associated function in lysosome biology as novel strategies to target LSC and AML Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. An enhanced inverted encoding model for neural reconstructions Here we present a more interpretable and flexible approach for reconstructing the contents of perception, attention, and memory from neuroimaging data. Our enhanced inverted encoding model (eIEM) incorporates methodological improvements including proper accounting of population-level tuning functions and a trial-by-trial prediction error-based metric where reconstruction quality is measured in meaningful units. Improved flexibility is further gained via eIEMs novel goodness-of-fit feature: for trial-by-trial reconstructions, goodness-of-fits are obtained independently (non-circularly) to prediction error and can be applied to any IEM procedure or decoding metric, resulting in improved reconstruction quality and brain-behavior correlations. We validate eIEM from methodological principles, simulated neuroimaging datasets, and three pre-existing fMRI datasets spanning perception, attention, and working memory. Notably, eIEM is easy to apply and broadly accessible - our Python package (https://pypi.org/project/inverted-encoding) implements eIEM in one line of code - and is easily modifiable to compare performance metrics and/or scale up to more complex models. Juvenile Gouldian Finches (Erythrura gouldiae) form sibling sub-groups during social integration The formation of social relationships in complex groups is critical in shaping patterns of social organization and behavioral development. In many birds, young individuals remain dependent on their parents for extended periods but must abruptly transition to navigating interactions in the wider group after independence. While lack of social relationships during this period is detrimental in the development of later social skills, little is known about the social relationships juveniles form after independence in many bird species. In this study we describe patterns of social interactions in juvenile Gouldian Finches (Erythrura gouldiae) during transitions from family groups into flocks of unfamiliar individuals. A group of 20 juveniles from 4 families were introduced into two flocks. After introductions juveniles showed a gradient of approach rates with most approaches directed towards siblings, followed by juvenile peers, adult females, and lastly adult males. Significant preferences for siblings resulted in the emergence of sibling subgroups within the larger social network. This active self-assortment of siblings suggests that sibling sub-groups are an important bridge linking social connections within the family and the wider group. Such findings suggests that sibling relationships have a critical role in the socialization after independence, as well as structuring the social organization of Gouldian finch flocks. Assessment of Sex Bias in Housekeeping Gene Expression in Adipose Tissue Through the Massive Analysis of Transcriptomics Data As the housekeeping genes (HKGs) generally involved in maintaining essential cell functions are typically assumed to exhibit constant expression levels across cell types, they are commonly employed as internal controls in gene expression studies. Nevertheless, multiple studies indicate that not all HKGs display stable expression across cells and tissues and under various healthy and diseased conditions, which can introduce systematic errors into experimental results. The selection and validation of HKGs as controls for each studied condition represent crucial steps in ensuring the validity of obtained results; however, up till now, sex has not been typically considered as a biological variable.

In this study, we evaluate the expression profiles of six classical HKGs (four metabolic: GAPDH, HPRT, PPIA, and UBC, and two ribosomal: 18S and RPL19) to determine expression stability in adipose tissues of Homo sapiens and Mus musculus and check sex bias and their overall suitability as internal controls. We also assess the stabile expression of genes included in distinct whole-transcriptome microarrays available from the Gene Expression Omnibus database (GEO) to identify sex-unbiased HKGs suitable for use as internal controls. We perform a sex-based analysis to describe any sexual dimorphisms in mRNA expression stability.

A novel computational strategy based on meta-analysis techniques proves that certain classical HKGs fail to function adequately as controls when analyzing human adipose tissue (HAT) considering sex as a variable. The extensively used 18S gene displays sex-based variability in adipose tissue, although PPIA and RPL19 do not, and hence, represent robust HKGs. We propose new sex-unbiased human and mouse HKGs (suHKG) derived from sex-specific expression profiles, such as RPS8 and UBB. All results generated during this study are readily available by accessing an open web resource (https://bioinfo.cipf.es/metafun-HKG) for consultation and reuse in further studies. Proton-driven alternating access in a spinster transporter, an emerging family of broad-specificity efflux pumps Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. We employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange. Furthermore, our resistance assays reveal substrate polyspecificity and HnSpns multidrug resistance (MDR) activity that underscore the previously unknown role of Spns proteins in MDR, beyond their activity in sphingolipid transport and signaling. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity. The analysis of single-cell proteomes has recently become a viable complement to transcriptomics and genomics studies. Proteins are the primary driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have lately attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lag. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE(R), reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1,500 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,600 proteins across 170 multiplexed single cells from two highly similar human cell types. This dedicated loss-less workflow allows distinguishing in vitro co-differentiated cell types of self-organizing cardiac organoids based on indicative markers across 150 single cells. In-depth characterization revealed enhanced cellular motility of cardiac endothelial cells and sarcomere organization in cardiomyocytes. Our versatile and automated sample preparation has not only proven to be easily adaptable but is also sufficiently sensitive to drive biological applications of single-cell proteomics. TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart, but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of cardiomyocytes in DMD patient cardiac tissues and in a mouse model of DMD we generated that manifests dilated cardiomyopathy. We also found that this telomere shortening is recapitulated in DMD cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Here we further characterize that DMD cardiomyocytes exhibit reduced cell size, nuclear size, and sarcomere density compared to healthy isogenic controls. The telomere-binding protein, TRF2, is a core component of the shelterin complex, which protects chromosome ends and is expressed at lower levels in DMD cardiomyocytes compared to controls. We investigated whether preservation of telomere length could be achieved with upregulation of TRF2. We found that TRF2 upregulation in DMD cardiomyocytes increased telomere lengths, cell size, nuclear size, sarcomere density, and cell survival. These data suggest TRF2 gene therapy has the potential to delay the onset of dilated cardiomyopathy. Bringing TrackMate into the era of machine-learning and deep-learning. TrackMate is an automated tracking software used to analyze bioimages and distributed as a Fiji plugin. Here we introduce a new version of TrackMate rewritten to improve performance and usability, and integrating several popular machine and deep learning algorithms to improve versatility. We illustrate how these new components can be used to efficiently track objects from brightfield and fluorescence microscopy images across a wide range of bio-imaging experiments. ART duration and immunometabolic state determine efficacy of DC-based treatment restoring functional HIV- specific CD8+ T cells in PLWH. Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated in detail. Here, we studied the association of ART duration with memory subsets, exhaustion and metabolic profiles of CD8+ T cells from PLWH and improvement of polyfunctional and effector HIV-1 specific responses after stimulation with Gag-adjuvant-primed DC. HIV-1-specific CD8+ T cell responses from a larger proportion PLWH on ART for more than 10 years (LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, CD8+ T cells from PLWH on ART for less than a decade (ST-ARTp) were less responsive to DC treatment and functional improvement was limited in this group. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolytic induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+PD1-cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines. P5A-ATPase controls the ER translocation of Wnt in neuronal migration Wnt family are conserved secretory proteins required for developmental patterning and tissue homeostasis. The mechanisms underlying intracellular maturation and intercellular signal transduction of Wnt proteins have been extensively studied. However, how Wnt is targeted to the endoplasmic reticulum (ER) for processing and secretion remains elusive. Here we report that CATP-8/P5A-ATPase directs neuronal migration non-cell autonomously in C. elegans by regulating EGL-20/Wnt biogenesis. CATP-8 functions as a translocase to translocate EGL-20/Wnt nascent polypeptide into the ER by interacting with the hydrophobic core region of EGL-20 signal sequence. Such regulation of Wnt biogenesis by P5A-ATPase is conserved in human cells. These findings reveal physiological roles of P5A-ATPase in neural development and identify Wnt proteins as direct substrates of P5A-ATPase for ER translocation. 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. Fixed or random? On the reliability of mixed-effects models for a small number of levels in grouping variables O_LIBiological data are often intrinsically hierarchical. Due to their ability to account for such dependencies, mixed-effects models have become a common analysis technique in ecology and evolution. While many questions around their theoretical foundations and practical applications are solved, one fundamental question is still highly debated: When facing a low number of levels should we model a grouping (blocking, clustering) variable as a random or fixed effect? In such situation, the variance of the random effect is imprecise, but whether this affects the statistical properties of the population effect is unclear.
C_LIO_LIHere, we analyzed the consequences of including a grouping variable as fixed or random effect in the correctly specified and other possible miss-specified models (too simple or too complex models) for data with small number of levels (2 - 8). For all these options, we calculated type I error rates and power. Moreover, we show how these statistical properties change with the study design.
C_LIO_LIWe found that the model choice does not influence the statistical properties of the population effect when the effect is the same at all levels However, if an ecological effect differs among levels, using a random slope and intercept model, and switching to a fixed-effect model only in case of a singular fit, avoids overconfidence in the results. Additionally, power and type I error are strongly influenced by the number of and difference between levels.
C_LIO_LIWe conclude that inferring the correct random effect structure is of high importance to get correct statistical properties. When in doubt, we recommend starting with the simpler model and using model diagnostics to identify missing components. When having identified the correct structure, we encourage to start with a mixed-effects model independent of the number of levels and switch to a fixed-effect model only in case of a singular fit. With these recommendations, we allow for more informative choices about study design and data analysis and thus make ecological inference with mixed-effects models more robust for small number of levels.
C_LI IRI-CCE: A Platform for Evaluating CRISPR-based Base Editing Tools and Its Components Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs. GCN5L1 promotes diastolic dysfunction by inhibiting cardiac pyruvate oxidation Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model and quantitative acetylproteomics in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits and diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.