Full Spectrum Flow Cytometry for High-Dimensional Immunophenotyping of Mouse Innate Lymphoid Cells This 25-parameter, 22-colour full spectrum flow cytometry panel was designed and optimised for the comprehensive enumeration and functional characterisation of innate lymphoid cell (ILC) subsets in mouse tissues (Table 1). The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ ILC3 and mature natural killer (NK) cell populations. Further characterisation of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimisation of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin, brefeldin A and pro-ILC activating cytokines.

O_TBL View this table:
org.highwire.dtl.DTLVardef@8df9d7org.highwire.dtl.DTLVardef@10dc5d4org.highwire.dtl.DTLVardef@136f44dorg.highwire.dtl.DTLVardef@e10165org.highwire.dtl.DTLVardef@22a0cd_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary Table

C_TABLECAPTION C_TBL
Surface-associated antigen induces B-cell permeabilization and lysosome exocytosis facilitating antigen uptake and presentation to T-cells B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.
A Physiometric Investigation of Inflammatory Composites: Comparison of A Priori Aggregates, Empirically-identified Factors, and Individual Proteins Most research testing the association between inflammation and health outcomes (e.g., heart disease, diabetes, depression) has focused on individual proteins; however, some studies have used summed composites of inflammatory markers without first investigating dimensionality. Using two different samples (MIDUS-2: N = 1,255 adults, MIDUS-R: N = 863 adults), this study investigates the dimensionality of eight inflammatory proteins (C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor- (TNF-), fibrinogen, E-selectin, and intercellular adhesion molecule (ICAM)-1) and compared the resulting factor structure to a) an "a priori"/tau-equivalent factor structure in which all inflammatory proteins equally load onto a single dimension (comparable to the summed composites) and b) proteins modeled individually (i.e., no latent variable) in terms of model fit, replicability, reliability, and their associations with health outcomes. An exploratory factor analysis indicated a two-factor structure (Factor 1: CRP and fibrinogen; Factor 2: IL-8 and IL-10) in MIDUS-2 and was replicated in MIDUS-R. Results did not clearly indicate whether the empirically-identified factor structure or the individual proteins modeled without a latent variable had superior model fit, but both strongly outperformed the "a priori"/tau-equivalent structure (which did not achieve acceptable model fit in any models). Modeling the empirically-identified factors and individual proteins (without a latent factor) as outcomes of medical diagnoses resulted in comparable conclusions. However, modeling individual proteins resulted in findings more robust to correction for multiple comparisons despite more conservative adjustments. Further, reliability for all latent variables was poor. These results indicate that modeling inflammation as a unidimensional construct equally associated with all available proteins does not fit the data well. Instead, individual inflammatory proteins or, potentially (if empirically supported and biologically-plausible) empirically-identified inflammatory factors should be used in accordance with theory. Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment. BackgroundMenthol, through its cooling and pleasant sensory effects, facilitates smoking and tobacco product initiation, resulting in the high popularity of mint/menthol-flavored E-cigarettes. More recently, E-cigarette vendors started marketing synthetic cooling agents as additives that impart a cooling effect but lack a characteristic minty odor. Knowledge about content of synthetic coolants in US-marketed E-cigarette products and associated health risks is limited.

MethodsE-liquid vendor sites were searched with the terms "koolada", "kool/cool", "ice", or WS-3/WS-23, denoting individual cooling agents, and relevant refill E-liquids were purchased. "Ice" flavor varieties of Puffbar, the most popular disposable E-cigarette brand, were compared with non-"Ice" varieties. E-liquids were characterized, and synthetic coolants quantified using GC/MS. Margin of exposure (MOE), a risk assessment parameter, was calculated to assess the risk associated with synthetic coolant exposure from E-cigarette use.

ResultsWS-3 was detected in 24/25 refill E-liquids analyzed. All Puffbar flavor varieties contained either WS-23 (13/14) or WS-3 (5/14), in both "Ice"- and non-"Ice" flavors. Modeling consumption of WS-3 from vaped E-liquids, resulted in MOEs below the safe margin of 100 for most daily use scenarios. MOEs for WS-23 were <100 for 10/13 Puffbar flavors in all use scenarios. Puffbar power specifications are identical to Juul devices.

ConclusionsSynthetic cooling agents (WS-3/WS-23) were present in US-marketed E-cigarettes, at levels that may result in consumer exposures exceeding safety thresholds set by regulatory agencies. Synthetic coolants are not only found in mint-or menthol-flavored products, but also in fruit- and candy-flavored products, including popular disposable E-cigarette products such as Puffbar.

ImplicationsSynthetic cooling agents are widely used in "kool/cool"- and "ice"-flavored E-liquids and in E-liquids without these labels, both as a potential replacement for menthol or to add cooling  notes to non-menthol flavors. These agents may be used to bypass current and future regulatory limits on menthol content in tobacco products, and not just E-cigarettes. Since synthetic cooling agents are odorless, they may not fall under the category of "characterizing flavor", potentially circumventing regulatory measures based on this concept. Regulators need to consider the additional health risks associated with exposure to synthetic cooling agents. Versatile Cell-Based Assay for Measuring DNA Alkylation Damage and its repair DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair. Construction of a new chromosome-scale, long-read reference genome assembly for the Syrian hamster, Mesocricetus auratus BackgroundThe Syrian hamster (Mesocricetus auratus) has been suggested as a useful mammalian model for a variety of diseases and infections, including infection with respiratory viruses such as SARS-CoV-2. The MesAur1.0 genome assembly was generated in 2013 using whole-genome shotgun sequencing with short-read sequence data. Current more advanced sequencing technologies and assembly methods now permit the generation of near-complete genome assemblies with higher quality and greater continuity.

FindingsHere, we report an improved assembly of the M. auratus genome (BCM_Maur_2.0) using Oxford Nanopore Technologies long-read sequencing to produce a chromosome-scale assembly. The total length of the new assembly is 2.46 Gbp, similar to the 2.50 Gbp length of a previous assembly of this genome, MesAur1.0. BCM_Maur_2.0 exhibits significantly improved continuity with a scaffold N50 that is 6.7 times greater than MesAur1.0. Furthermore, 21,616 protein coding genes and 10,459 noncoding genes are annotated in BCM_Maur_2.0 compared to 20,495 protein coding genes and 4,168 noncoding genes in MesAur1.0. This new assembly also improves the unresolved regions as measured by nucleotide ambiguities, where approximately 17.11% of bases in MesAur1.0 were unresolved compared to BCM_Maur_2.0 in which the number of unresolved bases is reduced to 3.00%.

ConclusionsAccess to a more complete reference genome with improved accuracy and continuity will facilitate more detailed, comprehensive, and meaningful research results for a wide variety of future studies using Syrian hamsters as models. A Multi-omics Method Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem Cell-derived Cardiomyocytes Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show immense promise for patient-specific disease modeling, cardiotoxicity screening, and regenerative therapy development. However, hPSC-CMs in culture have not recapitulated the structural or functional properties of adult CMs in vivo thus far. To gain global insight into hPSC-CM biology, we established a multi-omics method for analyzing the hPSC-CM metabolome and proteome from the same cell culture, creating multi-dimensional profiles of hPSC-CMs. Specifically, we developed a sequential extraction to capture metabolites and proteins from the same hPSC-CM monolayer cultures, and analyzed these extracts using high-resolution mass spectrometry (MS). Using this method, we annotated 205 metabolites/lipids and 4,008 proteins from 106 cells with high reproducibility. We further integrated the proteome and metabolome measurements to create network profiles of molecular phenotypes for hPSC-CMs. Out of 310 pathways identified using metabolomics and proteomics, 40 pathways were considered significantly overrepresented (FDR-corrected p [&le;] 0.05). Highly populated pathways included those involved in protein synthesis (ribosome, spliceosome), ATP generation (oxidative phosphorylation), and cardiac muscle contraction. This multi-omics method achieves deep coverage of metabolites and proteins, creating a multidimensional view of the hPSC-CM phenotype, which provides a strong technological foundation to advance the understanding of hPSC-CM biology. 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. 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. Hierarchical autoencoder-based integration improves performance in multi-omics cancer survival models through soft modality selection With decreasing costs of high-throughput sequencing, more and more datasets providing omics profiles of cancer patients become available. Thus, novel survival analysis approaches integrating these differently sized and heterogeneous molecular and clinical groups of variables start being developed. Due to the difficulty of the task of multi-omics data integration, the Cox Proportional-Hazards (PH) model using clinical data has remained one of the best-performing techniques, barely outperformed by models using molecular data modalities. There is therefore a need for methods that can successfully perform multi-omics integration in survival analysis and outperform the clinical Cox PH model. Moreover, while certain deep learning methods have been shown to provide state-of-the-art accuracy of cancer survival prediction, most of them show no benefit or even decay in performance when integrating a larger number of modalities, further motivating a need to investigate how modality-specific representations should be integrated when using neural networks for multi-omics integration. We benchmarked multiple integration techniques for a neural network architecture, revealing that hierarchical autoencoder-based integration of modality-specific representations outperformed other methods such as max-pooling and was comparable with state-of-the-art statistical approaches for multi-omics integration. Further, we showed that the hierarchical autoencoder-based integration of modality-specific representations achieved increased performance through a soft modality selection mechanism, focusing on the most informative modalities for each cancer. We thus framed multiomics integration as a partial group-wise feature selection problem, highlighting that only those models performed well that could adequately weight important modalities in the presence of the high noise imposed by less important modalities. Epidural Electrical Stimulation of the Cervical Dorsal Roots Restores Voluntary Upper Limb Control In Paralyzed Monkeys Recovering arm control is a top priority for people with paralysis. Unfortunately, the complexity of the neural mechanisms underlying arm control practically limited the effectiveness of neurotechnology approaches. Here, we exploited the neural function of surviving spinal circuits to restore voluntary arm and hand control in three monkeys with spinal cord injury using spinal cord stimulation. Our neural interface leverages the functional organization of the dorsal roots to convey artificial excitation via electrical stimulation to relevant spinal segments at appropriate movement phases. Stimulation bursts targeting specific spinal segments produced sustained arm movements enabling monkeys with arm paralysis to perform an unconstrained reach-and-grasp task. Stimulation specifically improved strength, task performances and movement quality. Electrophysiology suggested that residual descending inputs were necessary to produce coordinated movements. The efficacy and reliability of our approach hold realistic promises of clinical translation. From GWAS Variant to Function: a Study of ~148,000 Variants for Blood Cell Traits. Genome-wide association studies (GWAS) have identified hundreds of thousands of genetic variants associated with complex diseases and traits. However, most variants are noncoding and not clearly linked to genes, making it challenging to interpret these GWAS signals. We present a systematic variant-to-function study, prioritizing the most likely functional elements of the genome for experimental follow-up, for >148,000 variants identified for hematological traits. Specifically, we developed VAMPIRE: Variant Annotation Method Pointing to Interesting Regulatory Effects, an interactive web application implemented in R Shiny (http://shiny.bios.unc.edu/vampire/). This tool efficiently integrates and displays information from multiple complementary sources, including epigenomic signatures from blood cell relevant tissues or cells, functional and conservation summary scores, variant impact on protein and gene expression, chromatin conformation information, as well as publicly available GWAS and phenome-wide association study (PheWAS) results. Leveraging data generated from independently performed functional validation experiments, we demonstrate that our prioritized variants, genes, or variant-gene links are significantly more likely to be experimentally validated. This study not only has important implications for systematic and efficient revelation of functional mechanisms underlying GWAS variants for hematological traits, but also provides a prototype that can be adapted to many other complex traits, paving the path for efficient variant to function (V2F) analyses. 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. Text has been revised, image definition has been revised. Maize is a staple food all over the world. Models for climate change suggest that, in the future, cloud formation might be reduced in the tropics increasing the exposure to Ultraviolet-B (UV-B) radiation, a DNA-damaging agent. UV-B (290 to 320 nm) has been shown to affect yield in maize. In this project we have determined the differences in DNA repair efficiencies between U.S. inbred lines B73 and Mo17, and Central American purple landraces from Guatemala and Costa Rica. Our results from single cell electrophoresis experiments (Comet Assay) suggest that the landrace Pujagua Santa Cruz (P1, Costa Rican) was resistant to damage caused by the radiomimetic agent zeocin (24h/100 micrograms per mL), while landrace Pujagua La Cruz (P2, Costa Rican) was able to repair DNA damage after one hour. On the other hand, line Mo17 (Missouri, USA) was unable to repair the damage, while B73 (Iowa, USA) and the landraces Jocopilas (Guatemalan), Orotina Congo (Costa Rican) and Talamanca (Costa Rican) were partially able to repair DNA damage. High Resolution Melting (HRM) curve analysis of putative homologous DNA repair gene ZeaATM1 showed that both P1 and P2 had differences in the melting temperatures for this gene compared to B73 and Mo17, while P1 showed additional differences in ZeaSOG1, ZeaRAD51 and ZeaBRCA1, suggesting that in this landrace the presence of polymorphisms may be common among key genes for this pathway. Taken together our results suggest that key adaptive differences in DNA repair efficiencies exist between inbred lines and landraces of maize and that some Central America landraces could be used as a valuable pool of alleles for plant breeding aiming to increase tolerance to radiation. Mouse lemur, a new primate model to investigate cardiac pacemaker activity. BackgroundGray mouse lemurs (Microcebus murinus) are one of the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since Microcebus murinus replicates similar senescence processes of humans, it constitutes a useful model also for studying cardiovascular dysfunctions. However, its cardiac physiology is unknown. Thus, we investigated sinoatrial node (SAN) pacemaker activity of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.

Methods and ResultsWe recorded the heart automaticity in freely moving mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of electrical impulses in the SAN tissue. Conversely, their ventricular depolarization is more similar to large mammals. Mouse lemur SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (ICa,L) and T-type (ICa,T) calcium currents. Moreover, mouse lemur SAN myocytes display spontaneous ryanodine receptor (RyR)-dependent Ca2+ transients. Using muscle-derived stem cells (MDSCs) from the Microcebus murinus skeletal muscle, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).

ConclusionsIn conclusion, our characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans, and for pharmacologic testing of molecules that could assist in modulating age-related dysfunctions of automaticity. Chromosome breaks in breast cancers occur near herpes tumor virus sequences and explain why cancer drugs stop working One explanation for why breast cancer has a relentless tendency to come back after treatment is that breast cancer phenomes are fluid, so it is difficult for therapy to target new phenotypes that arise. In breast cancer, viral interactions cause chromosome breakage and fusion, producing anaphase bridges containing two centromeres. Cell division then leads to more chromosome breaks, more mutations, and new cancer phenotypes. Analyses of public data from about 2100 breast cancers implicate Epstein-Barr virus (EBV or Human Herpes Virus 4) in the cycles that break chromosomes and produce anaphase bridges. EBV generates breakage cycles with highly fragmented chromosomes (chromothripsis) as an inherent feature. These episodes continually produce new cancer driver mutations. DNA at chromothripsis boundaries in breast cancers consistently matches EBV tumor variants. Breakpoints are often near known EBV binding sites, and breast cancer shares an EBV methylation signature with other known EBV cancers. Breast cancer breakpoints on all chromosomes cluster around the same positions as in cancers definitively associated with EBV infection (nasopharyngeal cancer and Burkitts lymphoma). The status of EBV in the microbiome and the presence of anaphase bridges are phenotypic markers of breast cancer that are not explicitly targeted by chemotherapy. The breast cancer phenome continually supplies new cancer driver genes that foster breast cancer recurrence.

SummaryBreaks in breast cancer chromosomes and in fragments of anaphase bridges occur mainly near regions that resemble Epstein-Barr virus (EBV) tumor variants

Extensive breaks are an inherent outcome of anaphase bridges and the reassembly of highly fragmented chromosomes continually generates new cancer driver mutations and new cancer phenotypes.

Treatment does not explicitly target the underlying breakage and reassembly cycles so breast cancer often comes back.

The breast cancer phenome is not fixed and can continuously generate new phenotypes

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/467751v4_ufig1.gif" ALT="Figure 1">
View larger version (38K):
org.highwire.dtl.DTLVardef@6537cforg.highwire.dtl.DTLVardef@132fa50org.highwire.dtl.DTLVardef@1cfcb04org.highwire.dtl.DTLVardef@11979aa_HPS_FORMAT_FIGEXP  M_FIG C_FIG Differential regulation of alternative promoters emerges from unified kinetics of enhancer-promoter interaction Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. Here, we apply single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, we find that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, we show that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the kinetic mechanisms of complex eukaryotic gene regulation. Selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes. Naming of human diseases on the wrong side of history BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Robust and efficient online auditory psychophysics Most human auditory psychophysics research has historically been conducted in carefully controlled environments with calibrated audio-equipment, and over potentially hours of repetitive testing with expert listeners. Here, we operationally define such conditions as having high  auditory hygiene. Hereof, conducting auditory psychophysical paradigms online presents a serious challenge, in that results may hinge on absolute sound presentation level, reliably estimated perceptual thresholds, and sustained motivation and attention. We introduce a set of procedures that address these challenges and facilitate auditory hygiene for online auditory psychophysics. First, we establish a simple means of setting sound presentation levels. Across a set of four level-setting conditions conducted in person, we demonstrate the stability and robustness of this amplitude setting procedure in open air and controlled settings. Second, we test participants tone-in-noise thresholds using widely adopted online experiment platforms and demonstrate that reliable threshold estimates can be derived online in approximately one minute of testing. Third, using these amplitude and threshold setting procedures to establish participant-specific stimulus conditions, we show that an online implementation of the classic probe-signal paradigm can be used to demonstrate frequency-selective attention on an individual participant basis, using a third of the trials used in recent in-lab experiments. Finally, we show how threshold and attentional measures relate to well-validated assays of online participants in-task motivation, fatigue, and confidence. This demonstrates the promise of online auditory psychophysics for asking new auditory neuroscience questions quickly, efficiently, and with more diverse samples. Code for the tests is publicly available through Pavlovia and Gorilla. First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI Development of a Novel PROTAC using the Nucleic Acid Aptamer as a Targeting Ligand for Tumor Selective Degradation of Nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. Hierarchical clustering optimizes the tradeoff between compositionality and expressivity of task structures for flexible reinforcement learning A hallmark of human intelligence, but challenging for reinforcement learning (RL) agents, is the ability to compositionally generalise, that is, to recompose familiar knowledge components in novel ways to solve new problems. For instance, when navigating in a city, one needs to know the location of the destination and how to operate a vehicle to get there, whether it be pedalling a bike or operating a car. In RL, these correspond to the reward function and transition function, respectively. To compositionally generalize, these two components need to be transferable independently of each other: multiple modes of transport can reach the same goal, and any given mode can be used to reach multiple destinations. Yet there are also instances where it can be helpful to learn and transfer entire structures, jointly representing goals and transitions, particularly whenever these recur in natural tasks (e.g., given a suggestion to get ice cream, one might prefer to bike, even in new towns). Prior theoretical work has explored how, in model-based RL, agents can learn and generalize task components (transition and reward functions). But a satisfactory account for how a single agent can simultaneously satisfy the two competing demands is still lacking. Here, we propose a hierarchical RL agent that learns and transfers individual task components as well as entire structures (particular compositions of components) by inferring both through a non-parametric Bayesian model of the task. It maintains a factorised representation of task components through a hierarchical Dirichlet process, but it also represents different possible covariances between these components through a standard Dirichlet process. We validate our approach on a variety of navigation tasks covering a wide range of statistical correlations between task components and show that it can also improve generalisation and transfer in more complex, hierarchical tasks with goal/subgoal structures. Finally, we end with a discussion of our work including how this clustering algorithm could conceivably be implemented by cortico-striatal gating circuits in the brain. Comparative infection and pathogenesis of SARS-CoV-2 Omicron and Delta variants in aged and young Syrian hamsters Coronavirus disease 2019 continues to batter the world with the unceasing introduction of new variants of the causative virus, SARS-CoV-2. In order to understand differences in disease caused by variants of concern and to develop variant-specific vaccines, suitable small animal models are required that mimic disease progression in humans at various stages of life. In this study, we compared the dynamics of infection with two SARS-CoV-2 variants of concern (Delta and Omicron) in aged (>1 year 3 months old) and young (<5 weeks old) Syrian hamsters (Mesocricetus auratus). We show that no weight loss occurred in Omicron infected groups regardless of age, while infection with the Delta variant caused weight loss of up to 10% by day 7 post-infection with slower and incomplete recovery in the aged group. Omicron replicated to similar levels as Delta in the lungs, trachea and nasal turbinates, with no significant differences in the tissue viral loads of aged versus young animals for either variant. In contrast to rare necrosis observed in Omicron-infected animals regardless of age, severe necrosis was observed in the olfactory epithelium in Delta-infected animals. Omicron infection also resulted in mild pulmonary disease in both young and aged animals compared to the moderate acute necrotizing bronchointerstitial pneumonia seen in Delta-infected animals. These results suggest that Omicron infection results in an attenuated clinical disease outlook in Syrian hamsters compared to infection with the Delta variant irrespective of age. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement.