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.
Limitations of composability of cis-regulatory elements in messenger RNA Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoters and terminators, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cisregulatory motifs within terminator regions display similar context dependence, using transcriptomewide datasets of mRNA decay. To test the extent of composability, we construct reporter genes consisting of combinations of motifs within various terminator contexts, paired with different promoters. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. This emphasises the need for improved motif inference that includes both local and global context effects, which in turn could aid in the accurate use of CREs for the engineering of synthetic genetic constructs.
Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. First detection of herpesvirus and prevalence of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9 CRISPR-mediated genome editing has become a powerful tool for genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve the HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP Kinase gene to generate a BleoR-MKK2 fusion protein. Based on exogenous BleoR expression, the HDR-mediated knock-in efficiency was up to [~]40-fold greater when using a XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared to no HDR enhancement or NHEJ silencing. Furthermore, this corporation of HDR enhancer overexpression and NHEJ repression also resulted in 7-fold fewer CRISPR-induced Insertions and Deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammalians for improving CRISPR-mediated gene knock-in efficiency. Potential geographic distribution and ecological niche of New World dobsonflies (Megaloptera, Corydalidae): the case of the Neartic-Neotropical transition zone The dobsonflies (Megaloptera, Corydalidae) are an interesting group of insects, but among the New World dobsonflies, it is not known the effect of the Neartic-Neotropical transition zone on their biogeographic distribution. Here we studied at the species level, the records on the geographic range of the dobsonflies that occurred in and near the transition zone. We presented potential geographic distributions based on ecological niche models for several species of dobsonflies. Results suggested that the geographic range of dobsonflies in the transition zone is associated to mountainous formations and that most species favor warm climates with higher precipitation rates. Climate types tend to be important for species that show narrow geographic ranges, but precipitation tends to be the most important variable to explain species dispersion. Overall, our models support the dispersion of dobsonflies from the Neotropics to North America and explain the two endemic species in Mexico as the result of the formation of the transition zone. 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. OrganoID: a versatile deep learning platform for tracking and analysis of single-organoid dynamics Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses, however these metrics are difficult and labor-intensive to obtain for high-throughput image datasets. Here, we present OrganoID, a robust image analysis platform that automatically recognizes, labels, and tracks single organoids, pixel-by-pixel, in brightfield and phase-contrast microscopy experiments. The platform was trained on images of pancreatic cancer organoids and validated on separate images of pancreatic, lung, colon, and adenoid cystic carcinoma organoids, which showed excellent agreement with manual measurements of organoid count (96%) and size (95%) without any parameter adjustments. Single-organoid tracking accuracy remained above 89% over a four-day time-lapse microscopy study. Automated single-organoid morphology analysis of a chemotherapy dose-response experiment identified decreased organoid circularity as an important morphological feature reflecting drug response. OrganoID enables straightforward, detailed, and accurate image analysis to accelerate the use of organoids in high-throughput, data-intensive biomedical applications. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. The Tabula Sapiens: a multiple organ single cell transcriptomic atlas of humans Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual. Swine H1N1 influenza virus variants with enhanced polymerase activity and HA stability promote airborne transmission in ferrets Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor HA stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits.

IMPORTANCEDiverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both -2,6- and -2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern. Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. Identification of Antimalarial Compounds that Inhibit Apicomplexan AP2 Proteins in the Human Malaria Parasite Plasmodium falciparum Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified compounds that interact with this domain. Four compounds were found to compete for DNA binding with AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that are targeted against the ApiAP2 family of proteins. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics.

Author SummaryPlasmodium parasites are the causative agent of malaria, which resulted in over 600,000 deaths in 2021. Due to resistance arising for every antimalarial therapeutic deployed to date, new drug targets and druggable pathways must be explored. To address this concern, we used a molecular docking screen to predict competitors of DNA binding by the parasite specific family of Apicomplexan AP2 (ApiAP2) transcription factor proteins for testing in vitro and in vivo. We find that ApiAP2 competing compounds have antimalarial activity consistent with the disruption of gene regulation. This work will further our understanding of both the biological role and targetability of parasite transcriptional regulation. 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. Uncovering non-random binary patterns within sequences of intrinsically disordered proteins Sequence-ensemble relationships of intrinsically disordered proteins (IDPs) are governed by binary patterns such as the linear clustering or mixing of specific residues or residue types with respect to one another. To enable the discovery of potentially important, shared patterns across sequence families, we describe a computational method referred to as NARDINI for Non-random Arrangement of Residues in Disordered Regions Inferred using Numerical Intermixing. This work was partially motivated by the observation that parameters that are currently in use for describing different binary patterns are not interoperable across IDPs of different amino acid compositions and lengths. In NARDINI, we generate an ensemble of scrambled sequences to set up a composition-specific null model for the patterning parameters of interest. We then compute a series of pattern-specific z-scores to quantify how each pattern deviates from a null model for the IDP of interest. The z-scores help in identifying putative non-random linear sequence patterns within an IDP. We demonstrate the use of NARDINI derived z-scores by identifying sequence patterns in three well-studied IDP systems. We also demonstrate how NARDINI can be deployed to study archetypal IDPs across homologs and orthologs. Overall, NARDINI is likely to aid in designing novel IDPs with a view toward engineering new sequence-function relationships or uncovering cryptic ones. We further propose that the z-scores introduced here are likely to be useful for theoretical and computational descriptions of sequence-ensemble relationships across IDPs of different compositions and lengths. Using total RNA quality metrics for time since deposition estimates in degrading bloodstains Determining the time since deposition (TSD) of bloodstains would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. The physicochemical changes occurring to biomolecules as a bloodstain degrades can be used to approximate the TSD of bloodstains. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has previously targeted mRNA molecules. Whole bovine blood was stored in plastic microcentrifuge tubes at 21{degrees}C or 4{degrees}C and tested over different timepoints spanning one week. Nine RNA metrics were visually assessed and quantified using linear and mixed models; the RNA Integrity Number equivalent (RINe) and the DV200 demonstrated strong negative trends over time and statistical independence. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200 metrics. Importantly, this suggests that these standardized metrics can likely be directly compared between scenarios and individuals, with DV200 having an inflection point at [~]28 hrs. This study provides a novel approach for blood TSD estimates, producing metrics that are not affected by inter-individual variation and improving our understanding of the rapid degradation occurring in bloodstains.

HIGHLIGHTSO_LIAmplification-free analysis of total RNA in degrading bloodstains.
C_LIO_LIShort-term RNA degradation assessment using high-resolution size measurements.
C_LIO_LITotal RNA quality and quantity metrics were assessed across a one-week.
C_LIO_LITotal RNA quality metrics demonstrated the strongest timewise trends.
C_LIO_LIBiological replicates produced similar results for RNA quality metrics.
C_LI "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Vesalius: high-resolution in silico anatomization of Spatial Transcriptomic data using Image Analysis Characterization of tissue architecture promises to deliver insights into development, cell communication and disease. In silico spatial domain retrieval methods have been developed for spatial transcriptomics (ST) data assuming transcriptional similarity of neighboring barcodes. However, domain retrieval approaches with this assumption cannot work in complex tissues composed of multiple cell types. This task becomes especially challenging in cellular resolution ST methods. We developed Vesalius to decipher tissue anatomy from ST data by applying image processing technology. Vesalius uniquely detected territories composed of multiple cell types and successfully recovered tissue structures in high-resolution ST data including in mouse brain, embryo, liver, and colon. Utilizing this tissue architecture, Vesalius identified tissue morphology specific gene expression and regional specific gene expression changes for astrocytes, interneuron, oligodendrocytes, and entorhinal cells in the mouse brain. HSP90 modulates T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis. 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. Integrated analysis of cervical squamous cell carcinoma cohorts from three continents reveals conserved subtypes of prognostic significance. Human papillomavirus (HPV)-associated cervical cancer represents one of the leading causes of cancer death worldwide. Although low-middle income countries are disproportionately affected, our knowledge of the disease predominantly originates from populations in high-income countries. Using the largest multi-omic analysis of cervical squamous cell carcinoma (CSCC) to date, totalling 643 tumours and representing patient populations from the USA, Europe and Sub-Saharan Africa, we identify two CSCC subtypes (C1 and C2) with differing prognosis. C1 tumours are largely HPV16-driven, display increased cytotoxic T-lymphocyte infiltration and frequently harbour PIK3CA and EP300 mutations. C2 tumours are associated with shorter overall survival, are frequently driven by HPVs from the HPV18-containing alpha-7 clade, harbour alterations in the Hippo signalling pathway and increased expression of immune checkpoint genes, B7-H3 (also known as CD276) and NT5E (also known as CD73) and PD-L2 (also known as PDCD1LG2). In conclusion, we identify two novel, therapy-relevant CSCC subtypes that share the same defining characteristics across three geographically diverse cohorts. Different contribution of the monkey prefrontal and premotor dorsal cortex in decision making during a transitive inference task Several studies have reported similar neural modulations between brain areas of the frontal cortex, such as the dorsolateral prefrontal (DLPFC) and the premotor dorsal (PMd) cortex, in tasks requiring encoding of the abstract rules for selecting the proper action. Here we compared the neuronal modulation of the DLPFC and PMd of monkeys trained to choose the higher rank from a pair of abstract images (target item), selected from an arbitrarily rank-ordered set (A>B>C>D>E>F) in the context of a transitive inference task. Once acquired by trial-and-error, the ordinal relationship between pairs of adjacent images (i.e., A>B; B>C; C>D; D>E; E>F), monkeys were tested in indicating the ordinal relation between items of the list not paired during learning. During these decisions, we observed that the choice accuracy increased and the reaction time decreased as the rank difference between the compared items enhanced. This result is in line with the hypothesis that after learning, the monkeys built an abstract mental representation of the ranked items, where rank comparisons correspond to the items position comparison on this representation. In both brain areas, we observed higher neuronal activity when the target item appeared in a specific location on the screen with respect to the opposite position and that this difference was particularly enhanced at lower degrees of difficulty. By comparing the time evolution of the activity of the two areas, we observed that the neural encoding of target item spatial position occurred earlier in the DLPFC than in the PMd. Introspection confidence predicts EEG decoding of self-generated thoughts and meta-awareness The neurophysiological bases of mind wandering (MW) - an experiential state wherein attention is disengaged from the external environment in favour of internal thoughts, and state meta-awareness are poorly understood. In parallel, the relationship between introspection confidence in experiential state judgements and neural representations remains unclear. Here, we recorded EEG whilst participants completed a listening task within which they made experiential state judgments and rated their confidence. Alpha power was reliably greater during MW episodes, with unaware MW further associated with greater delta and theta power. Multivariate pattern classification analysis revealed that MW, and meta-awareness can be decoded from the distribution of power in these three frequency bands. Critically, we show that individual decoding accuracies positively correlate with introspection confidence. Our results reaffirm the role of alpha oscillations in MW, implicate lower frequencies in meta-awareness, and are consistent with the proposal that introspection confidence indexes neurophysiological discriminability of representational states. Accurate estimation of cell-type resolution transcriptome in bulk tissue through matrix completion Single cell RNA-seq (scRNA-seq) has been widely used to uncover cellular heterogeneity, however, the constraints of cost make it impractical as a routine on large patient cohorts. Here we present ENIGMA, a method that accurately deconvolute bulk tissue RNA-seq into single cell-type resolution given the knowledge gained from scRNA-seq. ENIGMA applies a matrix completion strategy to minimize the distance between mixture transcriptome and weighted combination of cell type-specific expression, allowing quantification of cell type proportions and reconstruction of cell type-specific transcriptome. The superior performance of ENIGMA was validated in simulated and realistic datasets, including disease-related tissues, demonstrating its ability in novel biological findings. GPI-anchored FGF directs cytoneme-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling. In vitro evolution of uracil glycosylase towards DnaKJ and GroEL binding evolves different misfolded states Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK+DnaJ+ATP (DnaKJ) or E.coli cell extract (CE). Using binding to the eUNG inhibitor Ugi as probe for native foldedess, the CE selected population was further divided to Ugi binders (+U) (native) or non-binders (-U). We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection, as well as CE+U and natural evolution of eUNG has its own trajectory. While GroEL and CE-U selected for mutants highly sensitive to protease cleavage, DnaKJ selected for partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. CE+U selected for more neutral mutations than natural evolution. In a more general context, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds mutants misfolding-prone species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL and DnaKJ selected populations, while harboring also unique properties, explained by the existence of additional chaperones in CE, such as Tig, HtpG and ClpB.