Miracle-dz/UniFAR-FTUs huggingface dataset:
Nuclear Hormone Receptor NHR-49 controls a HIF-1-independent hypoxia adaptation pathway in Caenorhabditis elegans

The response to insufficient oxygen (hypoxia) is orchestrated by the conserved Hypoxia-Inducible Factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel to HIF to mediate the physiological hypoxia response. Here, we describe a HIF-independent hypoxia response pathway controlled by Caenorhabditis elegans Nuclear Hormone Receptor NHR-49, an orthologue of mammalian Peroxisome Proliferator-Activated Receptor alpha (PPARα). We show that nhr-49 is required for worm survival in hypoxia and is synthetic lethal with hif-1 in this context, demonstrating that these factors act independently. RNA-seq analysis shows that in hypoxia nhr-49 regulates a set of genes that are hif-1-independent, including autophagy genes that promote hypoxia survival. We further show that Nuclear Hormone Receptor nhr-67 is a negative regulator and Homeodomain-interacting Protein Kinase hpk-1 is a positive regulator of the NHR-49 pathway. Together, our experiments define a new, essential hypoxia response pathway that acts in parallel to the well-known HIF-mediated hypoxia response.
Genes with Specificity for Expression in the Round Cell Layer of the Growth Plate are Enriched in GWAS of Human Height

Human adult height reflects the outcome of childhood skeletal growth. Growth plate (epiphyseal) chondrocytes are key determinants of height. As epiphyseal chondrocytes mature and proliferate, they pass through three developmental stages, which are organized into three distinct layers in the growth plate: 1) resting (round), 2) proliferative (flat), and 3) hypertrophic. Recent genome-wide association studies (GWAS) of human height identified numerous associated loci, which are enriched for genes expressed in growth plate chondrocytes. However, it remains unclear which specific genes expressed in which layers of the growth plate regulate skeletal growth and human height. To connect the genetics of height and growth plate biology, we analyzed GWAS data through the lens of gene expression in the three dissected layers of murine newborn tibial growth plate. For each gene, we derived a specificity score for each growth plate layer and regressed these scores against gene-level p-values from recent height GWAS data. We found that specificity for expression in the round cell layer, which contains chondrocytes early in maturation, is significantly associated with height GWAS p-values (p=8.5×10−9); this association remains after conditioning on specificity for the other cell layers. The association also remains after conditioning on membership in an “OMIM gene set” (genes known to cause monogenic skeletal growth disorders, p<9.7×10−6). We replicated the association in RNA-seq data from maturing chondrocytes sampled at early and late time points during differentiation in vitro: we found that expression early in differentiation is significantly associated with p-values from height GWAS (p=6.1×10−10) and that this association remains after conditioning on expression at 10 days in culture and on the OMIM gene set (p<0.006). These findings newly implicate genes highlighted by GWAS of height and specifically expressed in the round cell layer as being potentially important regulators of skeletal biology.
Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.
Acyloxyacyl Hydrolase Regulates Microglia-Mediated Pelvic Pain Through Toll-Like Receptor-4

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a devastating condition of chronic pelvic pain and urinary dysfunction. We have shown that mice deficient for the lipase acyloxyacyl hydrolase (AOAH) develop pelvic allodynia and exhibit symptoms and comorbidities consistent with IC/BPS, as well as gut dysbiosis. Microglia are resident immune cells of the central nervous system (CNS) that respond to changes in the gut microbiome, and studies have linked microglial activation to neuropathic pain. Additionally, microglia express toll-like receptors (TLRs), including TLR4, which are activated by microbial components. We have previously shown that AOAH-deficient mice exhibit increased gut permeability, suggesting a possible mechanism of microglial TLR4 activation via translocation of microbial products across the intestinal barrier to the brain. Here, we assessed the role of AOAH and TLR4 in microglial activation and pelvic pain. AOAH immunoreactivity co-localized with the microglial marker P2YR12 but not astrocytes, suggesting a functional role for AOAH in microglia. Pharmacologic ablation of CNS microglia with PLX5622 resulted in decreased pelvic allodynia in AOAH-deficient mice and resurgence of pelvic pain upon drug washout. Aligned with microglial activation, we observed altered cytokine abundance in Aoah−/− cortex that was reduced in Aoah/Tlr4−/− cortex. Consistent with our hypothesis of TLR4 activation by gut microbes, we observed microbiome-dependent activation of cultured BV2 microglial cells. Skeletal analyses revealed that AOAH-deficient mice have an activated microglia morphology in brain regions associated with neuropathic pain, independent of TLR4. Compared to Aoah−/− mice, Aoah/Tlr4−/− mice exhibited decreased pelvic pain and microglial cytokine expression. Together, these findings demonstrate differential roles for AOAH and TLR4 in microglial activation and pelvic pain and thus identify novel therapeutic targets for IC/BPS.
Stability of ligand-induced protein conformation influences affinity in maltose-binding protein

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MPB) from Escherichia coli. We used singlemolecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.
Widespread ripples synchronize human cortical activity during sleep, waking, and memory recall

Declarative memory encoding, consolidation, and retrieval require the integration of elements encoded in widespread cortical locations. The mechanism whereby such ‘binding’ of different components of mental events into unified representations occurs is unknown. The ‘binding-bysynchrony’ theory proposes that distributed encoding areas are bound by synchronous oscillations enabling enhanced communication. However, evidence for such oscillations is sparse. Brief high-frequency oscillations (‘ripples’) occur in the hippocampus and cortex, and help organize memory recall and consolidation. Here, using intracranial recordings in humans, we report that these ~70ms duration 90Hz ripples often couple (within ±500ms), co-occur (≥25ms overlap), and crucially, phase-lock (have consistent phase-lags) between widely distributed focal cortical locations during both sleep and waking, even between hemispheres. Cortical ripple co-occurrence is facilitated through activation across multiple sites, and phaselocking increases with more cortical sites co-rippling. Ripples in all cortical areas co-occur with hippocampal ripples but do not phase-lock with them, further suggesting that cortico-cortical synchrony is mediated by cortico-cortical connections. Ripple phase-lags vary across sleep nights, consistent with participation in different networks. During waking, we show that hippocampo-cortical and cortico-cortical co-ripples increase preceding successful delayed memory recall, when binding between the cue and response is essential. Ripples increase and phase-modulate unit firing, and co-ripples increase high-frequency correlations between areas, suggesting synchronized unit-spiking facilitating information exchange. Co-occurrence, phasesynchrony, and high-frequency correlation are maintained with little decrement over very long distances (25cm). Hippocampo-cortico-cortical co-ripples appear to possess the essential properties necessary to support binding-by-synchrony during memory retrieval, and perhaps generally in cognition. Significance Statement Different elements of a memory, or any mental event, are encoded in locations distributed across the cortex. A prominent hypothesis proposes that widespread networks are integrated with bursts of synchronized high-frequency oscillations called ‘ripples,’ but evidence is limited. Here, using recordings inside the human brain, we show that ripples occur simultaneously in multiple lobes in both cortical hemispheres, and the hippocampus, generally during sleep and waking, and especially during memory recall. Ripples phase-lock local cell firing, and phase-synchronize with little decay between locations separated by up to 25cm, enabling long-distance integration. Indeed, co-rippling sites have increased correlation of very high-frequency activity which reflects cell firing. Thus, ripples may help bind information across the cortex in memory and other mental events.
AIM-CICs: automatic identification method for Cell-in-cell structures based on convolutional neural network

Whereas biochemical markers are available for most types of cell death, current studies on non-autonomous cell death by entosis relays strictly on the identification of cell-in-cell structure (CICs), a unique morphological readout that can only be quantified manually at present. Moreover, the manual CICs quantification is generally over-simplified as CICs counts, which represents a major hurdle against profound mechanistic investigations. In this study, we take advantage of artificial intelligence (AI) technology to develop an automatic identification method for CICs (AIM-CICs), which performs comprehensive CICs analysis in an automated and efficient way. The AIM-CICs, developed on the algorithm of convolutional neural network (CNN), can not only differentiate between CICs and non-CICs (AUC > 0.99), but also accurately categorize CICs into five subclasses based on CICs stages and cell number involved (AUC > 0.97 for all subclasses). The application of AIM-CICs would systemically fuel researches on CICs-mediated cell death such as high-throughput screening.
Tissue-resident, extravascular CD64-Ly6C- population forms a critical barrier for inflammation in the synovium

Monocytes are one of the most abundant immune cells infiltrating the inflamed organs. However, the majority of studies on monocytes focus on circulating cells, rather than those in the tissue. Here, we identify and characterize an intravascular (i.v.) and extravascular (e.v.) synovial population (Syn Ly6C- cells) which lack cell surface markers of classical monocytes (Ly6C and CD62) or tissue macrophages (CD64 and Tim4), are transcriptionally distinct and conserved in RA patients. e.v. Syn Ly6C- cells are independent of NR4A1 and CCR2, long-lived and embryonically derived while the i.v. Syn Ly6C- cells are dependent on NR4A1, short lived and derived from circulating NCM. e.v. Syn Ly6C- cells undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. These findings uncover a new facet of mononuclear cell biology and are imperative to understanding tissue-resident myeloid cell function in RA.
TRPV1 in arteries enables a rapid myogenic tone

Arterioles maintain blow flow by adjusting their diameter in response to changes in local blood pressure. In this process called the myogenic response, a vascular smooth muscle mechanosensor controls tone predominantly through altering the membrane potential. In general, myogenic responses occur slowly, reaching a plateau in minutes. In the heart and skeletal muscle, however, myogenic tone is rapid; activation occurs in tens of seconds and arterial constrictions or raised extravascular pressure as brief as 100 ms remove tone. Previously, we identified extensive expression of TRPV1 in the smooth muscle of arterioles supplying skeletal muscle, heart and the adipose. Here we reveal a critical role for TRPV1 in the myogenic tone of these tissues. TRPV1 antagonists dilated skeletal muscle arterioles in vitro and in vivo, increased coronary flow in isolated hearts, and transiently decreased blood pressure. All of these effects of TRPV1 antagonists were abolished by genetic disruption of TRPV1. Stretch of isolated vascular smooth muscle cells, or raised intravascular pressure in arteries (with or without endothelium), triggered Ca2+ signaling and vasoconstriction. The majority of these stretch-responses were TRPV1-mediated, with the remaining tone being inhibited by the TRPM4 antagonist, 9-phenantrol. Notably, tone developed more quickly in arteries from wild-type compared with TRPV1-null mice. Furthermore, the rapid vasodilation following brief constriction of arterioles was also dependent on TRPV1, consistent with a rapid deactivation or inactivation of TRPV1. Pharmacologic experiments revealed that membrane stretch activates a phospholipase C/protein kinase C signaling pathway to activate TRPV1, and in turn, L-type Ca2+ channels. These results suggest a critical role, for TRPV1 in the dynamic regulation of myogenic tone and blood flow in the heart and skeletal muscle.
Local Accumulation of Axonal Mitochondria in the Optic Nerve Glial Lamina Precedes Myelination

Mitochondria are essential for neurons and must be optimally distributed along their axon to fulfil local functions. A high density of mitochondria has been observed in retinal ganglion cell (RGC) axons of an unmyelinated region of the optic nerve, called the glial lamina (GL) in mouse (lamina cribrosa in human). In glaucoma, the world’s leading cause of irreversible blindness, the GL is the epicenter of RGC degeneration and is connected to mitochondrial dysfunction. It is generally accepted that the local accumulation of mitochondria in the GL is established due to the higher energy requirement of unmyelinated axons. Here we revisit the connection between mitochondrial positioning and myelin in RGC axons. We show that the high density of mitochondria in the GL is restricted to larger axons and is established before myelination. Thus, contrary to a longstanding belief in the field, the myelination pattern is not responsible for the establishment of the local accumulation of mitochondria in GL axons. Our findings open new research avenues likely critical to understanding the pathophysiology of glaucoma.
Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. Since the particular sequence of a DNA segment influences both base stacking and hydrogen bonding, the conformational dynamics of B-to-L transitions should be tunable. To test this idea, DNA with diaminopurine replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded A:T base pairs. Triple hydrogen bonding stiffened the DNA by 30% flexurally. In addition, DAP-substituted DNA formed plectonemes with larger gyres for both B- and L-form helices. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing by DNA stretched and unwound by enzymatic activity. The intramolecular nature and ease of this transition likely prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length indicating it is much more contractile and prone to sharp bends and kinks. However, left-handed DAP DNA was twice as stiff as unmodified L-DNA. Thus, significantly doubly and triply hydrogen bonded segments have very distinct mechanical dynamics at physiological levels of negative supercoiling and tension.
Dual mechanisms of opioid-induced respiratory depression in the inspiratory rhythm-generating network

The analgesic utility of opioid-based drugs is limited by the life-threatening risk of respiratory depression. Opioid-induced respiratory depression (OIRD), mediated by the μ-opioid receptor (MOR), is characterized by a pronounced decrease in the frequency and regularity of the inspiratory rhythm, which originates from the medullary preBötzinger Complex (preBӧtC). To unravel the cellular- and network-level consequences of MOR activation in the preBötC, MOR-expressing neurons were optogenetically identified and manipulated in transgenic mice in vitro and in vivo. Based on these results, a model of OIRD was developed in silico. We conclude that hyperpolarization of MOR-expressing preBötC neurons alone does not phenocopy OIRD. Instead, the effects of MOR activation are twofold: 1) pre-inspiratory spiking is reduced and 2) excitatory synaptic transmission is suppressed, thereby disrupting network-driven rhythmogenesis. These dual mechanisms of opioid action act together to make the normally robust inspiratory-rhythm-generating network particularly prone to collapse when challenged with exogenous opioids.
Big fish, little fish: N-terminal acetyltransferase Naa40p proteoforms caught in the act

NatD consist of the evolutionary conserved N-alpha acetyltransferase catalytic subunit Naa40p and is among the most selective N-terminal acetyltransferase (NAT) complexes identified to date. Here we identified a conserved 22 amino acid N-terminally truncated Naa40 proteoform named Naa40p25 or short Naa40 (Naa40S). Intriguingly, while upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the well characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40 (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Further, since Naa40L was identified as being N-terminally myristoylated and shown to interact with N-myristoyltransferase 1 (NMT1), NMT1 may steer Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.
NADPH Oxidase 2 Derived Reactive Oxygen Species Promote CD8+ T cell Effector Function

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ cytotoxic T lymphocytes (CTL). However, the molecular mechanisms impacting CTL function remain unknown. Here, we studied the role of NOX2 in both non-obese diabetic (NOD) mouse and human CTL function. Genetic ablation or chemical inhibition of NOX2 in CTL significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effectors genes such as IFNγ and granzyme B. Inhibition of NOX2 in both human and mouse CTL prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CTL. Further, we show that NOX2-generated oxidants deactivate the Tumor Suppressor Complex leading to mTOR complex 1 activation and CTL effector function. These results indicate that NOX2 plays a non-redundant role in T cell receptor-mediated CTL effector function.
PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors

The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+ progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+ progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in vivo and in lung explants in vitro resulted in an expansion of the SOX9+ EPC population and impaired epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA-Seq and ATAC-Seq in defining regulatory networks in development.