Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity.

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.

Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans.

Multiple sensory cues emanating from humans are thought to guide blood-feeding female mosquitoes to a host. To determine the relative contribution of carbon dioxide (CO2) detection to mosquito host-seeking behavior, we mutated the AaegGr3 gene, a subunit of the heteromeric CO2 receptor in Aedes aegypti mosquitoes. Gr3 mutants lack electrophysiological and behavioral responses to CO2. These mutants also fail to show CO2-evoked responses to heat and lactic acid, a human-derived attractant, suggesting that CO2 can gate responses to other sensory stimuli. Whereas attraction of Gr3 mutants to live humans in a large semi-field environment was only slightly impaired, responses to an animal host were greatly reduced in a spatial-scale-dependent manner. Synergistic integration of heat and odor cues likely drive host-seeking behavior in the absence of CO2 detection. We reveal a networked series of interactions by which multimodal integration of CO2, human odor, and heat orchestrates mosquito attraction to humans.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Molecular design of hypothalamus development.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.

Intestinal bile acids provide a surmountable barrier against C. difficile TcdB-induced disease pathogenesis.

Intestinal bile acids play an essential role in the Clostridioides difficile lifecycle having been shown in vitro to modulate various aspects of pathogenesis, including spore germination, vegetative growth, and more recently the action of the primary virulence determinant, TcdB. Here, we investigated whether physiological levels of the total pool of intestinal bile acids in mice and humans protect against TcdB action. Small molecules extracted from the lumenal contents of the small intestine, cecum, colon, and feces were found to inhibit TcdB in accordance with the differential amounts of total bile acids in each compartment. Extracts from antibiotic-treated and germ-free mice, despite harboring dramatically altered bile acid profiles, unexpectedly also prevented TcdB-induced cell rounding to similar extents. We show that protection, however, is surmountable and can be overcome at higher doses of TcdB-typical to those seen during severe C. difficile infection-suggesting that the protective properties of intestinal bile acids are operant primarily under low to moderate toxin levels. Taken together, these findings demonstrate a role for intestinal bile acids in attenuating virulence, provide insights into asymptomatic carriage of toxigenic C. difficile, and inform strategies to manipulate bile acid levels for therapeutic benefit.

Neuronal diversity of neuropeptide signaling, including galanin, in the mouse locus coeruleus.

The locus coeruleus (LC) is a small nucleus in the pons from which ascending and descending projections innervate major parts of the central nervous system. Its major transmitter is norepinephrine (NE). This system is evolutionarily conserved, including in humans, and its functions are associated with wakefulness and related to disorders, such as depression. Here, we performed single-cell ribonucleic acid-sequencing (RNA-seq) to subdivide neurons in the LC (24 clusters in total) into 3 NE, 17 glutamate, and 5 γ-aminobutyric acid (GABA) subtypes, and to chart their neuropeptide, cotransmitter, and receptor profiles. We found that NE neurons expressed at least 19 neuropeptide transcripts, notably galanin (Gal) but not Npy, and >30 neuropeptide receptors. Among the galanin receptors, Galr1 was expressed in ~19% of NE neurons, as was also confirmed by in situ hybridization. Unexpectedly, Galr1 was highly expressed in GABA neurons surrounding the NE ensemble. Patch-clamp electrophysiology and cell-type-specific Ca2+-imaging using GCaMP6s revealed that a GalR1 agonist inhibits up to ~35% of NE neurons. This effect is direct and does not rely on feed-forward GABA inhibition. Our results define a role for the galanin system in NE functions, and a conceptual framework for the action of many other peptides and their receptors.

Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis.

A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.

Translocation expands the scope of the large clostridial toxin family.

Large clostridial toxins (LCTs) are a family of six homologous disease-causing proteins characterised by their large size (>200 kDa) and conserved multidomain architectures. Using their central translocation and receptor-binding domain (T domain), LCTs bind host cell receptors and translocate their upstream glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol. The recent discovery of hundreds of LCT-like T domains in diverse genomic contexts and domain architectures from bacteria other than clostridia has provided significant new insights into the enigmatic process of LCT translocation, but also has put the definition of what constitutes an LCT into question. In this opinion article, we discuss how these findings have expanded our understanding of LCT translocation and reshaped the scope of the LCT family.

In silico prediction of heme binding in proteins.

The process of heme binding to a protein is prevalent in almost all forms of life to control many important biological properties, such as O2-binding, electron transfer, gas sensing or to build catalytic power. In these cases, heme typically binds tightly (irreversibly) to a protein in a discrete heme binding pocket, with one or two heme ligands provided most commonly to the heme iron by His, Cys or Tyr residues. Heme binding can also be used as a regulatory mechanism, for example in transcriptional regulation or ion channel control. When used as a regulator, heme binds more weakly, with different heme ligations and without the need for a discrete heme pocket. This makes the characterization of heme regulatory proteins difficult, and new approaches are needed to predict and understand the heme-protein interactions. We apply a modified version of the ProFunc bioinformatics tool to identify heme-binding sites in a test set of heme-dependent regulatory proteins taken from the Protein Data Bank and AlphaFold models. The potential heme binding sites identified can be easily visualized in PyMol and, if necessary, optimized with RosettaDOCK. We demonstrate that the methodology can be used to identify heme-binding sites in proteins, including in cases where there is no crystal structure available, but the methodology is more accurate when the quality of the structural information is high. The ProFunc tool, with the modification used in this work, is publicly available at https://www.ebi.ac.uk/thornton-srv/databases/profunc and can be readily adopted for the examination of new heme binding targets.

Analysis of the Human Protein Atlas Weakly Supervised Single-Cell Classification competition.

While spatial proteomics by fluorescence imaging has quickly become an essential discovery tool for researchers, fast and scalable methods to classify and embed single-cell protein distributions in such images are lacking. Here, we present the design and analysis of the results from the competition Human Protein Atlas - Single-Cell Classification hosted on the Kaggle platform. This represents a crowd-sourced competition to develop machine learning models trained on limited annotations to label single-cell protein patterns in fluorescent images. The particular challenges of this competition include class imbalance, weak labels and multi-label classification, prompting competitors to apply a wide range of approaches in their solutions. The winning models serve as the first subcellular omics tools that can annotate single-cell locations, extract single-cell features and capture cellular dynamics.

The effects of pathogenic and likely pathogenic variants for inherited hemostasis disorders in 140 214 UK Biobank participants.

Rare genetic diseases affect millions, and identifying causal DNA variants is essential for patient care. Therefore, it is imperative to estimate the effect of each independent variant and improve their pathogenicity classification. Our study of 140 214 unrelated UK Biobank (UKB) participants found that each of them carries a median of 7 variants previously reported as pathogenic or likely pathogenic. We focused on 967 diagnostic-grade gene (DGG) variants for rare bleeding, thrombotic, and platelet disorders (BTPDs) observed in 12 367 UKB participants. By association analysis, for a subset of these variants, we estimated effect sizes for platelet count and volume, and odds ratios for bleeding and thrombosis. Variants causal of some autosomal recessive platelet disorders revealed phenotypic consequences in carriers. Loss-of-function variants in MPL, which cause chronic amegakaryocytic thrombocytopenia if biallelic, were unexpectedly associated with increased platelet counts in carriers. We also demonstrated that common variants identified by genome-wide association studies (GWAS) for platelet count or thrombosis risk may influence the penetrance of rare variants in BTPD DGGs on their associated hemostasis disorders. Network-propagation analysis applied to an interactome of 18 410 nodes and 571 917 edges showed that GWAS variants with large effect sizes are enriched in DGGs and their first-order interactors. Finally, we illustrate the modifying effect of polygenic scores for platelet count and thrombosis risk on disease severity in participants carrying rare variants in TUBB1 or PROC and PROS1, respectively. Our findings demonstrate the power of association analyses using large population datasets in improving pathogenicity classifications of rare variants.

SpotLight Proteomics Identifies Variable Sequences of Blood Antibodies Specific Against Deamidated Human Serum Albumin.

Spontaneous deamidation of asparaginyl residues in proteins, if not repaired or cleared, can set in motion a cascade that leads to deteriorated health. Previously, we have discovered that deamidated human serum albumin (HSA) is elevated in the blood of patients with Alzheimer's disease and other neurodegenerative diseases, while the level of endogenous antibodies against deamidated HSA is significantly diminished, creating an imbalance between the risk factor and the defense against it. Endogenous antibodies against deamidated proteins are still unexplored. In the current study, we employed the SpotLight proteomics approach to identify novel amino acid sequences in antibodies specific to deamidated HSA. The results provide new insights into the clearance mechanism of deamidated proteins, a possible avenue for prevention of neurodegeneration.

Disrupted Cacna1c gene expression perturbs spontaneous Ca2+ activity causing abnormal brain development and increased anxiety.

The L-type voltage-gated Ca2+ channel gene CACNA1C is a risk gene for various psychiatric conditions, including schizophrenia and bipolar disorder. However, the cellular mechanism by which CACNA1C contributes to psychiatric disorders has not been elucidated. Here, we report that the embryonic deletion of Cacna1c in neurons destined for the cerebral cortex using an Emx1-Cre strategy disturbs spontaneous Ca2+ activity and causes abnormal brain development and anxiety. By combining computational modeling with electrophysiological membrane potential manipulation, we found that neural network activity was driven by intrinsic spontaneous Ca2+ activity in distinct progenitor cells expressing marginally increased levels of voltage-gated Ca2+ channels. MRI examination of the Cacna1c knockout mouse brains revealed volumetric differences in the neocortex, hippocampus, and periaqueductal gray. These results suggest that Cacna1c acts as a molecular switch and that its disruption during embryogenesis can perturb Ca2+ handling and neural development, which may increase susceptibility to psychiatric disease.

Human sodium current voltage-dependence at physiological temperature measured by coupling a patch-clamp experiment to a mathematical model.

Voltage-gated Na+ channels are crucial to action potential propagation in excitable tissues. Because of the high amplitude and rapid activation of the Na+ current, voltage-clamp measurements are very challenging and are usually performed at room temperature. In this study, we measured Na+ current voltage-dependence in stem cell-derived cardiomyocytes at physiological temperature. While the apparent activation and inactivation curves, measured as the dependence of current amplitude on voltage, fall within the range reported in previous studies, we identified a systematic error in our measurements. This error is caused by the deviation of the membrane potential from the command potential of the amplifier. We demonstrate that it is possible to account for this artifact using computer simulation of the patch-clamp experiment. We obtained surprising results through patch-clamp model optimization: a half-activation of -11.5 mV and a half-inactivation of -87 mV. Although the half-activation deviates from previous research, we demonstrate that this estimate reproduces the conduction velocity dependence on extracellular potassium concentration. KEY POINTS: Voltage-gated Na+ currents play a crucial role in excitable tissues including neurons, cardiac and skeletal muscle. Measurement of Na+ current is challenging because of its high amplitude and rapid kinetics, especially at physiological temperature. We have used the patch-clamp technique to measure human Na+ current voltage-dependence in human induced pluripotent stem cell-derived cardiomyocytes. The patch-clamp data were processed by optimization of the model accounting for voltage-clamp experiment artifacts, revealing a large difference between apparent parameters of Na+ current and the results of the optimization. We conclude that actual Na+ current activation is extremely depolarized in comparison to previous studies. The new Na+ current model provides a better understanding of action potential propagation; we demonstrate that it explains propagation in hyperkalaemic conditions.

Mitochondrial permeability transition regulator, cyclophilin D, is transcriptionally activated by C/EBP during adipogenesis.

Age-related bone loss is associated with decreased bone formation, increased bone resorption, and accumulation of bone marrow fat. During aging, differentiation potential of bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) is shifted toward an adipogenic lineage and away from an osteogenic lineage. In aged bone tissue, we previously observed pathological opening of the mitochondrial permeability transition pore (MPTP) which leads to mitochondrial dysfunction, oxidative phosphorylation uncoupling, and cell death. Cyclophilin D (CypD) is a mitochondrial protein that facilitates opening of the MPTP. We found earlier that CypD is downregulated during osteogenesis of BMSCs leading to lower MPTP activity and, thus, protecting mitochondria from dysfunction. However, during adipogenesis, a fate alternative to osteogenesis, the regulation of mitochondrial function and CypD expression is still unclear. In this study, we observed that BMSCs have increased CypD expression and MPTP activity, activated glycolysis, and fragmented mitochondrial network during adipogenesis. Adipogenic C/EBPα acts as a transcriptional activator of expression of the CypD gene, Ppif, during this process. Inflammation-associated transcription factor NF-κB shows a synergistic effect with C/EBPα inducing Ppif expression. Overall, we demonstrated changes in mitochondrial morphology and function during adipogenesis. We also identified C/EBPα as a transcriptional activator of CypD. The synergistic activation of CypD by C/EBPα and the NF-κB p65 subunit during this process suggests a potential link between adipogenic signaling, inflammation, and MPTP gain-of-function, thus altering BMSC fate during aging.

Toward New Horizons in Verdazyl-Nitroxide High-Spin Systems: Thermally Robust Tetraradical with Quintet Ground State.

High-spin organic tetraradicals with significant intramolecular exchange interactions have high potential for advanced technological applications and fundamental research, but examples reported to date exhibit limited stability and processability. In this work, we designed the first tetraradical based on an oxoverdazyl core and nitronyl nitroxide radicals and successfully synthesized it using a palladium-catalyzed cross-coupling reaction of an oxoverdazyl radical bearing three iodo-phenylene moieties with a gold(I) nitronyl nitroxide-2-ide complex in the presence of a recently developed efficient catalytic system. The molecular and crystal structures of the tetraradical were confirmed by single crystal X-ray diffraction analysis. The tetraradical possesses good thermal stability with decomposition onset at ∼125 °C in an inert atmosphere; in a toluene solution upon prolonged heating at 90 °C in air, no decomposition was observed. The resulting unique verdazyl-nitroxide conjugate was thoroughly studied using a range of experimental and theoretical techniques, such as SQUID magnetometry of polycrystalline powders, EPR spectroscopy in various matrices, cyclic voltammetry, and high-level quantum chemical calculations. All collected data confirm the high thermal stability of the resulting tetraradical and quintet multiplicity of its ground state, which makes the synthesis of this important paramagnet a new milestone in the field of creating high-spin systems.

Life-long impairment of glucose homeostasis upon prenatal exposure to psychostimulants.

Maternal drug abuse during pregnancy is a rapidly escalating societal problem. Psychostimulants, including amphetamine, cocaine, and methamphetamine, are amongst the illicit drugs most commonly consumed by pregnant women. Neuropharmacology concepts posit that psychostimulants affect monoamine signaling in the nervous system by their affinities to neurotransmitter reuptake and vesicular transporters to heighten neurotransmitter availability extracellularly. Exacerbated dopamine signaling is particularly considered as a key determinant of psychostimulant action. Much less is known about possible adverse effects of these drugs on peripheral organs, and if in utero exposure induces lifelong pathologies. Here, we addressed this question by combining human RNA-seq data with cellular and mouse models of neuroendocrine development. We show that episodic maternal exposure to psychostimulants during pregnancy coincident with the intrauterine specification of pancreatic ß cells permanently impairs their ability of insulin production, leading to glucose intolerance in adult female but not male offspring. We link psychostimulant action specifically to serotonin signaling and implicate the sex-specific epigenetic reprogramming of serotonin-related gene regulatory networks upstream from the transcription factor Pet1/Fev as determinants of reduced insulin production.

Comparative proteomics of a versatile, marine, iron-oxidizing chemolithoautotroph.

This study conducted a comparative proteomic analysis to identify potential genetic markers for the biological function of chemolithoautotrophic iron oxidation in the marine bacterium Ghiorsea bivora. To date, this is the only characterized species in the class Zetaproteobacteria that is not an obligate iron-oxidizer, providing a unique opportunity to investigate differential protein expression to identify key genes involved in iron-oxidation at circumneutral pH. Over 1000 proteins were identified under both iron- and hydrogen-oxidizing conditions, with differentially expressed proteins found in both treatments. Notably, a gene cluster upregulated during iron oxidation was identified. This cluster contains genes encoding for cytochromes that share sequence similarity with the known iron-oxidase, Cyc2. Interestingly, these cytochromes, conserved in both Bacteria and Archaea, do not exhibit the typical ß-barrel structure of Cyc2. This cluster potentially encodes a biological nanowire-like transmembrane complex containing multiple redox proteins spanning the inner membrane, periplasm, outer membrane, and extracellular space. The upregulation of key genes associated with this complex during iron-oxidizing conditions was confirmed by quantitative reverse transcription-PCR. These findings were further supported by electromicrobiological methods, which demonstrated negative current production by G. bivora in a three-electrode system poised at a cathodic potential. This research provides significant insights into the biological function of chemolithoautotrophic iron oxidation.

Electrochemical Nanopipette Sensor for In Vitro/In Vivo Detection of Cu2+ Ions.

In vitro/in vivo detection of copper ions is a challenging task but one which is important in the development of new approaches to the diagnosis and treatment of cancer and hereditary diseases such as Alzheimer's, Wilson's, etc. In this paper, we present a nanopipette sensor capable of measuring Cu2+ ions with a linear range from 0.1 to 10 µM in vitro and in vivo. Using the gold-modified nanopipette sensor with a copper chelating ligand, we evaluated the accumulation ability of the liposomal form of an anticancer Cu-containing complex at three levels of biological organization. First, we detected Cu2+ ions in a single cell model of human breast adenocarcinoma MCF-7 and in murine melanoma B16 cells. The insertion of the nanoelectrode did not result in leakage of the cell membrane. We then evaluated the distribution of the Cu-complex in MCF-7 tumor spheroids and found that the diffusion-limited accumulation was a function of the depth, typical for 3D culture. Finally, we demonstrated the use of the sensor for Cu2+ ion detection in the brain of an APP/PS1 transgenic mouse model of Alzheimer's disease and tumor-bearing mice in response to injection (2 mg kg-1) of the liposomal form of the anticancer Cu-containing complex. Enhanced stability and selectivity, as well as distinct copper oxidation peaks, confirmed that the developed sensor is a promising tool for testing various types of biological systems. In summary, this research has demonstrated a minimally invasive electrochemical technique with high temporal resolution that can be used for the study of metabolism of copper or copper-based drugs in vitro and in vivo.