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.

The clammy grip of parasitic tumors.

An epidemic of leukemia among bivalve molluscs is spreading along the Atlantic coast of North America, with a serious population decline of soft-shelled clams. In this issue of Cell, Metzger et al. use forensic DNA markers to demonstrate that the leukemia cells have a clonal origin and appear to be transmitted through sea water.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

Structure, substrate recognition and initiation of hyaluronan synthase.

Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.

Small-molecule inhibition of MAP2K4 is synergistic with RAS inhibitors in KRAS-mutant cancers.

The Kirsten rat sarcoma viral oncogene homologue KRAS is among the most commonly mutated oncogenes in human cancers, thus representing an attractive target for precision oncology. The approval for clinical use of the first selective inhibitors of G12C mutant KRAS therefore holds great promise for cancer treatment. However, despite initial encouraging clinical results, the overall survival benefit that patients experience following treatment with these inhibitors has been disappointing to date, pointing toward the need to develop more powerful combination therapies. Here, we show that responsiveness to KRASG12C and pan-RAS inhibitors in KRAS-mutant lung and colon cancer cells is limited by feedback activation of the parallel MAP2K4-JNK-JUN pathway. Activation of this pathway leads to elevated expression of receptor tyrosine kinases that reactivate KRAS and its downstream effectors in the presence of drug. We find that the combination of sotorasib, a drug targeting KRASG12C, and the MAP2K4 inhibitor HRX-0233 prevents this feedback activation and is highly synergistic in a panel of KRASG12C-mutant lung and colon cancer cells. Moreover, combining HRX-0233 and sotorasib is well-tolerated and resulted in durable tumor shrinkage in mouse xenografts of human lung cancer cells, suggesting a therapeutic strategy for KRAS-driven cancers.

Injury-induced myosin-specific tissue-resident memory T cells drive immune checkpoint inhibitor myocarditis.

Cardiac myosin-specific (MyHC) T cells drive the disease pathogenesis of immune checkpoint inhibitor-associated myocarditis (ICI-myocarditis). To determine whether MyHC T cells are tissue-resident memory T (TRM) cells, we characterized cardiac TRM cells in naive mice and established that they have a distinct phenotypic and transcriptional profile that can be defined by their upregulation of CD69, PD-1, and CXCR6. We then investigated the effects of cardiac injury through a modified experimental autoimmune myocarditis mouse model and an ischemia-reperfusion injury mouse model and determined that cardiac inflammation induces the recruitment of autoreactive MyHC TRM cells, which coexpress PD-1 and CD69. To investigate whether the recruited MyHC TRM cells could increase susceptibility to ICI-myocarditis, we developed a two-hit ICI-myocarditis mouse model where cardiac injury was induced, mice were allowed to recover, and then were treated with anti-PD-1 antibodies. We determined that mice who recover from cardiac injury are more susceptible to ICI-myocarditis development. We found that murine and human TRM cells share a similar location in the heart and aggregate along the perimyocardium. We phenotyped cells obtained from pericardial fluid from patients diagnosed with dilated cardiomyopathy and ischemic cardiomyopathy and established that pericardial T cells are predominantly CD69+ TRM cells that up-regulate PD-1. Finally, we determined that human pericardial macrophages produce IL-15, which supports and maintains pericardial TRM cells.

Feedback information transfer in the human brain reflects bistable perception in the absence of report.

In the search for the neural basis of conscious experience, perception and the cognitive processes associated with reporting perception are typically confounded as neural activity is recorded while participants explicitly report what they experience. Here, we present a novel way to disentangle perception from report using eye movement analysis techniques based on convolutional neural networks and neurodynamical analyses based on information theory. We use a bistable visual stimulus that instantiates two well-known properties of conscious perception: integration and differentiation. At any given moment, observers either perceive the stimulus as one integrated unitary object or as two differentiated objects that are clearly distinct from each other. Using electroencephalography, we show that measures of integration and differentiation based on information theory closely follow participants' perceptual experience of those contents when switches were reported. We observed increased information integration between anterior to posterior electrodes (front to back) prior to a switch to the integrated percept, and higher information differentiation of anterior signals leading up to reporting the differentiated percept. Crucially, information integration was closely linked to perception and even observed in a no-report condition when perceptual transitions were inferred from eye movements alone. In contrast, the link between neural differentiation and perception was observed solely in the active report condition. Our results, therefore, suggest that perception and the processes associated with report require distinct amounts of anterior-posterior network communication and anterior information differentiation. While front-to-back directed information is associated with changes in the content of perception when viewing bistable visual stimuli, regardless of report, frontal information differentiation was absent in the no-report condition and therefore is not directly linked to perception per se.

Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Zibotentan in Microvascular Angina: A Randomized, Placebo-Controlled, Crossover Trial.

Background: Microvascular angina is associated with dysregulation of the endothelin system and impairments in myocardial blood flow, exercise capacity, and health-related quality of life. The G allele of the noncoding single nucleotide polymorphism RS9349379 enhances expression of the endothelin-1 gene (EDN1) in human vascular cells, potentially increasing circulating concentrations of Endothelin-1 (ET-1). Whether zibotentan, an oral ET-A receptor selective antagonist, is efficacious and safe for the treatment of microvascular angina is unknown. Methods: Patients with microvascular angina were enrolled in this double-blind, placebo-controlled, sequential crossover trial of zibotentan (10 mg daily for 12 weeks). The trial population was enriched to ensure a G allele frequency of 50% for the RS9349379 single nucleotide polymorphism. Participants and investigators were blinded to genotype. The primary outcome was treadmill exercise duration (seconds) using the Bruce protocol. The primary analysis estimated the mean within-participant difference in exercise duration after treatment with zibotentan versus placebo. Results: A total of 118 participants (mean ±SD; years of age 63.5 [9.2 ]; 71 [60.2% ] females; 25 [21.2% ] with diabetes) were randomized. Among 103 participants with complete data, the mean exercise duration with zibotentan treatment compared with placebo was not different (between-treatment difference, -4.26 seconds [95 ] CI, -19.60 to 11.06] P=0.5871). Secondary outcomes showed no improvement with zibotentan. Zibotentan reduced blood pressure and increased plasma concentrations of ET-1. Adverse events were more common with zibotentan (60.2%) compared with placebo (14.4%; P<0.001). Conclusions: Among patients with microvascular angina, short-term treatment with a relatively high dose (10 mg daily) of zibotentan was not beneficial. Target-related adverse effects were common.

Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance.

BACKGROUND: Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. METHODS: A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. RESULTS: Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%. CONCLUSION: The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.

Membrane protein biogenesis by the EMC.

The endoplasmic reticulum (ER) membrane protein complex (EMC) was identified over a decade ago in a genetic screen for ER protein homeostasis. The EMC inserts transmembrane domains (TMDs) with limited hydrophobicity. Two recent cryo-EM structures, and a third model based on partial high- and low-resolution structures, suggest how this is accomplished.

The soluble factor milieu in idiopathic pulmonary fibrosis dysregulates epithelial differentiation.

In idiopathic pulmonary fibrosis (IPF), epithelial abnormalities are present including bronchiolization and alveolar cell dysfunction. We hypothesized that the IPF microenvironment disrupts normal epithelial growth and differentiation. We mimicked the soluble factors within an IPF microenvironment using an IPF cocktail (IPFc), composed of nine factors which are increased in IPF lungs (CCL2, IL-1ß, IL-4, IL-8, IL-13, IL-33, TGF-ß, TNFα, and TSLP). Using IPFc, we asked whether the soluble factor milieu in IPF affects epithelial growth and differentiation and how IPFc compares to TGF-ß alone. Epithelial growth and differentiation were studied using mouse lung organoids (primary Epcam+ epithelial cells co-cultured with CCL206 fibroblasts). Organoids exposed to IPFc and TGF-ß were re-sorted into epithelial and fibroblast fractions and subjected to RNA sequencing. IPFc did not affect the number of organoids formed. However, pro-surfactant protein C expression was decreased. On these parameters, TGF-ß alone had similar effects. However, RNA sequencing of re-sorted organoids revealed that IPFc and TGF-ß had distinct effects on both epithelial cells and fibroblasts. IPFc upregulated goblet cell markers, whereas these were inhibited by TGF-ß. Although both IPFc and TGF-ß increased extracellular matrix gene expression, only TGF-ß increased myofibroblast markers. VEGF-C and Wnt signaling were among the most differentially regulated signaling pathways by IPFc versus TGF-ß. Interestingly, Wnt pathway activation rescued Sftpc downregulation induced by IPFc. In conclusion, IPFc alters epithelial differentiation in a way that is distinct from TGF-ß. Alterations in Wnt signaling contribute to these effects. IPFc may be a more comprehensive representation of the soluble factor microenvironment in IPF.

Novel loss-of-function variants expand ABCC9-related intellectual disability and myopathy syndrome.

Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS). Here we identify nine additional subjects, from seven unrelated families, harbouring different homozygous loss-of-function variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels. Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intra-uterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 loss-of-function in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility. Our findings define an ABCC9 loss-of-function-related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction.

High sorbic acid resistance of Penicillium roqueforti is mediated by the SORBUS gene cluster.

Penicillium roqueforti is a major food-spoilage fungus known for its high resistance to the food preservative sorbic acid. Here, we demonstrate that the minimum inhibitory concentration of undissociated sorbic acid (MICu) ranges between 4.2 and 21.2 mM when 34 P. roqueforti strains were grown on malt extract broth. A genome-wide association study revealed that the six most resistant strains contained the 180 kbp gene cluster SORBUS, which was absent in the other 28 strains. In addition, a SNP analysis revealed five genes outside the SORBUS cluster that may be linked to sorbic acid resistance. A partial SORBUS knock-out (>100 of 180 kbp) in a resistant strain reduced sorbic acid resistance to similar levels as observed in the sensitive strains. Whole genome transcriptome analysis revealed a small set of genes present in both resistant and sensitive P. roqueforti strains that were differentially expressed in the presence of the weak acid. These genes could explain why P. roqueforti is more resistant to sorbic acid when compared to other fungi, even in the absence of the SORBUS cluster. Together, the MICu of 21.2 mM makes P. roqueforti among the most sorbic acid-resistant fungi, if not the most resistant fungus, which is mediated by the SORBUS gene cluster.

The photobiology of the human circadian clock.

SignificanceThe function of our biological clock is dependent on environmental light. Rodent studies have shown that there are multiple colors that affect the clock, but indirect measures in humans suggest blue light is key. We performed functional MRI studies in human subjects with unprecedented spatial resolution to investigate color sensitivity of our clock. Here, we show that narrowband blue, green, and orange light were all effective in changing neuronal activity of the clock. While the clock of nocturnal rodents is excited by light, the human clock responds with a decrease in neuronal activity as indicated by a negative BOLD response. The sensitivity of the clock to multiple colors should be integrated in light therapy aimed to strengthen our 24-h rhythms.

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Cbx3 maintains lineage specificity during neural differentiation.

Chromobox homolog 3 (Cbx3/heterochromatin protein 1γ [HP1γ]) stimulates cell differentiation, but its mechanism is unknown. We found that Cbx3 binds to gene promoters upon differentiation of murine embryonic stem cells (ESCs) to neural progenitor cells (NPCs) and recruits the Mediator subunit Med26. RNAi knockdown of either Cbx3 or Med26 inhibits neural differentiation while up-regulating genes involved in mesodermal lineage decisions. Thus, Cbx3 and Med26 together ensure the fidelity of lineage specification by enhancing the expression of neural genes and down-regulating genes specific to alternative fates.

Network Communications Flexibly Predict Visual Contents That Enhance Representations for Faster Visual Categorization.

Models of visual cognition generally assume that brain networks predict the contents of a stimulus to facilitate its subsequent categorization. However, understanding prediction and categorization at a network level has remained challenging, partly because we need to reverse engineer their information processing mechanisms from the dynamic neural signals. Here, we used connectivity measures that can isolate the communications of a specific content to reconstruct these network mechanisms in each individual participant (N = 11, both sexes). Each was cued to the spatial location (left vs right) and contents [low spatial frequency (LSF) vs high spatial frequency (HSF)] of a predicted Gabor stimulus that they then categorized. Using each participant's concurrently measured MEG, we reconstructed networks that predict and categorize LSF versus HSF contents for behavior. We found that predicted contents flexibly propagate top down from temporal to lateralized occipital cortex, depending on task demands, under supervisory control of prefrontal cortex. When they reach lateralized occipital cortex, predictions enhance the bottom-up LSF versus HSF representations of the stimulus, all the way from occipital-ventral-parietal to premotor cortex, in turn producing faster categorization behavior. Importantly, content communications are subsets (i.e., 55-75%) of the signal-to-signal communications typically measured between brain regions. Hence, our study isolates functional networks that process the information of cognitive functions.SIGNIFICANCE STATEMENT An enduring cognitive hypothesis states that our perception is partly influenced by the bottom-up sensory input but also by top-down expectations. However, cognitive explanations of the dynamic brain networks mechanisms that flexibly predict and categorize the visual input according to task-demands remain elusive. We addressed them in a predictive experimental design by isolating the network communications of cognitive contents from all other communications. Our methods revealed a Prediction Network that flexibly communicates contents from temporal to lateralized occipital cortex, with explicit frontal control, and an occipital-ventral-parietal-frontal Categorization Network that represents more sharply the predicted contents from the shown stimulus, leading to faster behavior. Our framework and results therefore shed a new light of cognitive information processing on dynamic brain activity.

High-quality lipid suppression and B0 shimming for human brain 1H MRSI.

Magnetic Resonance Spectroscopic Imaging (MRSI) is a powerful technique that can map the metabolic profile in the brain non-invasively. Extracranial lipid contamination and insufficient B0 homogeneity however hampers robustness, and as a result has hindered widespread use of MRSI in clinical and research settings. Over the last six years we have developed highly effective extracranial lipid suppression methods with a second order gradient insert (ECLIPSE) utilizing inner volume selection (IVS) and outer volume suppression (OVS) methods. While ECLIPSE provides > 100-fold in lipid suppression with modest radio frequency (RF) power requirements and immunity to B1+ field variations, axial coverage is reduced for non-elliptical head shapes. In this work we detail the design, construction, and utility of MC-ECLIPSE, a pulsed second order gradient coil with Z2 and X2Y2 fields, combined with a 54-channel multi-coil (MC) array. The MC-ECLIPSE platform allows arbitrary region of interest (ROI) shaped OVS for full-axial slice coverage, in addition to MC-based B0 field shimming, for robust human brain proton MRSI. In vivo experiments demonstrate that MC-ECLIPSE allows axial brain coverage of 92-95 % is achieved following arbitrary ROI shaped OVS for various head shapes. The standard deviation (SD) of the residual B0 field following SH2 and MC shimming were 25 ± 9 Hz and 18 ± 8 Hz over a 5 cm slab, and 18 ± 5 Hz and 14 ± 6 Hz over a 1.5 cm slab, respectively. These results demonstrate that B0 magnetic field shimming with the MC array supersedes second order harmonic capabilities available on standard MRI systems for both restricted and large ROIs. Furthermore, MC based B0 shimming provides comparable shimming performance to an unrestricted SH5 shim set for both restricted, and 5-cm slab shim challenges. Phantom experiments demonstrate the high level of localization performance achievable with MC-ECLIPSE, with ROI edge chemical shift displacements ranging from 1-3 mm with a median value of 2 mm, and transition width metrics ranging from 1-2.5 mm throughout the ROI edge. Furthermore, MC based B0 shimming is comparable to performance following a full set of unrestricted spherical harmonic fields up to order 5. Short echo time MRSI and GABA-edited MRSI acquisitions in the human brain following MC-shimming and arbitrary ROI shaping demonstrate full-axial slice coverage and extracranial lipid artifact free spectra. MC-ECLIPSE allows full-axial coverage and robust MRSI acquisitions, while allowing interrogation of cortical tissue proximal to the skull, which has significant value in a wide range of neurological and psychiatric conditions.