Plasmodium falciparum parasites cause the most virulent form of malaria, a disease that remains a major global health burden. The appearance of resistance to first line artemisinin-based therapies emphasizes the need to identify new parasite vulnerabilities to develop new therapeutics. Phosphoinositides are central regulators of membrane identity, vesicular trafficking, and signaling, and their synthesis depends on tightly controlled phosphatidylinositol transfer by Sec14-like phosphatidylinositol transfer proteins in many eukaryotes, yet their roles in P. falciparum remain poorly defined. Here, we analyzed six P. falciparum Sec14 domain-containing proteins: PfSec14-1 (PF3D7_0626400), PfSec14-2 (PF3D7_0629900), PfSec14-3 (PF3D7_0717100), PfSec14-4 (PF3D7_0920700), PfSec14-5 (PF3D7_1007200), and PfSec14-6 (PF3D7_1127600). Domain organization segregates these proteins into a BNIP-2 and Cdc42GAP homology (BCH) subfamily (PfSec14-3, PfSec14-5) and a canonical Sec14 subfamily (PfSec14-1, PfSec14-2, PfSec14-4, and PfSec14-6). Yeast complementation assays showed that PfSec14-1, PfSec14-4, and PfSec14-6 partially rescue growth of a temperature-sensitive sec14 mutant, suggesting phosphatidylinositol and phosphatidylcholine transfer activity. Gene disruption revealed that PfSec14-1 is important for asexual blood-stage proliferation, whereas PfSec14-2 is dispensable under standard culture conditions. In contrast, mislocalization of PfSec14-2 and PfSec14-4 using a knock-sideways approach did not impair asexual growth. Subcellular localization indicates distinct distributions for PfSec14-1, PfSec14-2, and PfSec14-4. Together, these findings reveal potential functional and spatial diversification of Sec14-like phosphatidylinositol transfer proteins in P. falciparum.

Postnatal mouse retinal vascular development is a widely used model for studying retinal vascular diseases and evaluating candidate therapies. This is particularly relevant for inherited disorders such as familial exudative vitreoretinopathy (FEVR), in which impaired vascular growth and organization are central to disease pathogenesis. Numerous approaches have been used to assess retinal vasculature in mouse flat mounts, ranging from qualitative descriptions to limited quantitative measurements of vascular growth. However, phenotypic variability across genetic models, including different models of FEVR, complicates comparisons and underscores the need for standardized, comprehensive multi-parameter analyses that are suitable for rapid and cost-effective screening studies. We describe a standardized morphometric protocol using ImageJ software to quantitatively analyze mouse retinal vasculature in a reproducible manner. The protocol begins with measurement of areas of vascular disorganization (meshes) as well as total vascular and retinal area. Two defined regions in the peripheral and midperipheral retina are then selected to quantify cell clusters, followed by image processing, binarization, and skeletonization. From these processed images, vascular density, branch number, branch length and thickness, junction number, triple points, and box-counting fractal dimension and lacunarity are quantified. Overall, this protocol provides a rapid, cost-effective, and standardized framework for quantifying retinal vascular phenotypes across diverse mouse models. By capturing multiple structural features and accommodating phenotypic variability, it is well-suited for comparative studies and therapeutic screening in retinal vascular disease.

Loss-of-function variants of the CHKB gene cause an autosomal recessive disease described as an early onset congenital megaconial (large peripheral mitochondria) muscular dystrophy. CHKB encodes choline kinase β, the first enzyme in the biochemical pathway for synthesis of the major membrane phospholipid phosphatidylcholine. Chkb -/- mice recapitulate the human disease with affected skeletal muscle displaying a decrease in strength, myofiber atrophy, megaconial mitochondria, fat accumulation within muscle cells, and an increase in muscle injury. Here, we assessed the therapeutic potential of an AAV therapy for the treatment of CHKB-mediated muscular dystrophy. Chkb -/- mice were injected once suborbitally with three different doses of recombinant AAV9 (rAAV9) encoding human CHKB under control of a constitutive and ubiquitous promoter (AAV9-CHKB). The AAV9-CHKB-treated mice were biochemically and phenotypically indistinguishable from the wild type mice. In the Chkb -/- mouse model, all doses resulted in expression of the CHKB protein and restored choline kinase β enzyme activity, body and muscle weight, and normal muscle cell physiology, and they prevented lipid metabolism imbalance and increased the capacity to walk. These findings point to AAV9-mediated gene therapy as a potential treatment for CHKB-mediated disease.

Biallelic variants in CHKA, which encodes the first enzyme in the CDP-choline pathway for the synthesis of phosphatidylcholine, cause an inherited disorder characterized by epilepsy, microcephaly, and intellectual disability. How a deficiency in CHKA activity manifests these neurological symptoms is poorly understood. In this study, we investigated patient-derived fibroblasts with CHKA missense variants to elucidate the molecular and biochemical mechanisms underlying the associated pathologies. CHKA variant fibroblasts exhibited impaired phospholipid and triacylglycerol synthesis, altered mitochondrial morphology and function, elevated reactive oxygen species (ROS) levels, and increased lipid peroxidation, suggesting a mechanism by which defective CHKA activity leads to lipid damage. Treatment with FCCP, a mitochondrial uncoupler, reduced ROS levels and attenuated lipid peroxidation in CHKA patient fibroblasts, suggesting a potential approach to therapeutic intervention.

Familial exudative vitreoretinopathy (FEVR) is an inherited childhood blinding disorder with close to 85% of molecularly determined cases due to rare variants in the genes encoding members of the frizzled 4 (FZD4) receptor complex. FEVR causes blindness due to complications arising from developmental peripheral non-perfusion of the retina. We sought to find a small molecule that could ameliorate FEVR models. In this study, we determine that the sphingosine1-phosphate receptor 2 (S1PR2) antagonist JTE-013 can ameliorate cellular and mouse models of FEVR. Using human primary retinal microvascular endothelial cells (hRMECs), we show that either knockdown of FZD4 expression using shRNA or expression of a known FEVR-causing dominant negative allele of FZD4 decreases the ability of hRMECs to tubularize. The addition of JTE-013 to both hRMEC models of FEVR resulted in the restoration of tubularization. In the well-established Fzd4-/- mouse model of FEVR, dosing animals with JTE-013 ameliorated the retinal vascularization defects in these mice. The implications of these findings are (i) a major contributor to abnormal retinal angiogenesis in FEVR is likely through a decrease in vascular formation/integrity, and (ii) treatment with a well-characterized S1PR2 inhibitor restores normal vascularization in cell and mouse models of FEVR. To our knowledge, this is the first study to implicate S1PR signaling in FEVR and to show that a small drug-like molecule can restore normal vascularization and prevent blinding complications.

Purpose:To investigate the gene variant spectrum in patients with familial exudative vitreoretinopathy (FEVR). Methods:Probands clinically diagnosed with FEVR and their relatives were enrolled and clinical information and DNA collected. An expanded FEVR panel was used, including six recognized FEVR genes (FZD4, NDP, LRP5, TSPAN12, ZNF408, and CTNNB1) and 19 genes previously associated with ocular features overlapping FEVR (FEVR-associated genes). Variants identified using targeted next-generation sequencing and/or Sanger sequencing were analyzed and classified using the American College of Medical Genetics and Clinical Genome Resource Sequence Variant Interpretation (ClinGen SVI) working group recommendations to detect disease-causing variants (DCVs). Results:Analyses of data from a cohort of 94 probands provided a molecular diagnosis for 39 (41.5%) probands: 34 (87.2%) had a single DCV, whereas 5 (12.8%) harbored more than 1 DCV. Of 41 total DCVs in solved probands, 33 (80.5%) were in 4 of the 6 recognized genes, LRP5, FZD4, TSPAN12, and NDP, whereas 8 were found in FEVR-associated genes, 6 in KIF11, and 2 (LAMA1 and DOCK6) each in association with a KIF11 DCV. Reanalyzing variants using the latest criteria impacted the variant classification in five probands (5.3%), changing variants that were once deemed likely pathogenic to variants of uncertain significance. Conclusions:The expanded FEVR gene panel detected DCVs in nearly one-half of our cohort. Including the criteria used in classification will improve transparency of variant calls as more data become available. Four FEVR genes account for most cases, and the role of rare FEVR genes and candidate genes requires further study.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane, where it plays important roles in membrane trafficking and cytoskeletal dynamics through proteins that directly bind to it. PI(4,5)P2 can be metabolized to other phosphorylated forms of phosphatidylinositol to regulate numerous processes such as cell growth and development. PI(4,5)P2 can also be hydrolyzed to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). Altered metabolism or mislocalization of PI(4,5)P2 can perturb one or more of its functions and contribute to disease states. Here, we present a protocol to visualize and quantify the localization of PI(4,5)P2 in live cells. The protocol uses a highly specific PI(4,5)P2 protein binding domain coupled to enhanced green fluorescence protein (PH-PLCD1-GFP), enabling localization and quantification of cytosol-facing PI(4,5)P2 to be determined. Localization and quantification of the PH-PLCD1-GFP, PI(4,5)P2 specific probe, is enabled by fluorescence imaging and confocal microscopy. This approach can be used to study the dynamics of PI(4,5)P2 localization temporally in live cells under both physiological and pathological conditions. Key features • Protocol for the quantification of PI(4,5)P2 membrane localization in live cells. • Uses the expression of the highly specific PH-PLCD1-GFP, PI(4,5)P2 probe, in cells, followed by fluorescence image acquisition using confocal microscopy and subsequent image processing. • Adaptable to various cell types and experimental conditions. • Presents detailed instructions for reagent preparation, fluorescence measurement, and quantification.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane, where it plays important roles in membrane trafficking and cytoskeletal dynamics through proteins that directly bind to it. PI(4,5)P2 can be metabolized to other phosphorylated forms of phosphatidylinositol to regulate numerous processes such as cell growth and development. PI(4,5)P2 can also be hydrolyzed to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). Altered metabolism or mislocalization of PI(4,5)P2 can perturb one or more of its functions and contribute to disease states. Here, we present a protocol to visualize and quantify the localization of PI(4,5)P2 in live cells. The protocol uses a highly specific PI(4,5)P2 protein binding domain coupled to enhanced green fluorescence protein (PH-PLCD1-GFP), enabling localization and quantification of cytosol-facing PI(4,5)P2 to be determined. Localization and quantification of the PH-PLCD1-GFP, PI(4,5)P2 specific probe, is enabled by fluorescence imaging and confocal microscopy. This approach can be used to study the dynamics of PI(4,5)P2 localization temporally in live cells under both physiological and pathological conditions. Key features • Protocol for the quantification of PI(4,5)P2 membrane localization in live cells. • Uses the expression of the highly specific PH-PLCD1-GFP, PI(4,5)P2 probe, in cells, followed by fluorescence image acquisition using confocal microscopy and subsequent image processing. • Adaptable to various cell types and experimental conditions. • Presents detailed instructions for reagent preparation, fluorescence measurement, and quantification.

Abstract The World Health Organization has identified antibiotic resistance as one of the three greatest threats to human health. The need for antibiotics is a pressing matter that requires immediate attention. Here, computer-aided drug design is used to develop a structurally unique antibiotic family targeting holo-acyl carrier protein synthase (AcpS). AcpS is a highly conserved enzyme essential for bacterial survival that catalyzes the first step in lipid synthesis. To the best of our knowledge, there are no current antibiotics targeting AcpS making this drug development program of high interest. We synthesize a library of > 700 novel compounds targeting AcpS, from which 33 inhibit bacterial growth in vitro at ≤ 2 μg/mL. We demonstrate that compounds from this class have stand-alone activity against a broad spectrum of Gram-positive organisms and synergize with colistin to enable coverage of Gram-negative species. We demonstrate efficacy against clinically relevant multi-drug resistant strains in vitro and in animal models of infection in vivo including a difficult-to-treat ischemic infection exemplified by diabetic foot ulcer infections in humans. This antibiotic family could form the basis for several multi-drug-resistant antimicrobial programs.

PurposeTo report a rare clinical finding of preretinal granules associated with atypical familial exudative vitreoretinopathy (FEVR) and perform a review of the literature.ObservationsAn asymptomatic 18-year-old male was referred for unilateral peripheral avascular retina evaluation in association with presumed FEVR. He was first noted to have white preretinal granules on fundus examination at five years of age. The lesions remained unchanged over the subsequent years. Genetic testing did not reveal a pathogenic or likely pathogenic variant in a known FEVR gene. A review of the literature revealed five other cases of FEVR with similar findings.Conclusionsand Importance: Literature review suggests preretinal granules may present rarely in FEVR. Negative genetic screening of known FEVR genes in our patient with atypical FEVR suggests either a molecularly distinct etiology supporting the rarity of this association with FEVR or, alternatively, the presence of granules in developmental retinal vascular anomalies that are not specific to FEVR. Future study and genetic testing is necessary to better understand the cause of these preretinal granules and the clinical manifestations of FEVR.

Familial exudative vitreoretinopathy (FEVR) is a rare genetic disease that impedes the vascularization of the retina. Retinal vascularization occurs in utero, and many cases are diagnosed after the occurrence of vision complications. Early treatment before the development of complications is critical for the prevention of vision loss in moderate to severe cases. For this, a genetic diagnosis is essential, however, only 50% of FEVR patients have a variant in a known FEVR gene. Over 25 genes have been associated with the FEVR phenotype, with the majority of cases being in FZD4, LRP5, TSPAN12, and NDP.

Achieving a diagnosis for Indigenous people living with a rare, often genetic, disease is crucial for equitable healthcare. The International Rare Disease Research Consortium convened a global Task Force to bridge the gap in diagnosing Indigenous rare diseases, and identify solutions to tackle the health inequity faced by Indigenous people.

Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants inCHKB, encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that inChkb−/−mice there is a failure of the α7β1 integrin complex that is specific to affected muscle. We observed that inChkb−/−hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P2binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P2reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression ofCHKB.

It has been shown that atrial natriuretic peptide (ANP) and its high affinity receptor (NPRA) are involved in the formation of ventricular conduction system (VCS). Inherited genetic variants in fatty acid oxidation (FAO) genes are known to cause conduction abnormalities in newborn children. Although the effect of ANP on energy metabolism in noncardiac cell types is well documented, the role of lipid metabolism in VCS cell differentiation via ANP/NPRA signaling is not known. In this study, histological sections and primary cultures obtained from E11.5 mouse ventricles were analyzed to determine the role of metabolic adaptations in VCS cell fate determination and maturation. Exogenous treatment of E11.5 ventricular cells with ANP revealed a significant increase in lipid droplet accumulation, FAO and higher expression of VCS marker Cx40. Using specific inhibitors, we further identified PPARγ and FAO as critical downstream regulators of ANP-mediated regulation of metabolism and VCS formation.

In Saccharomyces cerevisiae, the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid synthesis responding to the abundance of the phospholipid precursor phosphatidic acid at the endoplasmic reticulum. We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function mutations and the characterization of the strongest variant identified, Opi1N150D. LZ modeling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1N150D variant is dominant, and its phenotype is attenuated when loss of function mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region, we performed in vitro analyses to explore interdomain interactions. An Opi11-190 probe interacted with Opi1291-404, the C terminus that bears the activator interacting domain (AID). LZ or AID loss of function mutations attenuated the interaction of the probes but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane-binding events and LZ dimerization antagonizes intramolecular LZ-AID interaction and transcriptional repression.

The CHKB gene encodes choline kinase β, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb-/- mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb+/- mice. Unlike wildtype mice, 60% of the Chkb+/- and all Chkb-/- mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb+/- and Chkb-/- hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb-/- hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb+/- and Chkb-/- mice.