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Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

The role of phosphinositide metabolism, an essential role, in synaptic vesicle recycling is discussed relative to results of a study. Synaptojanin 1-deficient mice were found to show neurological defects and die soon after birth. In neurons of mutants, clathrin-coated vesicles accumulate int he cytomatrix-rich area surrounding the synaptic vesicle cluster in nerve endings.

Several inositol-containing compounds play key roles in receptor-mediated cell signaling events. Here, we describe a function for a specific inositol polyphosphate, D-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)[P.sub.4]], that is produced acutely in response to a receptor-independent process. Thus, infection of intestinal epithelial cells with the enteric pathogen Salmonella, but not with other invasive bacteria, induced a multifold increase in Ins(1,4,5,6)[P.sub.4] levels. To define a specific function of Ins(1,4,5,6)[P.sub.4], a membrane-permeant, hydrolyzable ester was used to deliver it to the intracellular compartment, where it antagonized epidermal growth factor (EGF)-induced inhibition of calcium-mediated chloride ([Cl.sup.-]) secretion (CaMCS) in intestinal epithelia. This EGF function is likely mediated through a phosphoinositide 3-kinase (PtdIns3K)-dependent mechanism because the EGF effects are abolished by wortmannin, and three different membrane-permeant esters of the PtdIns3K product phosphatidylinositol 3,4,5-trisphosphate mimicked the EGF effect on CaMCS. We further demonstrate that Ins(1,4,5,6)[P.sub.4] antagonized EGF signaling downstream of PtdIns3K because Ins(1,4,5,6)[P.sub.4] interfered with the PtdIns[P.sub.3] effect on CaMCS without affecting PtdIns3K activity. Thus, elevation of Ins(1,4,5,6)[P.sub.4] in Salmonella-infected epithelia may promote [Cl.sup.-] flux by antagonizing EGF inhibition mediated through PtdIns3K and PtdIns[P.sub.3].

A study of concomitant inositol phosphate metabolism to understand the dissociation between Cl- secretion and intracellular calcium levels revealed that the uncoupling was influenced by D-myo-inositol 3,4,5, 6-tetrakisphosphate (Ins(3,4,5,6)P4). Intracellular administration of Ins(3,4, 5,6)P4 by a new membrane-permeant ester confirmed the inhibiting quality of the inositol phosphate on Cl- flux. The study also revealed the long lasting effect of agonists that extended beyond the duration of (Ca2+)i influence on Cl- secretion.

Identification of common patterns of cancer metabolic reprogramming could assist the development of new therapeutic strategies. Recent attention in this field has focused on identifying and targeting signal transduction pathways that interface directly with major metabolic control processes. In the current study we demonstrate the importance of signaling by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to the metabolism and proliferation of the HCT116 colonic tumor cell line. We observed reciprocal cross talk between PPIP5K catalytic activity and glucose metabolism, and we show that CRISPR-mediated PPIP5K deletion suppresses HCT116 cell proliferation in glucose-limited culture conditions that mimic the tumor cell microenvironment. We conducted detailed, global metabolomic analyses of wild-type and PPIP5K knockout (KO) cells by measuring both steady-state metabolite levels and by performing isotope tracing experiments. We attribute the growth-impaired phenotype to a specific reduction in the supply of precursor material for de novo nucleotide biosynthesis from the one carbon serine/glycine pathway and the pentose phosphate pathway. We identify two enzymatic control points that are inhibited in the PPIP5K KO cells: serine hydroxymethyltransferase and phosphoribosyl pyrophosphate synthetase, a known downstream target of AMP-regulated protein kinase, which we show is noncanonically activated independently of adenine nucleotide status. Finally, we show the proliferative defect in PPIP5K KO cells can be significantly rescued either by addition of inosine monophosphate or a nucleoside mixture or by stable expression of PPIP5K activity. Overall, our data describe multiple, farreaching metabolic consequences for metabolic supervision by PPIP5Ks in a tumor cell line. [ABSTRACT FROM AUTHOR]

Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP8 synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP8) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP8 levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP8 to the XPR1 N-terminus (Kd = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification. [ABSTRACT FROM AUTHOR]

E-cigarettes are noncombustible, electronic nicotinedelivery devices that aerosolize an e-liquid, i.e., nicotine, in a propylene glycol-vegetable glycerin vehicle that also contains flavors. While the effects of nicotine are relatively well understood, more information regarding the potential biological effects of the other e-liquid constituents is needed. This is a serious concern, because e-liquids are available in >7,000 distinct flavors. We previously demonstrated that many e-liquids affect cell growth/viability through an unknown mechanism. Since Ca2+ is a ubiquitous second messenger that regulates cell growth, we characterized the effects of e-liquids on cellular Ca2+ homeostasis. To better understand the extent of this effect, we screened e-liquids for their ability to alter cytosolic Ca2+ levels and found that 42 of 100 flavored e-liquids elicited a cellular Ca2+ response. Banana Pudding (BP) e-liquid, a representative e-liquid from this group, caused phospholipase C activation, endoplasmic reticulum (ER) Ca2+ release, store-operated Ca2+ entry (SOCE), and protein kinase C (PKCα) phosphorylation. However, longer exposures to BP e-liquid depleted ER Ca2+ stores and inhibited SOCE, suggesting that this e-liquid may alter Ca2+ homeostasis by short- and long-term mechanisms. Since dysregulation of Ca2+ signaling can cause chronic inflammation, ER stress, and abnormal cell growth, flavored e-cigarette products that can elicit cell Ca2+ responses should be further screened for potential toxicity. [ABSTRACT FROM AUTHOR]