Congenital red hair heterochromia in the background of blond scalp hair.

We report a case of isolated patchy red hair heterochromia of the scalp in a healthy 6-year-old boy. Isolated patchy scalp hair heterochromia is the presence of two or more colors of hair in the same individual, thought to be due to genetic mosaicism although no specific etiology has been widely accepted. We have outlined a basic approach to diagnosing different types of heterochromia for clinical application.

Updates in the Diagnosis and Management of Linear IgA Disease: A Systematic Review.

Background and Objectives: Linear IgA disease (LAD) is a rare autoimmune blistering disease with linear IgA deposits along the basement membrane zone. Direct immunofluorescence remains the gold standard for diagnosis, but other diagnostic measures reported in recent literature have proven useful in the setting of inconclusive preliminary results. Dapsone is a commonly used treatment, but many therapeutic agents have emerged in recent years. The objective of this study is to provide a comprehensive overview of updates on the diagnosis and management of LAD. Materials and Methods: A literature search was conducted from May to June of 2021 for articles published in the last 5 years that were related to the diagnosis and management of LAD. Results: False-negative results in cases of drug-induced LAD and the presence of IgG and IgM antibodies on immunofluorescence studies were reported. Serration pattern analysis has been reported to be useful in distinguishing LAD from sublamina densa-type LAD. Rituximab, omalizumab, etanercept, IVIg, sulfonamides, topical corticosteroids, and others have been used successfully in adult and pediatric patients with varying disease severity. Topical corticosteroids were preferred for pediatric patients while rituximab and IVIg were used in adults with recalcitrant LAD. Sulfonamides were utilized in places without access to dapsone. Conclusion: In cases where preliminary biopsy results are negative and clinical suspicion is high, repeat biopsy and additional diagnostic studies should be used. Patient factors such as age, medical comorbidities, and disease severity play a role in therapeutic selection.

A role for triglyceride lipase brummer in the regulation of sex differences in Drosophila fat storage and breakdown.

Triglycerides are the major form of stored fat in all animals. One important determinant of whole-body fat storage is whether an animal is male or female. Here, we use Drosophila, an established model for studies on triglyceride metabolism, to gain insight into the genes and physiological mechanisms that contribute to sex differences in fat storage. Our analysis of triglyceride storage and breakdown in both sexes identified a role for triglyceride lipase brummer (bmm) in the regulation of sex differences in triglyceride homeostasis. Normally, male flies have higher levels of bmm mRNA both under normal culture conditions and in response to starvation, a lipolytic stimulus. We find that loss of bmm largely eliminates the sex difference in triglyceride storage and abolishes the sex difference in triglyceride breakdown via strongly male-biased effects. Although we show that bmm function in the fat body affects whole-body triglyceride levels in both sexes, in males, we identify an additional role for bmm function in the somatic cells of the gonad and in neurons in the regulation of whole-body triglyceride homeostasis. Furthermore, we demonstrate that lipid droplets are normally present in both the somatic cells of the male gonad and in neurons, revealing a previously unrecognized role for bmm function, and possibly lipid droplets, in these cell types in the regulation of whole-body triglyceride homeostasis. Taken together, our data reveal a role for bmm function in the somatic cells of the gonad and in neurons in the regulation of male-female differences in fat storage and breakdown and identify bmm as a link between the regulation of triglyceride homeostasis and biological sex.

G0S2 represses PI3K/mTOR signaling and increases sensitivity to PI3K/mTOR pathway inhibitors in breast cancer.

G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target implicated in cancer biology and therapy based on frequent methylation-mediated silencing in diverse solid tumors. We recently reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, correlates with increased rates of recurrence, indicating that G0S2 plays a role in breast cancer progression. However, the function(s) and mechanism(s) of G0S2 tumor suppression remain unclear. In order to determine potential mechanisms of G0S2 anti-oncogenic activity, we performed genome-wide expression analysis that revealed an enrichment of gene signatures related to PI3K/mTOR pathway activation in G0S2 null cells as compared to G0S2 wild-type cells. G0S2 null cells also exhibited a dramatic decreased sensitivity to PI3K/mTOR pathway inhibitors. Conversely, restoring G0S2 expression in human ER+ breast cancer cells decreased basal mTOR signaling and sensitized the cells to pharmacologic mTOR pathway inhibitors. Notably, we provide evidence here that the increase in recurrence seen with low G0S2 expression is especially prominent in patients who have undergone antiestrogen therapy. Further, ER+ breast cancer cells with restored G0S2 expression had a relative increased sensitivity to tamoxifen. These findings reveal that in breast cancer G0S2 functions as a tumor suppressor in part by repressing PI3K/mTOR activity, and that G0S2 enhances therapeutic responses to PI3K/mTOR inhibitors. Recent studies implicate hyperactivation of PI3K/mTOR signaling as promoting resistance to antiestrogen therapies in ER+ breast cancer. Our data establishes G0S2 as opposing this form of antiestrogen resistance. This promotes further investigation of the role of G0S2 as an antineoplastic breast cancer target and a biomarker for recurrence and therapy response.

Refractory testicular germ cell tumors are highly sensitive to the second generation DNA methylation inhibitor guadecitabine.

Testicular germ cell tumors (TGCTs) are the most common cancers of young males. A substantial portion of TGCT patients are refractory to cisplatin. There are no effective therapies for these patients, many of whom die from progressive disease. Embryonal carcinoma (EC) are the stem cells of TGCTs. In prior in vitro studies we found that EC cells were highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). Here, as an initial step in bringing demethylation therapy to the clinic for TGCT patients, we evaluated the effects of the clinically optimized, second generation demethylating agent guadecitabine (SGI-110) on EC cells in an animal model of cisplatin refractory testicular cancer. EC cells were exquisitely sensitive to guadecitabine and the hypersensitivity was dependent on high levels of DNA methyltransferase 3B. Guadecitabine mediated transcriptional reprogramming of EC cells included induction of p53 targets and repression of pluripotency genes. As a single agent, guadecitabine completely abolished progression and induced complete regression of cisplatin resistant EC xenografts even at doses well below those required to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin in vivo. Genome-wide analysis indicated that in vivo antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. These preclinical findings suggest that guadecitabine alone or in combination with cisplatin is a promising strategy to treat refractory TGCT patients.

Neuronal porosome proteome: Molecular dynamics and architecture.

Porosomes are the universal secretory portals at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the outside during cell secretion. In the past decade, the neuronal porosome complex, a 10-15nm cup-shaped lipoprotein structure has been isolated, its partial composition and 3D contour map determined, and it has been functionally reconstituted into artificial lipid membrane. Here we further determine the composition of the neuronal porosome proteome using immunoisolation and gel filtration chromatography, followed by tandem mass spectrometry. Results from the study demonstrate nearly 40 proteins to constitute the neuronal porosome proteome. Furthermore, interaction of proteins within the porosome and their resulting arrangement is predicted. The association and dissociation of proteins at the porosome following stimulation of cell secretion demonstrate the dynamic nature of the organelle.

Lysophosphatidylcholine inhibits membrane-associated SNARE complex disassembly.

In cells, N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors called SNAREs are involved in membrane fusion. In neurons, for example, target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the pre-synaptic membrane, and a synaptic vesicle-associated membrane protein (VAMP) or v-SNARE, is part of the conserved protein complex involved in neurotransmission. Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes. In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly. Exposure of cholesterol-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP results in dissociated vesicles. In contrast, exposure of LPC-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP, results in inhibition of t-/v-SNARE disassembly and the consequent accumulation of clustered vesicles. Similarly, exposure of isolated rat brain slices and pancreas to cholesterol or LPC, also demonstrates LPC-induced inhibition of SNARE complex disassembly. Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer. The altered plasma LPC levels observed in various cancers may in part contribute to defects in SNARE assembly-disassembly and membrane fusion, consequently affecting protein maturation and secretion in cancer cells.

Membrane-directed molecular assembly of the neuronal SNARE complex.

Since the discovery and implication of N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full-length recombinant t-SNAREs and v-SNARE present in opposing liposomes, leading to the assembly of a t-/v-SNARE ring complex. Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution. Similar to the t-/v-SNARE ring complex formed when 50 nm v-SNARE liposomes meet a t-SNARE-reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t-SNARE-reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.

Involvement of ß-adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release.

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G(αo) -mediated water channel aquaporin-6 (AQP-6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G(o) protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α-helix that resembles the intracellular loops of G protein-coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous ß-adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using ß-adrenergic receptor antibody, and vesicle swelling experiments using ß-adrenergic agonists and antagonists, demonstrate the presence of functional ß-adrenergic receptors at the SV membrane. Since a recent study shows vH(+) -ATPase to be upstream of AQP-6 in the pathway leading from G(αo) -mediated swelling of SV, participation of an endogenous ß-adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.

Involvement of cholesterol in synaptic vesicle swelling.

Studies demonstrate that cholesterol plays a critical role in the regulation of neurotransmitter release and that secretory vesicle swelling is a requirement for the regulated expulsion of intravesicular contents during cell secretion. In view of this, the involvement of cholesterol in synaptic vesicle swelling was hypothesized and tested in the present study, using isolated synaptic vesicles from rat brain and the determination of their swelling competency in the presence and absence of cholesterol. The involvement of the water channel aquaporin-6 (AQP-6) and proton pump vH(+)-ATPase in GTP-G(alpha o)-mediated synaptic vesicle swelling has been reported previously. Mastoparan, the amphiphilic tetradecapeptide from wasp venom, known to activate the GTPase activity of G(alpha o/i) proteins, stimulates synaptic vesicle swelling in the presence of GTP. In the current study, using nanometer-scale precision measurements of isolated synaptic vesicles, we report for the first time that depletion of cholesterol from synaptic vesicle membrane results in a significant loss of GTP-mastoparan-stimulable synaptic vesicle swelling. In contrast, incorporation of cholesterol into the synaptic vesicle membrane potentiates GTP-mastoparan-stimulable vesicle swelling. Our study further demonstrates that this effect of cholesterol is due, in part, to its involvement in the interactions between AQP-6, vH(+)-ATPase and the GTP-binding G(alpha o) protein at the synaptic vesicle membrane.

Membrane lipids influence protein complex assembly-disassembly.

Approximately 11% smaller t-/v-SNARE ring complexes are generated using 50 nm cholesterol-associated vesicles as opposed to vesicles containing L-alpha-lysophosphatidylcholine (LPC), as observed using atomic force microscopy. Circular dichroism spectroscopy demonstrated that in the presence of LPC as opposed to cholesterol, N-ethylmaleimide-sensitive factor + adenosine triphosphate induces disassembly of beta-sheet structures but not the alpha-helical contents within the t-/v-SNARE complex.

Involvement of vH(+)-ATPase in synaptic vesicle swelling.

Secretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The G(alphai3)-PLA2-mediated involvement of water channel AQP-1 in the regulation of secretory vesicle swelling in exocrine pancreas and the G(alphao)-mediated AQP-6 involvement in synaptic vesicle swelling in neurons have previously been reported. Furthermore, the role of vH(+)-ATPase in neurotransmitter transport into synaptic vesicles has also been shown. Using nanometer-scale precision measurements of isolated synaptic vesicles, the present study reports for the first time the involvement of vH(+)-ATPase in GTP-G(alphao)-mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP-G(alphao)-mediated vesicle swelling is vH(+)-ATPase dependent and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate a bafilomycin-sensitive vesicle acidification, following the GTP-G(alphao)-induced swelling stimulus. Water channels are bidirectional and the vH(+)-ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP-mastoparan stimulated swelling, suggesting that vH(+)-ATPase is upstream of AQP-6, in the pathway leading from G(alphao)-stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP-6-mediated gating of water into synaptic vesicles.

Structure of membrane-associated neuronal SNARE complex: implication in neurotransmitter release.

To enable fusion between biological membranes, t-SNAREs and v-SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring-complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t-/v-SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE-ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40-50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12-17 nm in diameter cup-shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t-/v-SNARE complex, 50 nm t-and v-SNARE proteoliposomes were mixed, allowing t-SNARE-vesicles to interact with v-SNARE vesicles, followed by detergent solubilization and imaging of the resultant t-/v-SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3-4 nm base diameter, where 40-50 nm in diameter v-SNARE-associated synaptic vesicle transiently dock and fuse to release neurotransmitters.