Accumulation of Seminolipid in Sertoli Cells Is Associated with Increased Levels of Reactive Oxygen Species and Male Subfertility: Studies in Aging Arsa Null Male Mice

Seminolipid (also known as sulfogalactosylglycerolipid-SGG), present selectively in male germ cells, plays important roles in spermatogenesis and sperm–egg interaction. The proper degradation of SGG in apoptotic germ cells is also as important. Sertoli cells first phagocytose apoptotic germ cells, then Sertoli lysosomal arylsulfatase A (ARSA) desulfates SGG, the first step of SGG degradation. We have reported that aging male Arsa−/− mice become subfertile with SGG accumulation in Sertoli cell lysosomes, typical of a lysosomal storage disorder (LSD). Since reactive oxygen species (ROS) levels are increased in other glycolipid-accumulated LSDs, we quantified ROS in Arsa−/− Sertoli cells. Our analyses indicated increases in superoxide and H2O2 in Arsa−/− Sertoli cells with elevated apoptosis rates, relative to WT counterparts. Excess H2O2 from Arsa−/− Sertoli cells could travel into testicular germ cells (TGCs) to induce ROS production. Our results indeed indicated higher superoxide levels in Arsa−/− TGCs, compared with WT TGCs. Increased ROS levels in Arsa−/− Sertoli cells and TGCs likely caused the decrease in spermatogenesis and increased the abnormal sperm population in aging Arsa−/− mice, including the 50% decrease in sperm SGG with egg binding ability. In summary, our study indicated that increased ROS production was the mechanism through which subfertility manifested following SGG accumulation in Sertoli cells

Visualizing the localization of sulfoglycolipids in lipid raft domains in model membranes and sperm membrane extracts

Sulfogalactosylglycerolipid (SGG) is found in detergent-resistant lipid raft fractions isolated from sperm plasma membranes and has been shown to be important in sperm-egg adhesion. In order to provide more direct evidence for the association of sulfoglycolipids with lipid raft domains, we have examined the distribution of two sulfoglycolipids in supported membranes prepared from artificial lipid mixtures and cellular lipid extracts. Atomic force microscopy has been used to visualize the localization of SGG and sulfogalactosylceramide (SGC) in liquid-ordered domains in supported bilayers of ternary lipid mixtures comprised of dipalmitoylphosphatidylcholine, cholesterol and palmitoyldocosahexaenoylphosphatidylcholine. The localization of SGC/SGG in the liquid-ordered raft domains is demonstrated by changes in bilayer morphology in the presence of sulfoglycolipid, by selective antibody labeling of the domains with anti-SGC/SGG and by the effects of the cholesterol-sequestering agent, methyl-beta-cyclodextrin, on the supported membranes. In addition, we use a combination of atomic force microscopy and immunofluorescence to show that supported bilayers made from lipids extracted from sperm anterior head plasma membranes (APM) and isolated APM vesicles exhibit small SGG-rich domains that are similar to those observed in bilayers of artificial lipid mixtures. The possible implications of these results for the involvement of SGG-rich lipid rafts in modulating sperm-egg interactions in vivo and the utility of model membranes for studying the behavior of lipid rafts are discussed.NRC publication: Ye

Lipidomic Profiling of Mastoid Bone and Tissue from Patients with Chronic Otomastoiditis

Introduction Chronic otomastoiditis causes pain, otorrhea, and hearing loss resulting from the growth of tissue within the normally hollow mastoid cavity. Objectives In this report, we used a lipidomics approach to profile major mastoid bone and tissue lipids from patients with and without otomastoiditis. Methods The bone dust created during mastoidectomy, as well as the mastoid tissue, was analyzed from seven patients. Bone dust was also collected and analyzed in an additional four otologic cases (parotidectomy requiring mastoidectomy). Samples were subjected to a modified Bligh/Dyer lipid extraction, then high-performance thin-layer chromatography (HPTLC), combined gas chromatography/electron impact-mass spectrometry (GC/EI-MS), and flow-injection/electrospray ionization-tandem mass spectrometry (FI/ESI-MSMS). Data were analyzed for identification and profiling of major lipid components. Results HPTLC revealed the presence of various lipid classes, including phosphatidylcholines, cholesterol, and triacylglycerols. GC/EI-MS analysis revealed the presence of cholesterol and several fatty acids. FI/ESI-MSMS analysis revealed a host of phosphatidylcholines, phosphatidylethanolamines, and cholesteryl esters. Conclusion We used a lipidomics approach to develop an efficient (both in time and tissue amount) methodology for analysis of these tissues, identify the most abundant and common lipid species, and create a base of knowledge from which more focused endeavors in biomarker discovery can emerge. In an effort toward improved patient categorization and individualized intervention, the ultimate goal of this work is to correlate these lipid molecules to disease state and progression. This is the first reported study of its kind on these tissues

Potential Use of Antimicrobial Peptides as Vaginal Spermicides/Microbicides

The concurrent increases in global population and sexually transmitted infection (STI) demand a search for agents with dual spermicidal and microbicidal properties for topical vaginal application. Previous attempts to develop the surfactant spermicide, nonoxynol-9 (N-9), into a vaginal microbicide were unsuccessful largely due to its inefficiency to kill microbes. Furthermore, N-9 causes damage to the vaginal epithelium, thus accelerating microbes to enter the women’s body. For this reason, antimicrobial peptides (AMPs), naturally secreted by all forms of life as part of innate immunity, deserve evaluation for their potential spermicidal effects. To date, twelve spermicidal AMPs have been described including LL-37, magainin 2 and nisin A. Human cathelicidin LL-37 is the most promising spermicidal AMP to be further developed for vaginal use for the following reasons. First, it is a human AMP naturally produced in the vagina after intercourse. Second, LL-37 exerts microbicidal effects to numerous microbes including those that cause STI. Third, its cytotoxicity is selective to sperm and not to the female reproductive tract. Furthermore, the spermicidal effects of LL-37 have been demonstrated in vivo in mice. Therefore, the availability of LL-37 as a vaginal spermicide/microbicide will empower women for self-protection against unwanted pregnancies and STI

Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin

ABSTRACT Dengue virus (DENV) is a major human pathogen. An important pathogenicity factor is non-structural protein 1 (NS1) required for viral replication and secreted from infected cells. A clinical study indicated that the anti-parasitic drug ivermectin lowers NS1 blood levels without affecting viremia. Ivermectin targets nuclear transport by binding to importin-α, but how NS1 secretion in patients is suppressed by this drug is unknown. We show that ivermectin impairs folding and secretion of endoplasmic reticulum-luminal glycoproteins, including NS1. Proteomic analysis identified chaperones interacting with NS1, including GRp78 (78-kDa glucose-regulated protein, also known as HSPA5 or BIP). This chaperone increased in abundance on DENV infection via activation of the unfolded protein response (UPR). Ivermectin blocked the nuclear transport of transcription factors required for UPR, thus impairing GRp78 upregulation and NS1 secretion. Reduction of GRp78 and NS1 secretion was also observed in patients treated with ivermectin. These results link nuclear transport and its inhibition by ivermectin to folding and secretion of luminal glycoproteins, including DENV NS1. IMPORTANCE Dengue virus (DENV) is a major human pathogen that can cause hemorrhagic fever and shock syndrome. One important factor of DENV pathogenicity is non-structural protein 1 (NS1), a glycoprotein that is secreted from infected cells. Here we study the mode of action of the widely used drug ivermectin, used to treat parasitic infections and recently shown to lower NS1 blood levels in DENV-infected patients. We found that ivermectin blocks the nuclear transport of transcription factors required for the expression of chaperones that support the folding and secretion of glycoproteins, including NS1. Impairing nuclear transport of these transcription factors by ivermectin or depleting them from infected cells dampens NS1 folding and thus its secretion. These results reveal a novel mode of action of ivermectin that might apply to other flaviviruses as well

Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification

Dengue virus (DENV) infection is a significant global health problem. There are no specific therapeutics or widely available vaccines. Early diagnosis is critical for patient management. Viral RNA detection by multiplex RT-PCR using multiple pairs of primers/probes allowing the simultaneous detection of all four DENV serotypes is commonly used. However, increasing the number of primers in the RT-PCR reaction reduces the sensitivity of detection due to the increased possibility of primer dimer formation. Here, a one tube, singleplex real-time RT-PCR specific to DENV 3′-UTR was developed for the detection and quantification of pan-DENV with no cross reactivity to other flaviviruses. The sensitivity of DENV detection was as high as 96.9% in clinical specimens collected at the first day of hospitalization. Our assay provided equivalent PCR efficiency and RNA quantification among each DENV serotype. The assay’s performance was comparable with previously established real-time RT-PCR targeting coding sequences. Using both assays on the same specimens, our results indicate the presence of defective virus particles in the circulation of patients infected with all serotypes. Dual regions targeting RT-PCR enhanced the sensitivity of viral genome detection especially during the late acute phase when viremia rapidly decline and an incomplete viral genome was clinically evident

Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy.

Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects

Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design