Miracle-dz/UniFAR-FTUs huggingface dataset:
SPHK1/sphingosine kinase 1-mediated autophagy differs between neurons and SH-SY5Y neuroblastoma cells

ABSTRACT Although implicated in neurodegeneration, autophagy has been characterized mostly in yeast and mammalian non-neuronal cells. In a recent study, we sought to determine if SPHK1 (sphingosine kinase 1), implicated previously in macroautophagy/autophagy in cancer cells, regulates autophagy in neurons. SPHK1 synthesizes sphingosine-1-phosphate (S1P), a bioactive lipid involved in cell survival. In our study, we discovered that, when neuronal autophagy is pharmacologically stimulated, SPHK1 relocalizes to the endocytic and autophagic organelles. Interestingly, in non-neuronal cells stimulated with growth factors, SPHK1 translocates to the plasma membrane, where it phosphorylates sphingosine to produce S1P. Whether SPHK1 also binds to the endocytic and autophagic organelles in non-neuronal cells upon induction of autophagy has not been demonstrated. Here, we determined if the effect in neurons is operant in the SH-SY5Y neuroblastoma cell line. In both non-differentiated and differentiated SH-SY5Y cells, a short incubation of cells in amino acid-free medium stimulated the formation of SPHK1-positive puncta, as in neurons. We also found that, unlike neurons in which these puncta represent endosomes, autophagosomes, and amphisomes, in SH-SY5Y cells SPHK1 is bound only to the endosomes. In addition, a dominant negative form of SPHK1 was very toxic to SH-SY5Y cells, but cultured primary cortical neurons tolerated it significantly better. These results suggest that autophagy in neurons is regulated by mechanisms that differ, at least in part, from those in SH-SY5Y cells.
SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

ABSTRACT We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H+ pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.
PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

ABSTRACT Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.
A new probe to measure autophagic flux in vitro and in vivo

ABSTRACT Macroautophagy is a catabolic process that delivers cytoplasmic components via the autophagosome to lysosomes for degradation. Measuring autophagic activity is critical to dissect molecular mechanisms and functions of autophagy but remains challenging due to the lack of a definitive method. We have recently developed a new fluorescent probe, GFP-LC3-RFP-LC3ΔG, to assess autophagic flux. Upon intracellular expression, the probe is cleaved by ATG4 family proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. The former is degraded by autophagy while the latter persists as an internal control in the cytosol. Autophagic flux can thus be quantified by obtaining the ratio of GFP:RFP signals. Using this method, we have identified several autophagy-modulating drugs by screening an approved drug library. We have also demonstrated that induced and basal autophagic flux can be monitored in zebrafish and mice.
Histidine‐rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease

Pathogen‐ and injury‐related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver‐derived plasma protein, histidine‐rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to mediate the transition of alternatively activated (M2) to proinflammatory (M1) macrophages, which limit tumor growth and metastasis. We hypothesized that liver‐derived HRG is a critical endogenous modulator of hepatic macrophage functionality and investigated its implications for liver inflammation and fibrosis by comparing C57BL/6N wild‐type (WT) and Hrg−/− mice. In homeostatic conditions, hepatic macrophages were overall reduced and preferentially polarized toward the anti‐inflammatory M2 subtype in Hrg−/− mice. Upon chronic liver damage induced by CCl4 or methionine‐choline‐deficient (MCD) diet, liver injury and fibrosis were attenuated in Hrg−/−, compared to WT, mice. Macrophage populations were reduced and skewed toward M2 polarization in injured livers of Hrg−/− mice. Moreover, HRG‐deficient mice showed significantly enhanced hepatic vascularization by micro‐computed tomography and histology, corroborating proangiogenic activities of M2‐polarized liver macrophages. Purified HRG protein induced, but HRG‐deficient serum prevented, M1 macrophage differentiation in vitro. Accordingly, Hrg−/− mice transplanted with Hrg+/+ bone marrow, but not Hrg−/−‐transplanted Hrg+/+ mice, remained protected from experimental steatohepatitis. Consistent with these findings, patients with chronic hepatitis C and nonalcoholic steatohepatitis significantly up‐regulated hepatocytic HRG expression, which was associated with M1 polarization of adjacent macrophages. Conclusions: Liver‐derived HRG, similar to alarmins, appears to be an endogenous molecular factor promoting polarization of hepatic macrophages toward the M1 phenotype, thereby promoting chronic liver injury and fibrosis progression, but limiting angiogenesis. Therefore, controlling tissue levels of HRG or PGF might be a promising strategy in chronic inflammatory liver diseases. (Hepatology 2016;63:1310‐1324)
The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced, hippocampal-dependent impairment of learning and memory ability

ABSTRACT Central nervous system (CNS) inflammation and autophagy dysfunction are known to be involved in the pathology of neurodegenerative diseases. Manganese (Mn), a neurotoxic metal, has the potential to induce microglia-mediated neuroinflammation as well as autophagy dysfunction. NLRP3 (NLR family, pyrin domain containing 3)- CASP1 (caspase 1) inflammasome-mediated neuroinflammation in microglia has specific relevance to neurological diseases. However, the mechanism driving these phenomena remains poorly understood. We demonstrate that Mn activates the NLRP3-CASP1 inflammasome pathway in the hippocampus of mice and BV2 cells by triggering autophagy-lysosomal dysfunction. The autophagy-lysosomal dysfunction is induced by lysosomal damage caused by excessive Mn accumulation, damaging the structure and normal function of these organelles. Additionally, we show that the release of lysosomal CTSB (cathepsin B) plays an important role in Mn-induced NLRP3-CASP1 inflammasome activation, and that the increased autophagosomes in the cytoplasm are not the main cause of NLRP3-CASP1 inflammasome activation. The accumulation of proinflammatory cytokines, such as IL1B (interleukin 1 β) and IL18 (interleukin 18), as well as the dysfunctional autophagy pathway may damage hippocampal neuronal cells, thus leading to hippocampal-dependent impairment in learning and memory, which is associated with the pathogenesis of Alzheimer disease (AD).
Protein tyrosine phosphatase 1B dephosphorylates PITX1 and regulates p120RasGAP in hepatocellular carcinoma

The effective therapeutic targets for hepatocellular carcinoma remain limited. Pituitary homeobox 1 (PITX1) functions as a tumor suppressor in hepatocarcinogenesis by regulating the expression level of Ras guanosine triphosphatase‐activating protein. Here, we report that protein tyrosine phosphatases 1B (PTP1B) directly dephosphorylated PITX1 at Y160, Y175, and Y179 to further weaken the protein stability of PITX. The PTP1B‐dependent decline of PITX1 reduced its transcriptional activity for p120RasGAP (RASA1), a Ras guanosine triphosphatase‐activating protein. Both silencing of PTP1B and PTP1B inhibitor up‐regulated the PITX1‐p120RasGAP axis through hyperphosphorylation of PITX1. Sorafenib, the first and only targeted drug approved for hepatocellular carcinoma, directly decreased PTP1B activity and promoted the expression of PITX1 and p120RasGAP by PITX1 hyperphosphorylation. Molecular docking also supported the potential interaction between PTP1B and sorafenib. PTP1B overexpression impaired the sensitivity of sorafenib in vitro and in vivo, implying that PTP1B has a significant effect on sorafenib‐induced apoptosis. In sorafenib‐treated tumor samples, we further found inhibition of PTP1B activity and up‐regulation of the PITX1‐p120RasGAP axis, suggesting that PTP1B inhibitor may be effective for the treatment of hepatocellular carcinoma. By immunohistochemical staining of hepatic tumor tissue from 155 patients, the expression of PTP1B was significantly in tumor parts higher than nontumor parts (P = 0.02). Furthermore, high expression of PTP1B was significantly associated with poor tumor differentiation (P = 0.031). Conclusion: PTP1B dephosphorylates PITX1 to weaken its protein stability and the transcriptional activity for p120RasGAP gene expression and acts as a determinant of the sorafenib‐mediated drug effect; targeting the PITX1‐p120RasGAP axis with a PTP1B inhibitor may provide a new therapy for patients with hepatocellular carcinoma. (Hepatology 2016;63:1528‐1543)
HIF1A and NFAT5 coordinate Na+-boosted antibacterial defense via enhanced autophagy and autolysosomal targeting

ABSTRACT Infection and inflammation are able to induce diet-independent Na+-accumulation without commensurate water retention in afflicted tissues, which favors the pro-inflammatory activation of mouse macrophages and augments their antibacterial and antiparasitic activity. While Na+-boosted host defense against the protozoan parasite Leishmania major is mediated by increased expression of the leishmanicidal NOS2 (nitric oxide synthase 2, inducible), the molecular mechanisms underpinning this enhanced antibacterial defense of mouse macrophages with high Na+ (HS) exposure are unknown. Here, we provide evidence that HS-increased antibacterial activity against E. coli was neither dependent on NOS2 nor on the phagocyte oxidase. In contrast, HS-augmented antibacterial defense hinged on HIF1A (hypoxia inducible factor 1, alpha subunit)-dependent increased autophagy, and NFAT5 (nuclear factor of activated T cells 5)-dependent targeting of intracellular E. coli to acidic autolysosomal compartments. Overall, these findings suggest that the autolysosomal compartment is a novel target of Na+-modulated cell autonomous innate immunity. Abbreviations: ACT: actins; AKT: AKT serine/threonine kinase 1; ATG2A: autophagy related 2A; ATG4C: autophagy related 4C, cysteine peptidase; ATG7: autophagy related 7; ATG12: autophagy related 12; BECN1: beclin 1; BMDM: bone marrow-derived macrophages; BNIP3: BCL2/adenovirus E1B interacting protein 3; CFU: colony forming units; CM-H2DCFDA: 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; CTSB: cathepsin B; CYBB: cytochrome b-245 beta chain; DAPI: 4,6-diamidino-2-phenylindole; DMOG: dimethyloxallyl glycine; DPI: diphenyleneiodonium chloride; E. coli: Escherichia coli; FDR: false discovery rate; GFP: green fluorescent protein; GSEA: gene set enrichment analysis; GO: gene ontology; HIF1A: hypoxia inducible factor 1, alpha subunit; HUGO: human genome organization; HS: high salt (+ 40 mM of NaCl to standard cell culture conditions); HSP90: heat shock 90 kDa proteins; LDH: lactate dehydrogenase; LPS: lipopolysaccharide; Lyz2/LysM: lysozyme 2; NFAT5/TonEBP: nuclear factor of activated T cells 5; MΦ: macrophages; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MIC: minimum inhibitory concentration; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NaCl: sodium chloride; NES: normalized enrichment score; n.s. : not significant; NO: nitric oxide; NOS2/iNOS: nitric oxide synthase 2, inducible; NS: normal salt; PCR: polymerase chain reaction; PGK1: phosphoglycerate kinase 1; PHOX: phagocyte oxidase; RFP: red fluorescent protein; RNA: ribonucleic acid; ROS: reactive oxygen species; sCFP3A: super cyan fluorescent protein 3A; SBFI: sodium-binding benzofuran isophthalate; SLC2A1/GLUT1: solute carrier family 2 (facilitated glucose transporter), member 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like kinase 1; v-ATPase: vacuolar-type H+-ATPase; WT: wild type
Evaluation of antiinflammatory and antiadhesive effects of heparins in human endotoxemia

ObjectiveCytokines and adhesion molecules have a decisive role in the development of early inflammatory response as well as the late sequelae of sepsis. Because L-selectin-deficient mice are protected from lethal endotoxemia, blockade of L-selectin may provide a useful therapeutic option in human sepsis. Heparin has immunomodulatory properties and effectively inhibits L- and P-selectin binding in vitro. We therefore investigated whether clinically applied doses of unfractionated or low-molecular-weight heparin affect early inflammatory response in human endotoxemia. DesignThe study was randomized, double-blinded, placebo-controlled, in three parallel groups consisting of 30 healthy male volunteers. SettingUniversity medical center. InterventionsAll subjects received a 2-ng/kg intravenous bolus of lipopolysaccharide and 10 mins later unfractionated heparin, low-molecular-weight heparin, or placebo as bolus primed continuous infusion for 6 hrs. Measurements and Main ResultsLipopolysaccharide infusion induced similar increases of tumor necrosis factor-&agr;, interleukin-6, interleukin-8, C-reactive protein, and soluble E-selectin levels in all treatment groups. CD11b expression increased by approximately 400%, but L-selectin decreased by 41% in the placebo arm 6 hrs after lipopolysaccharide infusion. Interestingly, both heparins (in particular unfractionated heparin) decreased L-selectin down-regulation as compared with placebo. Similarly, the decrease in lymphocyte counts was significantly less in the unfractionated heparin group during the first 24 hrs (p < .05 vs. placebo) ConclusionsHeparins displayed little effects on cytokine production and endothelial cell activation in endotoxemia. Of note, however, unfractionated heparin reduced L-selectin down-regulation and lymphocytopenia. These could present novel mechanisms of action of unfractionated heparin.
CCL5 (RANTES) gene polymorphisms in pulmonary tuberculosis patients of south India

The chemokine CCL5 is known to play an important role in the formation of granuloma during infection with Mycobacterium tuberculosis. Production of CCL5 is influenced by polymorphisms in the CCL5 gene. Hence, in the present study, we investigated whether polymorphisms in the promoter and intron regions of CCL5 gene are associated with susceptibility or resistance to pulmonary tuberculosis in south Indian population. Polymorphisms in the promoter (−403G/A and −28C/G) and intron (In1.1T/C) regions of CCL5 gene were studied in 212 pulmonary tuberculosis (PTB) patients and 213 healthy controls (HCs). Allele and genotype frequencies of CCL5 gene polymorphisms were not different between PTB patients and HCs. When the haplotype and diplotype frequencies were compared, a significantly decreased frequencies of the haplotype A‐C‐C [P = 0.037; Odds ratio (OR): 0.57; 95% confidence interval (CI): 0.34–0.97] and the diplotype G/A‐T/C (P = 0.017; OR: 0.46; 95% CI: 0.24–0.88) were observed among PTB patients when compared with HCs. However, the significant differences observed for the haplotype and the diplotype were lost when corrected for multiple comparisons [Bonferroni correction: A‐C‐C P corrected (Pc) = 0.148 and G/A‐T/C Pc = 0.136]. Though the present results suggest that the CCL5 gene haplotype A‐C‐C and the diplotype G/A‐T/C may be associated with resistance to PTB, further studies with increased sample size may be useful to confirm this present finding as well as to understand the role of CCL5 haplotype and diplotype on genetic susceptibility to TB.
Tumor‐derived exosomes elicit tumor suppression in murine hepatocellular carcinoma models and humans in vitro

Hepatocellular carcinoma (HCC) remains a global challenge due to high morbidity and mortality rates and poor response to treatment. Immunotherapy, based on introduction of dendritic cells (DCs) activated by tumor cell lysates as antigens ex vivo, shows limited response rates in HCC patients. Here, we demonstrate that tumor cell–derived exosomes (TEXs), displaying an array of HCC antigens, can elicit a stronger immune response than cell lysates in vitro and in vivo. Significant tumor growth inhibition was achieved in ectopic and orthotopic HCC mice treated with TEX‐pulsed DCs. Importantly, the tumor immune microenvironment was significantly improved in orthotopic HCC mice treated by TEX‐pulsed DCs, demonstrated by increased numbers of T lymphocytes, elevated levels of interferon‐γ, and decreased levels of interleukin‐10 and tumor growth factor‐β in tumor sites. As expected, T cells played an essential role in the TEX‐pulsed DC‐mediated immune response. Notably, exosomes from HCC cells not only promoted HCC‐specific cytolysis but also provided cross‐protective effects against pancreatic cancer cells. Moreover, HCC‐specific cytolysis, elicited by DCs pulsed with human HepG2 cell–derived exosomes, was observed across different human HCC cells irrespective of human leukocyte antigen types. Conclusion: HCC TEXs can potently carry HCC antigens, trigger a strong DC‐mediated immune response, and improve the HCC tumor microenvironment. (Hepatology 2016;64:456‐472)
Spatial Epidemiology of the Syphilis Epidemic in Toronto, Canada

Background Urban centers across Canada and the United States have battled syphilis epidemics with high rates of human immunodeficiency virus (HIV) coinfection for over a decade. We examined the spatial epidemiology of syphilis over time for Toronto (Canada) with the intention of forming new insights and strategies for restoring low syphilis rates. Methods Syphilis incidence rates, HIV-syphilis coinfection, and sexual risk behavior prevalences were estimated and mapped from primary, secondary, early latent syphilis cases reported to Toronto Public Health between January 1, 2006, and December 31, 2010, using ArcGIS 9.0. Geographic clusters of significantly elevated syphilis incidence rates were identified using SaTScan 9.0. The relationship between syphilis incidence rates and sociocultural factors was modeled using the Besag, York, and Mollie model. Results Between 2006 and 2010, syphilis incidence rates were high in Toronto’s downtown core area, intensified, and spread outward initiating 3 independent outbreak areas. HIV coinfection was high (47%); however, no spatial clustering was identified. Syphilis incidence rates, HIV coinfection, and behavioral risk factors promoting sexually transmitted infection transmission were high outside the core area, suggesting that peripheral sexual networks may be influencing high syphilis infection rates both inside and outside the core. Conclusions Toronto’s syphilis epidemic is mature. Response, resources, and intervention activities should target core and noncore areas.
Could Home Sexually Transmitted Infection Specimen Collection With e-Prescription Be a Cost-Effective Strategy for Clinical Trials and Clinical Care?

Background Results of a recent demonstration project evaluating feasibility, acceptability, and cost of a Web-based sexually transmitted infection (STI) testing and e-prescription treatment program (eSTI) suggest that this approach could be a feasible alternative to clinic-based testing and treatment, but the results need to be confirmed by a randomized comparative effectiveness trial. Methods We modeled a decision tree comparing (1) cost of eSTI screening using a home collection kit and an e-prescription for uncomplicated treatment versus (2) hypothetical costs derived from the literature for referral to standard clinic-based STI screening and treatment. Primary outcome was number of STIs detected. Analyses were conducted from the clinical trial perspective and the health care system perspective. Results The eSTI strategy detected 75 infections, and the clinic referral strategy detected 45 infections. Total cost of eSTI was $94,938 ($1266/STI detected) from the clinical trial perspective and $96,088 ($1281/STI detected) from the health care system perspective. Total cost of clinic referral was $87,367 ($1941/STI detected) from the clinical trial perspective and $71,668 ($1593/STI detected) from the health care system perspective. Conclusions Results indicate that eSTI will likely be more cost-effective (lower cost/STI detected) than clinic-based STI screening, both in the context of clinical trials and in routine clinical care. Although our results are promising, they are based on a demonstration project and estimates from other small studies. A comparative effectiveness research trial is needed to determine actual cost and impact of the eSTI system on identification and treatment of new infections and prevention of their sequelae.