The protective effect of 3% diquafosol on meibomian gland morphology in glaucoma patients treated with prostaglandin analogs: a 12-month follow-up study.

BACKGROUND: To determine if 3% diquafosol (DQS) can preserve the meibomian gland morphology in glaucoma patients treated with prostaglandin analogs (PGA) for a 12-month follow-up period. METHODS: This study included 84 eyes of 46 normal tension glaucoma (NTG) patients who were treated with either preservative-containing PGA (PC-PGA; 16 patients, 28 eyes), preservative-free PGA (PF-PGA; 21 patients, 39 eyes), or a combination of PC-PGA and 3% DQS (PC-PGA + DQS; 9 patients, 17 eyes). The meibography of the upper eyelid was acquired using Keratograph® 5 M at baseline and at each follow-up (1, 3, 6, 9, and 12 months). Meibomian gland loss (MGL) was quantitatively analyzed by using ImageJ software. RESULTS: In the PC-PGA group, MGL increased significantly from baseline to month 9 and month 12, whereas no significant changes were observed in the PF-PGA and PC-PGA + DQS groups during the entire 12 months. All groups showed similar MGL at each follow-up time from baseline to six months. However, MGL in the PC-PGA group was significantly higher than those in the PF-PGA and PC-PGA + DQS groups at the 9 and 12 months. CONCLUSIONS: Combining 3% DQS with PC-PGA was as effective as PF-PGA in preserving the meibomian gland morphology for at least 12 months. Our results suggest that 3% DQS may be a promising strategy for managing glaucoma patients with a high risk of developing meibomian gland dysfunction due to preservative-containing topical medications.

Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders.

BACKGRAOUD & AIMS: Aberrant epithelial bicarbonate (HCO3-) secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with several diseases including cystic fibrosis and pancreatitis. Dynamically regulated ion channel activity and anion selectivity of CFTR by kinases sensitive to intracellular chloride concentration ([Cl-]i) play an important role in epithelial HCO3- secretion. However, the molecular mechanisms of how [Cl-]i-dependent mechanisms regulate CFTR are unknown. METHODS: We examined the mechanisms of the CFTR HCO3- channel regulation by [Cl-]i-sensitive kinases using an integrated electrophysiological, molecular, and computational approach including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. In addition, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. RESULTS: Among the WNK1, SPAK, and OSR1 kinases that constitute a [Cl-]i-sensitive kinase cascade, the expression of WNK1 alone was sufficient to increase the CFTR bicarbonate permeability (PHCO3/PCl) and conductance (GHCO3) in patch clamp recordings. Molecular dissection of the WNK1 domains revealed that the WNK1 kinase domain is responsible for CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions mediate the [Cl-]i-sensitivity of WNK1. Furthermore, the pancreatitis-causing R74Q and R75Q mutations in the elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. CONCLUSION: The CFTR HCO3- channel activity is regulated by [Cl-]i and a WNK1-dependent mechanism. Our results provide new insights into the regulation of the ion selectivity of CFTR and the pathogenesis of CFTR-related disorders.

Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR.

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl- channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.

ANO9/TMEM16J promotes tumourigenesis via EGFR and is a novel therapeutic target for pancreatic cancer.

BACKGROUND: Anoctamin (ANO)/transmembrane member 16 (TMEM16) proteins mediate diverse physiological and pathophysiological functions including cancer cell proliferation. The present study aimed to identify the role of ANOs in pancreatic cancer. METHODS: In an initial screen of ANOs, ANO9/TMEM16J was overexpressed in pancreatic cancer cells, and its role in the pathogenesis of pancreatic cancer was evaluated using an integrated in vitro and in vivo approach. To determine clinical relevance of the experimental findings, the prognostic value of ANO9 was evaluated in patients with pancreatic cancer. RESULTS: The ANO9 mRNA and protein levels were increased in pancreatic cancer-derived cells. Exogenous expression of ANO9 in PANC-1 cells significantly increased cell proliferation in cell cultures and in mice. In contrast, knockdown of ANO9 in AsPC-1, BxPC-3, and Capan-2 cells strongly inhibited cell proliferation. Mechanistic analysis suggested that physical association of ANO9 with epidermal growth factor receptor (EGFR) underlies ANO9-induced cell proliferation. Knockdown of ANO9 augmented the effects of the EGFR inhibitor and the cytotoxic agent on pancreatic cancer cell proliferation. In addition, high ANO9 expression is a poor prognostic factor in patients with pancreatic cancer. CONCLUSIONS: The ANO9/TMEM16J appears to be a clinically useful prognostic marker for pancreatic cancer and a potential therapeutic target.

Sec16A is critical for both conventional and unconventional secretion of CFTR.

CFTR is a transmembrane protein that reaches the cell surface via the conventional Golgi mediated secretion pathway. Interestingly, ER-to-Golgi blockade or ER stress induces alternative GRASP-mediated, Golgi-bypassing unconventional trafficking of wild-type CFTR and the disease-causing ΔF508-CFTR, which has folding and trafficking defects. Here, we show that Sec16A, the key regulator of conventional ER-to-Golgi transport, plays a critical role in the ER exit of protein cargos during unconventional secretion. In an initial gene silencing screen, Sec16A knockdown abolished the unconventional secretion of wild-type and ΔF508-CFTR induced by ER-to-Golgi blockade, whereas the knockdown of other COPII-related components did not. Notably, during unconventional secretion, Sec16A was redistributed to cell periphery and associated with GRASP55 in mammalian cells. Molecular and morphological analyses revealed that IRE1α-mediated signaling is an upstream regulator of Sec16A during ER-to-Golgi blockade or ER stress associated unconventional secretion. These findings highlight a novel function of Sec16A as an essential mediator of ER stress-associated unconventional secretion.

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR.

Induction of endoplasmic reticulum (ER)-to-Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)-mediated, Golgi-independent unconventional cell-surface trafficking of the folding-deficient ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation-dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508-CFTR, such as induction of ER-to-Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C-terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation-mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation-inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER-retained ΔF508-CFTR and mediates the ER stress-induced unconventional secretion pathway.

A forensic pathological study of eighty-two cases of adrenal hemorrhage / 法医学杂志

OBJECTIVE@#To analyze the relationship between adrenal hemorrhage and the cause of death, age and gender.@*METHODS@#Eighty-two cases of adrenal hemorrhage were statistically analyzed.@*RESULTS@#Adrenal hemorrhage occurred mostly in cases of sudden death, infection, trauma and asphyxia. Male had more chance than female to have adrenal hemorrhage. Adrenal hemorrhage caused by sudden death, trauma and poisoning was more frequently seen in young adults, whereas adrenal hemorrhage in children as well as in fetus and newborns was often caused by infection as well as sudden death and asphyxia respectively. Adrenal hemorrhage caused by sudden death and asphyxia was mainly located in medulla, while the infection usually induced hemorrhage in cortex. Adrenal hemorrhage caused by trauma showed an equal opportunity in either the cortex or medulla.@*CONCLUSION@#Our data indicate that adrenal hemorrhage might provide some clues in searching for the cause of death.