Mevalonate Cascade Regulation of Airway Mesenchymal Cell Autophagy and Apoptosis: A Dual Role for p53

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Nucleoporin's Like Charge Regions Are Major Regulators of FG Coverage and Dynamics Inside the Nuclear Pore Complex.

Nucleocytoplasmic transport has been the subject of a large body of research in the past few decades. Recently, the focus of investigations in this field has shifted from studies of the overall function of the nuclear pore complex (NPC) to the examination of the role of different domains of phenylalanine-glycine nucleoporin (FG Nup) sequences on the NPC function. In our recent bioinformatics study, we showed that FG Nups have some evolutionarily conserved sequence-based features that might govern their physical behavior inside the NPC. We proposed the 'like charge regions' (LCRs), sequences of charged residues with only one type of charge, as one of the features that play a significant role in the formation of FG network inside the central channel. In this study, we further explore the role of LCRs in the distribution of FG Nups, using a recently developed coarse-grained molecular dynamics model. Our results demonstrate how LCRs affect the formation of two transport pathways. While some FG Nups locate their FG network at the center of the NPC forming a homogeneous meshwork of FG repeats, other FG Nups cover the space adjacent to the NPC wall. LCRs in the former group, i.e. FG Nups that form an FG domain at the center, tend to regulate the size of the highly dense, doughnut-shaped FG meshwork and leave a small low FG density area at the center of the pore for passive diffusion. On the other hand, LCRs in the latter group of FG Nups enable them to maximize their interactions and cover a larger space inside the NPC to increase its capability to transport numerous cargos at the same time. Finally, a new viewpoint is proposed that reconciles different models for the nuclear pore selective barrier function

Wrong-Site Surgery in California, 2007-2014

Objective The implementation of a universal surgical safety protocol in 2004 was intended to minimize the prevalence of wrong-site surgery (WSS). However, complete elimination of WSS in the operating room continues to be a challenge. The purpose of this study is to evaluate the prevalence and etiology of WSS in the state of California. Study Design A retrospective study of all WSS reports investigated by the California Department of Public Health between 2007 and 2014. Methods Prevalence of overall and specialty-specific WSS, causative factors, and recommendations on further improvement are discussed. Results A total of 95 cases resulted in incident reports to the California Department of Public Health and were included in our study. The most common errors were operating on the wrong side of the patient's body (n = 60, 62%), performing the wrong procedure (n = 21, 21%), operating on the wrong body part (n = 12, 12%), and operating on the wrong patient (n = 2, 2%). WSS was most prevalent in orthopedic surgery (n = 33, 35%), followed by general surgery (n = 26, 27%) and neurosurgery (n = 16, 17%). All 3 otolaryngology WSS cases in California are associated with the ear. Conclusion WSS continues to surface despite national efforts to decrease its prevalence. Future research could establish best practices to avoid these "never events" in otolaryngology and other surgical specialties

Smartphone-Enabled Otoscopy in Neurotology/Otology

Objective A smartphone-enabled otoscope (SEO) can capture tympanic membrane (TM) images. We sought to compare a SEO to microscopic otoscopy in the detection and evaluation of TM pathology in an otology/neurotology practice. Study Design Prospective single-site study in adults presenting over a 3-month period. Setting Neurotology clinic within a tertiary care academic medical center. Subjects and Methods Following consent, 57 patients underwent a medical and microscopic ear examination. Afterward, clinicians photographed bilateral TMs using a SEO. A second "blinded" neurotologist received a SEO-acquired image of each TM and a brief patient history. Our primary end point was identification of TM pathology (or lack thereof) and the blinded neurotologists' corresponding diagnosis. Secondary end points included patient-reported SEO comfort levels. Results A single SEO-acquired TM image and brief patient history resulted in correct diagnosis of 96% (23/24) of normal TMs and identification of 100% (33/33) of microscope-confirmed abnormal TMs. When pathology was identified by the "blinded" physician, the diagnosis was identical to that made by the primary treating physician 82% (27/33) of the time. On patient surveys, 93% (53/57) of patients felt "very comfortable" with SEO utilization, and 88% (50/57) reported viewing acquired images was "very useful" in understanding their condition. Conclusion A SEO is 96% specific in identifying normal TMs and 100% sensitive in identifying pathology. Its 97% positive predictive value and small false-positive rate makes it a useful screening tool. Furthermore, patients are receptive to this technology and felt comfortable with its utilization in a health care or possible telemedicine setting

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway