Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

A joint modelling approach for clustered recurrent events and death events

In dental implant research studies, events such as implant complications including pain or infection may be observed recurrently before failure events, i.e. the death of implants. It is natural to assume that recurrent events and failure events are correlated to each other, since they happen on the same implant (subject) and complication times have strong effects on the implant survival time. On the other hand, each patient may have more than one implant. Therefore these recurrent events or failure events are clustered since implant complication times or failure times within the same patient (cluster) are likely to be correlated. The overall implant survival times and recurrent complication times are both interesting to us. In this paper, a joint modelling approach is proposed for modelling complication events and dental implant survival times simultaneously. The proposed method uses a frailty process to model the correlation within cluster and the correlation within subjects. We use Bayesian methods to obtain estimates of the parameters. Performance of the joint models are shown via simulation studies and data analysis. © 2013 Copyright Taylor and Francis Group, LLC

Implant Loading Protocols for Edentulous Patients with Fixed Prostheses: A Systematic Review and Meta-Analysis

Purpose: To report on the effect of immediate implant loading with fixed prostheses compared to early and conventional loading on implant and prosthesis survival, failure, and complications. Materials and Methods: An electronic and manual search was conducted to identify randomized controlled clinical trials (RCTs) as well as prospective and retrospective studies involving rough surface implants and implant fixed complete dental prostheses for edentulous patients. Results: The 62 studies that fulfilled the inclusion criteria featured 4 RCTs, 2 prospective case-control studies, 34 prospective cohort studies, and 22 retrospective cohort studies. These studies yielded data from 2,695 patients (2,757 edentulous arches) with 13,653 implants. Studies were grouped according to the loading protocol applied; 45 studies reported on immediate loading, 8 on early loading, and 11 on conventional loading. For the immediate loading protocol with flap surgery, the implant and prosthesis survival rates ranged from 90.1% to 100% and 93.75% to 100%, respectively (range of follow-up, 1 to 10 years). When immediate loading was combined with guided flapless implant placement, the implant survival rates ranged from 90% to 99.4%. For the early loading protocol, the implant and prosthesis survival rates ranged from 94.74% to 100% and 93.75% to 100%, respectively (range of follow-up, 1 to 10 years). For the conventional loading protocol, the implant and prosthesis survival rates ranged from 94.95% to 100% and 87.5% to 100%, respectively (range of follow-up, 2 to 15 years). No difference was identified between maxilla and mandible. Conclusions: When selecting cases carefully and using dental implants with a rough surface, immediate loading with fixed prostheses in edentulous patients results in similar implant and prosthesis survival and failure rates as early and conventional loading. For immediate loading, most of the studies recommended a minimal insertion torque of 30 Ncm. The estimated 1-year implant survival was above 99% with all three loading protocols. Caution is necessary when interpreting these results, as there are many confounding factors that affect treatment outcomes with each of the loading protocols

The healthcare burden and associated adverse events from total alloplastic temporomandibular joint replacement: a national United States perspective

© 2020 International Association of Oral and Maxillofacial SurgeonsThe purpose of this study was to provide a United States perspective on alloplastic total joint replacement. We sought to estimate the inpatient burden and report the most common adverse events using two administrative datasets. The National Inpatient Sample was queried from October 2015 to December 2016 for total joint replacement admissions using International Classification of Diseases 10th revision codes, and the Manufacturer and User Facility Device Experience registry was queried from January 2009 to September 2019 using manufacturer brands. The combined final sample included 114 inpatient admissions and 392 adverse events. Mean age was 43.1 years, and most patients were white (82.7%) and female (86.0%). The mean hospital charge was $108,709.43 and the mean length of stay was 2.6 days. The most common adverse events were infection (26.3%), heterotopic bone (20.9%), and poor intraoperative fit (14.0%). Fifty-four percent of cases had bilateral total joint replacements, 24.6% had simultaneous subcutaneous abdominal fat grafting, and 11.4% had simultaneous maxillary repositioning. Fat grafting and maxillary repositioning were not associated with any significant difference in the length of stay or cost. Compared to unilateral cases, bilateral total joint replacements carried significantly greater charges (P < 0.01), but no increased length of stay (P = 0.70), suggesting that bilateral and unilateral cases may experience a similar postoperative course

Microscopic changes in the condyle and disc in response to distraction osteogenesis of the minipig mandible

Unilateral mandibular distraction osteogenesis (DO) has been shown to cause gross changes in the mandibular condyle and articular disc. The purpose of this study was to correlate histologic findings with these gross changes in a minipig distraction model. Semiburied distractors were placed via submandibular incisions in 15 minipigs. Two unoperated animals served as controls. The protocol consisted of 0-day latency and rates of 1, 2, or 4 mm/day for a 12-mm gap. After the minipigs were killed (at 0, 24, or 90 days), ipsilateral and contralateral condyles and discs were harvested, decalcified, prepared for standard paraffin embedding, and evaluated to determine changes in 1) morphology and thickness of the articular cartilage and subchondral bone and 2) morphology of the disc. In control animals, there were no degenerative changes in the articular cartilage and underlying condylar bone; there were no significant differences in the mean articular cartilage thickness. The temporomandibular joint discs were normal. In experimental animals, distracted condyles showed increasing degenerative changes and mean articular cartilage thickness as the DO rate increased. The discs were thinner. These changes were present, but to a lesser degree, in the contralateral condyles. After 90 days, degenerative changes in the condyles and discs were reduced, after remodeling, except in the 4 mm/day DO group. Histologic changes in the condyles and temporomandibular joint discs in response to mandibular DO correlated with previously reported gross changes. These changes were greater at higher distraction rates and remodeling back to normal occurred in mandibular condyles distracted at 1 mm/day

Adverse events secondary to cetuximab therapy in head & neck cancer therapy and risk factors for serious outcomes

The objective of this study is to illustrate the adverse events secondary to cetuximab therapy for head and neck cancer and elucidate risk factors for serious outcomes. This retrospective study was conducted using the FDA Adverse Event Reporting System (FAERS). The predictor variables were patient characteristics, country of treatment, and adverse events. The outcome variable was the rate of serious outcomes. Multivariate logistic regression was created to identify all significant risk factors of the outcome. P < 0.05 was considered statistically significant. The final sample consisted of 3,086 reports of adverse events from cetuximab therapy in head and neck cancer treatment, of which 2,746 reports were considered serious (89.0%) per the FAERS criteria. Mucosal inflammation was the most common adverse event. The strongest risk factor for a serious outcome was cetuximab therapy in countries outside the US (OR 105.2, P < 0.01). Polytherapy (OR 7.6, P < 0.01) was also a risk factor for serious outcome. Health-care providers should be aware of potential complications following cetuximab administration, particularly when administered in countries outside the US and in conjunction with other medications

Health Insurance Affects Head and Neck Cancer Treatment Patterns and Outcomes

The purpose of this study is to examine the effect of insurance coverage on stage of presentation, treatment, and survival of head and neck cancer (HNC). A retrospective study was conducted using the Surveillance, Epidemiology, and End Results (SEER) program to identify patients diagnosed with HNC. The primary variable of interest was insurance analyzed as a dichotomous variable: Patients were considered uninsured if they were classified as "uninsured" by SEER, whereas patients were considered insured if they were defined by SEER as "any Medicaid," "insured," or "insured/no specifics." The outcomes of interest were cancer stage at presentation (M0 vs M1), receipt of definitive treatment, and HNC-specific mortality (HNCSM). Multivariable logistic regression modeled the association between insurance status and stage at presentation, as well as between insurance status and receipt of definitive treatment, whereas HNCSM was modeled using Fine and Gray competing risks. Sensitivity logistic regression analysis was used to determine whether observed interactions remained significant by insurance type (privately insured, Medicaid, and uninsured). Patients without medical insurance were more likely to present with metastatic cancer (adjusted odds ratio, 1.60; P < .001), were more likely to not receive definitive treatment (adjusted odds ratio, 1.64; P < .001), and had a higher risk of HNCSM (adjusted hazard ratio, 1.20; P = .002). Sensitivity analyses showed that when results were stratified by insurance type, significant interactions remained for uninsured patients and patients with Medicaid. Uninsured patients and patients with Medicaid are more likely to present with metastatic disease, are more likely to not be treated definitively, and are at a higher risk of HNCSM. The treatment gap between Medicaid and private insurance observed in this study should serve as an immediate policy target for health care reform

Impact of African–American race on presentation, treatment, and survival of head and neck cancer

To determine the associations between African American race and stage at diagnosis, receipt of definitive therapy, and cancer-specific mortality among patients with head and neck cancer. The Surveillance, Epidemiology and End Results (SEER) database was used to conduct a retrospective study on 34,437 patients diagnosed with head and neck cancer from 2007 to 2010. Multivariable logistic regression analyses were applied to determine the impact of race on cancer stage at presentation (metastatic vs. non-metastatic) and receipt of definitive treatment. Fine and Gray competing-risks regression modeled the association between race and head and neck cancer-specific mortality. African Americans were more likely to present with metastatic cancer compared to non-African Americans (Adjusted Odds Ratio [AOR] 1.76; CI 1.50–2.07; P<0.001). Among patients with non-metastatic disease, African Americans were less likely to receive definitive treatment (AOR 0.63; CI 0.55–0.72; P<0.001). After a median follow-up of 19months, African Americans with non-metastatic disease were found to have a higher risk of head and neck cancer specific mortality (AHR 1.19; 95% CI 1.09–1.29; P<0.001). African Americans with head and neck cancer are more likely to present with metastatic disease, less likely to be treated definitively, and are more likely to die from head and neck cancer. The unacceptably high rates of disparity found in this study should serve as immediate targets for urgent healthcare policy intervention