Bypassing the nuclear gate: A non-canonical entry pathway for the mitochondrial pyruvate dehydrogenase complex.

Zervopoulos et al. (2022) propose a non-canonical nuclear import pathway for the functional mitochondrial pyruvate dehydrogenase complex (PDC), facilitated by dynamic MFN2-mediated tethering of mitochondria to the nuclear envelope upon exposure to proliferative stimuli.

Lipid and protein content profiling of isolated native autophagic vesicles.

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.

Engineered microRNA-based regulatory element permits safe high-dose miniMECP2 gene therapy in Rett mice.

MECP2 gene transfer has been shown to extend the survival of Mecp2-/y knockout mice modelling Rett syndrome, an X-linked neurodevelopmental disorder. However, controlling deleterious overexpression of MECP2 remains the critical unmet obstacle towards a safe and effective gene therapy approach for Rett syndrome. A recently developed truncated miniMECP2 gene has also been shown to be therapeutic after AAV9-mediated gene transfer in knockout neonates. We show that AAV9/miniMECP2 has a similar dose-dependent toxicity profile to that of a published second-generation AAV9/MECP2 vector after treatment in adolescent mice. To overcome that toxicity, we developed a risk-driven viral genome design strategy rooted in high-throughput profiling and genome mining to rationally develop a compact, synthetic microRNA target panel (miR-responsive auto-regulatory element, 'miRARE') to minimize the possibility of miniMECP2 transgene overexpression in the context of Rett syndrome gene therapy. The goal of miRARE is to have a built-in inhibitory element responsive to MECP2 overexpression. The data provided herein show that insertion of miRARE into the miniMECP2 gene expression cassette greatly improved the safety of miniMECP2 gene transfer without compromising efficacy. Importantly, this built-in regulation system does not require any additional exogenous drug application, and no miRNAs are expressed from the transgene cassette. Although broad applications of miRARE have yet to be determined, the design of miRARE suggests a potential use in gene therapy approaches for other dose-sensitive genes.

Justice for Our Genes: The Case for Genetic Non-discrimination Regulations in the New Zealand Life Insurance Industry.

While most comparable jurisdictions have adopted more restrictive positions, life insurers in New Zealand remain permitted to request the disclosure of predictive genetic test results from applicants, driving up the cost to obtain life insurance for those with known susceptibilities to genetic disease. The permissive approach is now an outlier, and risks disincentivising health care and research innovation, facilitating irrational discrimination, and compounding existing health inequities. This article examines the New Zealand position through a consequentialist lens. It analyses justifications for the status quo, as well as international approaches, before concluding that genetic non-discrimination regulations governing New Zealand's life insurance industry should be introduced to enhance public wellbeing.

Combination fixed-dose beta agonist and steroid inhaler as required for adults or children with mild asthma.

BACKGROUND: Asthma affects 350 million people worldwide including 45% to 70% with mild disease. Treatment is mainly with inhalers containing beta2-agonists, typically taken as required to relieve bronchospasm, and inhaled corticosteroids (ICS) as regular preventive therapy. Poor adherence to regular therapy is common and increases the risk of exacerbations, morbidity and mortality. Fixed-dose combination inhalers containing both a steroid and a fast-acting beta2-agonist (FABA) in the same device simplify inhalers regimens and ensure symptomatic relief is accompanied by preventative therapy. Their use is established in moderate asthma, but they may also have potential utility in mild asthma. OBJECTIVES: To evaluate the efficacy and safety of single combined (fast-onset beta2-agonist plus an inhaled corticosteroid (ICS)) inhaler only used as needed in people with mild asthma. SEARCH METHODS: We searched the Cochrane Airways Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and Embase, ClinicalTrials.gov and the World Health Organization (WHO) trials portal. We contacted trial authors for further information and requested details regarding the possibility of unpublished trials. The most recent search was conducted on 19 March 2021. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and cross-over trials with at least one week washout period. We included studies of a single fixed-dose FABA/ICS inhaler used as required compared with no treatment, placebo, short-acting beta agonist (SABA) as required, regular ICS with SABA as required, regular fixed-dose combination ICS/long-acting beta agonist (LABA), or regular fixed-dose combination ICS/FABA with as required ICS/FABA. We planned to include cluster-randomised trials if the data had been or could be adjusted for clustering. We excluded trials shorter than 12 weeks. We included full texts, abstracts and unpublished data. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data. We analysed dichotomous data as odds ratios (OR) or rate ratios (RR) and continuous data as mean difference (MD). We reported 95% confidence intervals (CIs). We used Cochrane's standard methodological procedures of meta-analysis. We applied the GRADE approach to summarise results and to assess the overall certainty of evidence. Primary outcomes were exacerbations requiring systemic steroids, hospital admissions/emergency department or urgent care visits for asthma, and measures of asthma control. MAIN RESULTS: We included six studies of which five contributed results to the meta-analyses. All five used budesonide 200 µg and formoterol 6 µg in a dry powder formulation as the combination inhaler. Comparator fast-acting bronchodilators included terbutaline and formoterol. Two studies included children aged 12+ and adults; two studies were open-label. A total of 9657 participants were included, with a mean age of 36 to 43 years. 2.3% to 11% were current smokers. FABA / ICS as required versus FABA as required Compared with as-required FABA alone, as-required FABA/ICS reduced exacerbations requiring systemic steroids (OR 0.45, 95% CI 0.34 to 0.60, 2 RCTs, 2997 participants, high-certainty evidence), equivalent to 109 people out of 1000 in the FABA alone group experiencing an exacerbation requiring systemic steroids, compared to 52 (95% CI 40 to 68) out of 1000 in the FABA/ICS as-required group. FABA/ICS as required may also reduce the odds of an asthma-related hospital admission or emergency department or urgent care visit (OR 0.35, 95% CI 0.20 to 0.60, 2 RCTs, 2997 participants, low-certainty evidence). Compared with as-required FABA alone, any changes in asthma control or spirometry, though favouring as-required FABA/ICS, were small and less than the minimal clinically-important differences. We did not find evidence of differences in asthma-associated quality of life or mortality. For other secondary outcomes FABA/ICS as required was associated with reductions in fractional exhaled nitric oxide, probably reduces the odds of an adverse event (OR 0.82, 95% CI 0.71 to 0.95, 2 RCTs, 3002 participants, moderate-certainty evidence) and may reduce total systemic steroid dose (MD -9.90, 95% CI -19.38 to -0.42, 1 RCT, 443 participants, low-certainty evidence), and with an increase in the daily inhaled steroid dose (MD 77 µg beclomethasone equiv./day, 95% CI 69 to 84, 2 RCTs, 2554 participants, moderate-certainty evidence). FABA/ICS as required versus regular ICS plus FABA as required There may be little or no difference in the number of people with asthma exacerbations requiring systemic steroid with FABA/ICS as required compared with regular ICS (OR 0.79, 95% CI 0.59 to 1.07, 4 RCTs, 8065 participants, low-certainty evidence), equivalent to 81 people out of 1000 in the regular ICS plus FABA group experiencing an exacerbation requiring systemic steroids, compared to 65 (95% CI 49 to 86) out of 1000 FABA/ICS as required group. The odds of an asthma-related hospital admission or emergency department or urgent care visit may be reduced in those taking FABA/ICS as required (OR 0.63, 95% CI 0.44 to 0.91, 4 RCTs, 8065 participants, low-certainty evidence). Compared with regular ICS, any changes in asthma control, spirometry, peak flow rates (PFR), or asthma-associated quality of life, though favouring regular ICS, were small and less than the minimal clinically important differences (MCID). Adverse events, serious adverse events, total systemic corticosteroid dose and mortality were similar between groups, although deaths were rare, so confidence intervals for this analysis were wide. We found moderate-certainty evidence from four trials involving 7180 participants that FABA/ICS as required was likely associated with less average daily exposure to inhaled corticosteroids than those on regular ICS (MD -154.51 µg/day, 95% CI -207.94 to -101.09). AUTHORS' CONCLUSIONS: We found FABA/ICS as required is clinically effective in adults and adolescents with mild asthma. Their use instead of FABA as required alone reduced exacerbations, hospital admissions or unscheduled healthcare visits and exposure to systemic corticosteroids and probably reduces adverse events. FABA/ICS as required is as effective as regular ICS and reduced asthma-related hospital admissions or unscheduled healthcare visits, and average exposure to ICS, and is unlikely to be associated with an increase in adverse events. Further research is needed to explore use of FABA/ICS as required in children under 12 years of age, use of other FABA/ICS preparations, and long-term outcomes beyond 52 weeks.

Epileptiform activity in traumatic brain injury predicts post-traumatic epilepsy.

We hypothesize that epileptiform abnormalities (EAs) in the electroencephalogram (EEG) during the acute period following traumatic brain injury (TBI) independently predict first-year post-traumatic epilepsy (PTE1 ). We analyze PTE1 risk factors in two cohorts matched for TBI severity and age (n = 50). EAs independently predict risk for PTE1 (odds ratio [OR], 3.16 [0.99, 11.68]); subdural hematoma is another independent risk factor (OR, 4.13 [1.18, 39.33]). Differences in EA rates are apparent within 5 days following TBI. Our results suggest that increased EA prevalence identifies patients at increased risk for PTE1 , and that EAs acutely post-TBI can identify patients most likely to benefit from antiepileptogenesis drug trials. Ann Neurol 2018;83:858-862.

Continuous electroencephalography predicts delayed cerebral ischemia after subarachnoid hemorrhage: A prospective study of diagnostic accuracy.

OBJECTIVE: Delayed cerebral ischemia (DCI) is a common, disabling complication of subarachnoid hemorrhage (SAH). Preventing DCI is a key focus of neurocritical care, but interventions carry risk and cannot be applied indiscriminately. Although retrospective studies have identified continuous electroencephalographic (cEEG) measures associated with DCI, no study has characterized the accuracy of cEEG with sufficient rigor to justify using it to triage patients to interventions or clinical trials. We therefore prospectively assessed the accuracy of cEEG for predicting DCI, following the Standards for Reporting Diagnostic Accuracy Studies. METHODS: We prospectively performed cEEG in nontraumatic, high-grade SAH patients at a single institution. The index test consisted of clinical neurophysiologists prospectively reporting prespecified EEG alarms: (1) decreasing relative alpha variability, (2) decreasing alpha-delta ratio, (3) worsening focal slowing, or (4) late appearing epileptiform abnormalities. The diagnostic reference standard was DCI determined by blinded, adjudicated review. Primary outcome measures were sensitivity and specificity of cEEG for subsequent DCI, determined by multistate survival analysis, adjusted for baseline risk. RESULTS: One hundred three of 227 consecutive patients were eligible and underwent cEEG monitoring (7.7-day mean duration). EEG alarms occurred in 96.2% of patients with and 19.6% without subsequent DCI (1.9-day median latency, interquartile range = 0.9-4.1). Among alarm subtypes, late onset epileptiform abnormalities had the highest predictive value. Prespecified EEG findings predicted DCI among patients with low (91% sensitivity, 83% specificity) and high (95% sensitivity, 77% specificity) baseline risk. INTERPRETATION: cEEG accurately predicts DCI following SAH and may help target therapies to patients at highest risk of secondary brain injury. Ann Neurol 2018;83:958-969.

Electronic Health Data Predict Outcomes After Aneurysmal Subarachnoid Hemorrhage.

BACKGROUD: Using electronic health data, we sought to identify clinical and physiological parameters that in combination predict neurologic outcomes after aneurysmal subarachnoid hemorrhage (aSAH). METHODS: We conducted a single-center retrospective cohort study of patients admitted with aSAH between 2011 and 2016. A set of 473 predictor variables was evaluated. Our outcome measure was discharge Glasgow Outcome Scale (GOS). For laboratory and physiological data, we computed the minimum, maximum, median, and variance for the first three admission days. We created a penalized logistic regression model to determine predictors of outcome and a multivariate multilevel prediction model to predict poor (GOS 1-2), intermediate (GOS 3), or good (GOS 4-5) outcomes. RESULTS: One hundred and fifty-three patients met inclusion criteria; most were discharged with a GOS of 3. Multivariate analysis predictors of mortality (AUC 0.9198) included APACHE II score, Glasgow Come Scale (GCS), white blood cell (WBC) count, mean arterial pressure, variance of serum glucose, intracranial pressure (ICP), and serum sodium. Predictors of death/dependence versus independence (GOS 4-5)(AUC 0.9456) were levetiracetam, mechanical ventilation, WBC count, heart rate, ICP variance, GCS, APACHE II, and epileptiform discharges. The multiclass prediction model selected GCS, admission APACHE II, periodic discharges, lacosamide, and rebleeding as significant predictors; model performance exceeded 80% accuracy in predicting poor or good outcome and exceeded 70% accuracy for predicting intermediate outcome. CONCLUSIONS: Variance in early physiologic data can impact patient outcomes and may serve as targets for early goal-directed therapy. Electronically retrievable features such as ICP, glucose levels, and electroencephalography patterns should be considered in disease severity and risk stratification scores.

Patient-Specific Classification of ICU Sedation Levels From Heart Rate Variability.

OBJECTIVE: To develop a personalizable algorithm to discriminate between sedation levels in ICU patients based on heart rate variability. DESIGN: Multicenter, pilot study. SETTING: Several ICUs at Massachusetts General Hospital, Boston, MA. PATIENTS: We gathered 21,912 hours of routine electrocardiogram recordings from a heterogenous group of 70 adult ICU patients. All patients included in the study were mechanically ventilated and were receiving sedatives. MEASUREMENTS AND MAIN RESULTS: As "ground truth" for developing our method, we used Richmond Agitation Sedation Scale scores grouped into four levels denoted "comatose" (-5), "deeply sedated" (-4 to -3), "lightly sedated" (-2 to 0), and "agitated" (+1 to +4). We trained a support vector machine learning algorithm to calculate the probability of each sedation level from heart rate variability measures derived from the electrocardiogram. To estimate algorithm performance, we calculated leave-one-subject out cross-validated accuracy. The patient-independent version of the proposed system discriminated between the four sedation levels with an overall accuracy of 59%. Upon personalizing the system supplementing the training data with patient-specific calibration data, consisting of an individual's labeled heart rate variability epochs from the preceding 24 hours, accuracy improved to 67%. The personalized system discriminated between light- and deep-sedation states with an average accuracy of 75%. CONCLUSIONS: With further refinement, the methodology reported herein could lead to a fully automated system for depth of sedation monitoring. By enabling monitoring to be continuous, such technology may help clinical staff to monitor sedation levels more effectively and to reduce complications related to over- and under sedation.

Vibrational Spectroscopy of Fe3+(CH4)n (n = 1-3) and Fe4+(CH4)4.

Vibrational spectra are measured for Fe3+(CH4)n (n = 1-3) and Fe4+(CH4)4 in the C-H stretching region (2650-3100 cm-1) using photofragment spectroscopy, monitoring loss of CH4. All of the spectra are dominated by an intense peak at around 2800 cm-1 that is red-shifted by ∼120 cm-1 from free methane. This peak is due to the symmetric C-H stretch of the η3 hydrogen-coordinated methane ligands. For clusters with three iron atoms, the peak becomes less red-shifted as the number of methane ligands increases. For clusters with one methane ligand per iron atom, the red shift increases in going from Fe2+(CH4)2 (88 cm-1) to Fe3+(CH4)3 (108 cm-1) to Fe4+(CH4)4 (122 cm-1). This indicates increased covalency in the binding of methane to the larger iron clusters and parallels their increased reactivity. Density functional theory calculations, B3LYP, BPW91, and M11L, are used to identify possible structures and geometries and to predict the spectra. Results show that all three functionals tend to overestimate the methane binding energies. The M11L calculations provide the best match to the experimental spectra.

Post-Translational Modifications and Diabetes.

Diabetes and its associated complications have increasingly become major challenges for global healthcare. The current therapeutic strategies involve insulin replacement therapy for type 1 diabetes (T1D) and small-molecule drugs for type 2 diabetes (T2D). Despite these advances, the complex nature of diabetes necessitates innovative clinical interventions for effective treatment and complication prevention. Accumulative evidence suggests that protein post-translational modifications (PTMs), including glycosylation, phosphorylation, acetylation, and SUMOylation, play important roles in diabetes and its pathological consequences. Therefore, the investigation of these PTMs not only sheds important light on the mechanistic regulation of diabetes but also opens new avenues for targeted therapies. Here, we offer a comprehensive overview of the role of several PTMs in diabetes, focusing on the most recent advances in understanding their functions and regulatory mechanisms. Additionally, we summarize the pharmacological interventions targeting PTMs that have advanced into clinical trials for the treatment of diabetes. Current challenges and future perspectives are also provided.

Prevalence of carboxy-Δ8 -tetrahydrocannabiniol in antidoping samples.

Δ9 -Tetrahydrocannabinol (Δ9 -THC) is usually the primary psychoactive agent in cannabis preparations. Recently, products containing another isomer, Δ8 -tetrahydrocannabinol (Δ8 -THC), have become available for sale. Δ8 -THC exists naturally in the cannabis plant at very low concentrations; hence, the Δ8 -THC present in most of the above-mentioned products is likely to be manufactured synthetically. A surge in popularity of these products, coupled with little oversight to ensure purity and potency, has led to reports of adverse events. Workplace drug testing programs as well as many sporting organizations prohibit the use of cannabinoids. Carboxy-Δ9 -THC (Δ9 -THC-COOH) is the targeted urinary metabolite for detection of cannabis use. The proliferation of products containing Δ8 -THC, which metabolizes to Δ8 -THC-COOH, presents analytical complexity with respect to separation and quantification of the individual isomers as well as legal complexity with respect to lack of clarity around the legal status of Δ8 -THC. This study aims to estimate the prevalence of Δ8 -THC use in the athlete community by monitoring for Δ8 -THC-COOH in samples collected for antidoping. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was utilized to resolve Δ8 and Δ9 -THC-COOH. One thousand samples with a presumptive Δ9 -THC-COOH finding in routine screening were analyzed by the above LC-MS/MS method. Approximately 12% of samples contained Δ8 -THC-COOH at relative abundances between 5% and 100% of total carboxy-THC content.

Remote feedback in endovascular simulation training: a mixed-methods study.

BACKGROUND: There is an increasing need to increase simulation-based learning opportunities for vascular surgery residents in endovascular skills training. This study aims to explore the effectiveness of remote expert instructional feedback of endovascular simulation-based education, as a means of increasing training opportunities in this area for vascular surgery residents. METHODS: A mixed-methods study design was adopted. Twelve vascular surgery residents from Ireland were tasked with completing two endovascular renal artery procedures: one with in-person expert feedback and the other with remote instruction. Participants ranged in experience levels from second year to final year of residency. Following the training activities, interviews and a questionnaire were employed to gather information on the usefulness of remote feedback. RESULTS: There was no significant difference reported by participants using a post-event validated questionnaire between remote and in-person feedback. During the interviews, participants expressed mixed feelings about the presence of the educator while practicing, but they eventually saw no limiting factors to their practice when the trainer provided remote feedback. When receiving performance feedback remotely, clear communication and a shared knowledge of the task development are critical to success. CONCLUSIONS: We believe these findings can inform the design and development of remote learning and assessment of endovascular skills training and ultimately provide increased opportunities for more skills practice for vascular surgical residents.

Early outcomes for rectal cancer surgery in the republic of ireland following a national centralization program.

BACKGROUND: Following a national audit of rectal cancer management in 2007, a national centralization program in the Republic of Ireland was initiated. In 2010, a prospective evaluation of rectal cancer treatment and early outcomes was conducted. METHODS: A total of 29 colorectal surgeons in 14 centers prospectively collated data on all patients with rectal cancer who underwent curative surgery in 2010. RESULTS: Data were available on 447 patients who underwent proctectomy with curative intent for rectal cancer in 2010; 23.7 % of patients underwent abdominoperineal excision. The median number of lymph nodes identified was 12. The 30-day mortality rate was 1.1 %. Compared with 2007, there was a reduction in positive circumferential margin rate (15.8 vs 4.5 %, P < 0.001), clinical anastomotic leak rate (10.8 vs 4.3 %, P = 0.002), and postoperative radiotherapy use (17.8 vs 4.0 %, P < 0.001). Also, 53.9 % received preoperative radiotherapy in 2010. Four centers gave statistically more patients (high-administration), and four centers gave fewer patients (low-administration) preoperative radiotherapy for T2/T3 tumors (P < 0.05). On multivariate analysis, being treated in a "high-administration center" increased the likelihood (likelihood ratio [LR], 2.9; 95 % CI 1.7-4.8; P < 0.001) while attending a "low-administration center" (LR, 0.3; 95 % CI 0.2-0.5; P < 0.001) reduced the likelihood of receiving preoperative radiotherapy for a T2/T3 rectal cancer. CONCLUSIONS: Patients undergoing rectal cancer surgery in hospitals following a national centralization initiative received high-quality surgery. Significant heterogeneity exists in radiotherapy administration, and evidence-based guidelines should be developed and implemented.

Role of laparoscopy in penetrating abdominal trauma: a systematic review.

BACKGROUND: Debate remains regarding the optimum role of laparoscopy in the setting of trauma although it can offer advantages over traditional exploratory laparotomy. Laparoscopy can be a screening, diagnostic or therapeutic tool in trauma. The purpose of this review is to evaluate the role of laparoscopy in penetrating abdominal trauma METHODS: The PUBMED database was searched with the keywords "Laparoscopy AND Trauma". Additional citation searching and searching of the grey literature was conducted. Relevant studies were chosen on the basis of the defined inclusion and exclusion criteria and quality was assessed where appropriate using the Downs and Black checklist RESULTS: In total, 51 studies were included in the analysis of which only 13 were prospective. In most studies, laparoscopy was used as a screening, diagnostic or therapeutic tool. In total, 2569 patients underwent diagnostic laparoscopy (DL) for penetrating abdominal trauma (PAT), 1129 (43.95 %) were positive for injury. 13.8 % of those with injury had a therapeutic laparoscopy. In total 33.8 % were converted to laparotomy, 16 % of which were non-therapeutic and 11.5 % of them were negative. 1497 patients were spared a non-therapeutic laparotomy. Overall, 72 patients suffered complications, there were 3 mortalities and 83 missed injuries. Sensitivity ranged from 66.7-100 %, specificity from 33.3-100 % and accuracy from 50-100 %. 23 of the 50 studies reported sensitivity, specificity and accuracy of 100 %, including the four most recent studies. In general the quality of the reported studies was poor. When used for cohort studies, the mean Downs and Black checklist score was 13.25 out of a possible total of 28. CONCLUSIONS: In summary, laparoscopy in PAT may have an important role in a selected subgroup of patients, with surgeon expertise also an important factor. Laparoscopy has screening, diagnostic and therapeutic roles, particularly where diaphragm injury is suspected. It is extremely sensitive in determining need for laparotomy but detects hollow visceral injuries less reliably. It has potential as a therapeutic tool in centres with appropriate expertise. The development of specific guidelines or protocols may increase the value of laparoscopy in trauma but this would require more evidence of a higher quality.

Autophagy as a caretaker of nuclear integrity.

Due to their essential functions, dysregulation of nuclear pore complexes (NPCs) is strongly associated with numerous human diseases, including neurodegeneration and cancer. On a cellular level, longevity of scaffold nucleoporins in postmitotic cells of both C. elegans and mammals renders them vulnerable to age-related damage, which is associated with an increase in pore leakiness and accumulation of intranuclear aggregates in rat brain cells. Thus, understanding the mechanisms which underpin the homeostasis of this complex, as well as other nuclear proteins, is essential. In this review, autophagy-mediated degradation pathways governing nuclear components in yeast will be discussed, with a particular focus on NPCs. Furthermore, the various nuclear degradation mechanisms identified thus far in diverse eukaryotes will also be highlighted.

FACS-mediated isolation of native autophagic vesicles.

Autophagosome isolation enables the thorough investigation of structural components and engulfed materials. Recently, we introduced a novel antibody-based FACS-mediated method for isolation of native macroautophagic/autophagic vesicles and confirmed the quality of the preparations. We performed phospholipidomic and proteomic analyses to characterize autophagic vesicle-associated phospholipids and protein cargoes under different autophagy conditions. Lipidomic analyses identified phosphoglycerides and sphingomyelins within autophagic vesicles and revealed that the lipid composition was unaffected by different rates of autophagosome formation. Proteomic analyses identified more than 4500 potential autophagy substrates and showed that in comparison to autophagic vesicles isolated under basal autophagy conditions, starvation only marginally affected the cargo profile. Proteasome inhibition, however, resulted in the enhanced degradation of ubiquitin-proteasome system components. Taken together, the novel isolation method enriched large quantities of autophagic vesicles and enabled detailed analyses of their lipid and cargo composition.

Frequency of inter-specialty consensus decisions and adherence to advice following discussion at a weekly neurovascular multidisciplinary meeting.

BACKGROUND/AIMS: Data are limited on the frequency of 'consensus decisions' between sub-specialists attending a neurovascular multidisciplinary meeting (MDM) regarding management of patients with extracranial carotid/vertebral stenoses and post-MDM 'adherence' to such advice. METHODS: This prospective audit/quality improvement project collated prospectively-recorded data from a weekly Neurovascular/Stroke Centre MDM documenting the proportion of extracranial carotid/vertebral stenosis patients in whom 'consensus management decisions' were reached by neurologists, vascular surgeons, stroke physicians-geriatricians and neuroradiologists. Adherence to MDM advice was analysed in asymptomatic carotid stenosis (ACS), symptomatic carotid stenosis (SCS), 'indeterminate symptomatic status stenosis' (ISS) and vertebral artery stenosis (VAS) patients, including intervals between index event to MDM + / - intervention. RESULTS: One hundred fifteen patients were discussed: 108 with carotid stenosis and 7 with VAS. Consensus regarding management was noted in 96.5% (111/115): 100% with ACS and VAS, 96.2% with SCS and 92.9% with ISS. Adherence to MDM management advice was 96.4% (107/111): 100% in ACS, ISS and VAS patients; 92% (46/50) in SCS patients. The median interval from index symptoms to revascularisation in 50-99% SCS patients was 12.5 days (IQR: 9-18.3 days; N = 26), with a median interval from MDM to revascularisation of 5.5 days (IQR: 1-7 days). Thirty patients underwent revascularisation. Two out of twenty-nine patients (6.9%) with either SCS or ISS had a peri-procedural ipsilateral ischaemic stroke, with no further strokes/deaths during 3-months follow-up. CONCLUSIONS: The high frequency of inter-specialty consensus regarding management and adherence to proposed treatment supports a collaborative/multidisciplinary model of care in patients with extracranial arterial stenoses. Service development should aim to shorten times between MDM discussion-intervention and optimise prevention of stroke/death.

Assessment of the role of aptitude in the acquisition of advanced laparoscopic surgical skill sets: results from a virtual reality-based laparoscopic colectomy training programme.

PURPOSE: The surgeons of the future will need to have advanced laparoscopic skills. The current challenge in surgical education is to teach these skills and to identify factors that may have a positive influence on training curriculums. The primary aim of this study was to determine if fundamental aptitude impacts on ability to perform a laparoscopic colectomy. METHODS: A practical laparoscopic colectomy course was held by the National Surgical Training Centre at the Royal College of Surgeons in Ireland. The course consisted of didactics, warm-up and the performance of a laparoscopic sigmoid colectomy on thesimulator. Objective metrics such as time and motion analysis were recorded. Each candidate had their psychomotor and visual spatial aptitude assessed. The colectomy trays were assessed by blinded experts post procedure for errors. RESULTS: Ten trainee surgeons that were novices with respect to advanced laparoscopic procedures attended the course. A significant correlation was found between psychomotor and visual spatial aptitude and performance on both the warm-up session and laparoscopic colectomy (r > 0.7, p < 0.05). Performance on the warm-up session correlated with performance of the laparoscopic colectomy (r = 0.8, p = 0.04). There was also a significant correlation between the number of tray errors and time taken to perform the laparoscopic colectomy (r = 0.83, p = 0.001). CONCLUSION: The results have demonstrated that there is a relationship between aptitude and ability to perform both basic laparoscopic tasks and laparoscopic colectomy on a simulator. The findings suggest that there may be a role for the consideration of an individual's inherent baseline ability when trying to design and optimise technical teaching curricula for advanced laparoscopic procedures.

Re-amputation and survival following toe amputation: outcome data from a tertiary referral centre.

PURPOSE: Toe amputation is a commonly performed procedure for irreversible foot sepsis. However, outcome and predictors of outcome are poorly understood. Our aim was to determine survival and rate of progression to further amputation following index toe amputation. METHODS: Consecutive patients between 2010 and 2015 were included. Progression to further minor amputation, major amputation or death was recorded. Multivariable Cox regression analyses were undertaken to determine independent predictors of outcome and survival. RESULTS: One hundred forty-six patients were included, with mean age of 65 years. Fifty-five (37.7%) patients underwent hallux amputation, while 91 (62.3%) underwent amputation of non-hallux digit(s). Following index toe amputation, 63 (43.2%) patients progressed to further minor or major ipsilateral amputation, median time to which was 36 months. Twenty-one patients (14.4%) progressed to major ipsilateral amputation. Patients undergoing index non-hallux amputation were significantly more likely to require further minor amputation (P = 0.050); however, the rate of major amputation between hallux (14.5%) and non-hallux (14.3%) groups was similar. Overall, 5-year ipsilateral amputation-free (iAFS) was 39.6 ± 4.1%, ipsilateral major amputation-free (iMAFS) was 55.9 ± 4.1% and overall survival (OS) was 64.3 ± 4.0% and did not differ between index amputation sites. CONCLUSION: Almost half of patients undergoing toe amputation required further digital amputation. However, limb preservation rates are high, and a majority of patients are alive at 5-year follow-up. There was no significant difference in outcome between patients undergoing hallux and non-hallux primary procedures. Overall, increasing age remains the only independent predictor of iMAFS and OS.