BackgroundAutophagy is a dynamic process during which isolation membranes package substrates to form autophagosomes that are fused with lysosomes to form autolysosomes for degradation. Although it is agreed that the LC3II-associated mature autophagosomes move along microtubular tracks, it is still in dispute if the conversion of LC3I to LC3II before autophagosomes are fully mature and subsequent fusion of mature autophagosomes with lysosomes require microtubules.ResultsWe use biochemical markers of autophagy and a collection of microtubule interfering reagents to test the question. Results show that interruption of microtubules with either microtubule stabilizing paclitaxel or destabilizing nocodazole similarly impairs the conversion of LC3I to LC3II, but does not block the degradation of LC3II-associated autophagosomes. Acetylation of microtubules renders them resistant to nocodazole treatment. Treatment with vinblastine that causes depolymerization of both non-acetylated and acetylated microtubules results in impairment of both LC3I-LC3II conversion and LC3II-associated autophagosome fusion with lysosomes.ConclusionsAcetylated microtubules are required for fusion of autophagosomes with lysosomes to form autolysosomes.

The ubiquitously distributed MAP1S is a homologue of the exclusively neuronal distributed microtubule-associated protein 1A and 1B (MAP1A/B). They give rise to multiple isoforms through similar post-translational modification. Isoforms of MAP1S have been implicated in microtubule dynamics and mitotic abnormalities and mitotic cell death. Here we show that ablation of the Map1s gene in mice caused reduction in the B-cell CLL/lymphoma 2 or xL (Bcl-2/xL) and cyclin-dependent kinase inhibitor 1B (P27) protein levels, accumulation of defective mitochondria, and severe defects in response to nutritive stress, suggesting defects in autophagosomal biogenesis and clearance. Furthermore, MAP1S isoforms interacted with the autophagosome-associated light chain 3 of MAP1A/B (LC3), a homologue of yeast autophagy-related gene 8 (ATG8), and recruited it to stable microtubules in a MAP1S and LC3 isoformdependent mode. In addition, MAP1S interacted with mitochondrion-associated leucine-rich PPR-motif containing protein (LRPPRC) that interacts with the mitophagy initiator and Parkinson disease-related protein Parkin. The three-way interactions of MAP1S isoforms with LC3 and microtubules as well as the interaction of MAP1S with LRPPRC suggest that MAP1S isoforms may play positive roles in integration of autophagic components with microtubules and mitochondria in both autophagosomal biogenesis and degradation. For the first time, our results clarify roles of MAP1S in bridging microtubules and mitochondria with autophagic and mitophagic initiation, maturation, trafficking, and lysosomal clearance. Defects in the MAP1S-regulated autophagy may impact heart disease, cancers, neurodegenerative diseases, and a wide range of other diseases.Autophagy, or self-digestion, is a process that begins with the formation of isolation membranes that engulf substrates to form autophagosomes. Then autophagosomes fuse with lysosomes to generate autolysosomes in which substrates are degraded (1). The initiation of autophagy is regulated by the mammalian target of rapamycin (mTOR) pathway. Autophagy will be shut down through either the phosphatidylinositol 3-kinase (PI3K)-v-akt murine thymoma viral oncogene (AKT)-mTOR pathway in response to signals from growth factors or the serine/threonine kinase 11 (LKB1)-AMP-activated protein kinase (AMPK) 2 -mTOR pathway in response to signals from nutrients and metabolites. The anti-apoptotic protein Bcl-2 and Bcl-xL exhibit opposite functions in autophagy initiation. They inhibit autophagy initiation through the PI3K-AKTmTOR pathway by sequestering the BCL2 interacting protein (Beclin 1) or activate autophagy initiation through the LKB1-AMPK-mTOR pathway by increasing P27 levels (2, 3).After initiation, the metabolism of LC3 has emerged as a key biochemical marker for tracking of autophagy and autophagosomes (4, 5). The 22-kDa full-length LC3 (4) is proteolytically modified into LC3I form by ATG4, resulting in exposure of a C-terminal glycine (5). LC3I is first conjugated with the ubiquitin activating enzyme (E1)-...

ObjectivesExplore the experience of patients undergoing colorectal surgery within an Enhanced Recovery After Surgery (ERAS) programme. Use these experiential data to inform the development of a framework to support ongoing, meaningful patient engagement in ERAS.DesignQualitative patient-led study using focus groups and narrative interviews. Data were analysed iteratively using a Participatory Grounded Theory approach.SettingFive tertiary care centres in Alberta, Canada, following the ERAS programme.ParticipantsTwenty-seven patients who had undergone colorectal surgery in the last 12 months were recruited through purposive sampling. Seven patients participated in a codesign focus group to set and prioritise the research direction. Narrative interviews were conducted with 20 patients.ResultsPatients perceived that an ERAS programme should not be limited to the perioperative period, but should encompass the journey from diagnosis to recovery. Practical recommendations to improve the patient experience across the surgical continuum, and enhance patient engagement within ERAS included: (1) fully explain every protocol, and the purpose of the protocol, both before surgery and while in-hospital, so that patients can become knowledgeable partners in their recovery; (2) extend ERAS guidelines to the presurgery phase, so that patients can be ready emotionally, psychologically and physically for surgery; (3) extend ERAS guidelines to the recovery period at home to avoid stressful situations for patients and families; (4) consider activating a programme where experienced patients can provide peer support; (5) one size does not fit all; personalised adaptations within the standardised pathway are required.Drawing upon these data, and through consultation with ERAS Alberta stakeholders, the ERAS team developed a matrix to guide sustained patient involvement and action throughout the surgical care continuum at three levels: individual, unit and ERAS system.ConclusionThis patient-led study generated new insights into the needs of ERAS patients and informed the development of a framework to improve patient experiences and outcomes.

First generation, E1-deleted Adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen CEA, induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of pre-existing or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of pre-existing Ad5 immunity in a majority (61.3%) of patients. Importantly, there was minimal toxicity, and overall patient survival (48% at 12 months) was similar regardless of pre-existing Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.