Atg21 organizes Atg8 lipidation at the contact of the vacuole with the phagophore.

Coupling of Atg8 to phosphatidylethanolamine is crucial for the expansion of the crescent-shaped phagophore during cargo engulfment. Atg21, a PtdIns3P-binding beta-propeller protein, scaffolds Atg8 and its E3-like complex Atg12-Atg5-Atg16 during lipidation. The crystal structure of Atg21, in complex with the Atg16 coiled-coil domain, showed its binding at the bottom side of the Atg21 beta-propeller. Our structure allowed detailed analyses of the complex formation of Atg21 with Atg16 and uncovered the orientation of the Atg16 coiled-coil domain with respect to the membrane. We further found that Atg21 was restricted to the phagophore edge, near the vacuole, known as the vacuole isolation membrane contact site (VICS). We identified a specialized vacuolar subdomain at the VICS, typical of organellar contact sites, where the membrane protein Vph1 was excluded, while Vac8 was concentrated. Furthermore, Vac8 was required for VICS formation. Our results support a specialized organellar contact involved in controlling phagophore elongation. Abbreviations: FCCS: fluorescence cross correlation spectroscopy; NVJ: nucleus-vacuole junction; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol- 3-phosphate; VICS: vacuole isolation membrane contact site.

Mechanistic dissection of macro- and micronucleophagy.

Nucleophagy, the mechanism for autophagic degradation of nuclear material, occurs in both a macro- and micronucleophagic manner. Upon nitrogen deprivation, we observed, in an in-depth fluorescence microscopy study, the formation of micronuclei: small parts of superfluous nuclear components surrounded by perinuclear ER. We identified two types of micronuclei associated with a corresponding autophagic mode. Our results showed that macronucleophagy degraded these smaller micronuclei. Engulfed in Atg8-positive phagophores and containing cargo receptor Atg39, macronucleophagic structures revealed finger-like extensions when observed in 3-dimensional reconstitutions of fluorescence microscopy images, suggesting directional growth. Interestingly, in the late stages of phagophore elongation, the adjacent vacuolar membrane showed a reduction of integral membrane protein Pho8. This change in membrane composition could indicate the formation of a specialized vacuolar domain, required for autophagosomal fusion. Significantly larger micronuclei formed at nucleus vacuole junctions and were identified as a substrate of piecemeal microautophagy of the nucleus (PMN), by the presence of the integral membrane protein Nvj1. Micronuclei sequestered by vacuolar invaginations also contained Atg39. A detailed investigation revealed that both Atg39 and Atg8 accumulated between the vacuolar tips. These findings suggest a role for Atg39 in micronucleophagy. Indeed, following the degradation of Nvj1, an exclusive substrate of PMN, in immunoblots, we could confirm the essential role of Atg39 for PMN. Our study thus details the involvement of Atg8 in both macronucleophagy and PMN and identifies Atg39 as the general cargo receptor for nucleophagic processes.Abbreviations: DIC: Differential interference contrast, FWHM: Full width at half maximum, IQR: Interquartile range, MIPA: Micropexophagy-specific membrane apparatus, NLS: Nuclear localization signal, NVJ: Nucleus vacuole junction, PMN: Piecemeal microautophagy of the nucleus, pnER: Perinuclear ER.

Nucleophagy-Implications for Microautophagy and Health.

Nucleophagy, the selective subtype of autophagy that targets nuclear material for autophagic degradation, was not only shown to be a model system for the study of selective macroautophagy, but also for elucidating the role of the core autophagic machinery within microautophagy. Nucleophagy also emerged as a system associated with a variety of disease conditions including cancer, neurodegeneration and ageing. Nucleophagic processes are part of natural cell development, but also act as a response to various stress conditions. Upon releasing small portions of nuclear material, micronuclei, the autophagic machinery transfers these micronuclei to the vacuole for subsequent degradation. Despite sharing many cargos and requiring the core autophagic machinery, recent investigations revealed the aspects that set macro- and micronucleophagy apart. Central to the discrepancies found between macro- and micronucleophagy is the nucleus vacuole junction, a large membrane contact site formed between nucleus and vacuole. Exclusion of nuclear pore complexes from the junction and its exclusive degradation by micronucleophagy reveal compositional differences in cargo. Regarding their shared reliance on the core autophagic machinery, micronucleophagy does not involve normal autophagosome biogenesis observed for macronucleophagy, but instead maintains a unique role in overall microautophagy, with the autophagic machinery accumulating at the neck of budding vesicles.

The 4th St. Gallen EORTC Gastrointestinal Cancer Conference: Controversial issues in the multimodal primary treatment of gastric, junctional and oesophageal adenocarcinoma.

Multimodal primary treatment of localised adenocarcinoma of the stomach, the oesophagus and the oesophagogastric junction (AEG) was reviewed by a multidisciplinary expert panel in a moderated consensus session. Here, we report the key points of the discussion and the resulting recommendations. The exact definition of the tumour location and extent by white light endoscopy in conjunction with computed tomography scans is the backbone for any treatment decision. Their value is limited with respect to the infiltration depth, lymph node involvement and peritoneal involvement. Additional endoscopic ultrasound was recommended mainly for tumours of the lower oesophagogastric junction (i.e. AEG type II and III according to Siewert) and in early cancers before endoscopic resection. Laparoscopy to diagnose peritoneal involvement was thought to be necessary before the start of neoadjuvant treatment in all gastric cancers and in AEG type II and III. In general, perioperative multimodal treatment was suggested for all locally advanced oesophageal tumours and for gastric cancers with a clinical stage above T1N0. There was consensus that the combination of fluorouracil, folinic acid, oxaliplatin and docetaxel is now a new standard chemotherapy (CTx) regimen for fit patients. In contrast, the optimal choice of perioperative CTx versus neoadjuvant radiochemotherapy (neoRCTx), especially for AEG, was identified as an open question. Expert treatment recommendations depend on the tumour location, biology, the risk of incomplete (R1) resection, response to treatment, local or systemic recurrence risks, the predicted perioperative morbidity and patients' comorbidities. In summary, any treatment decision requires an interdisciplinary discussion in a comprehensive multidisciplinary setting.

3rd St. Gallen EORTC Gastrointestinal Cancer Conference: Consensus recommendations on controversial issues in the primary treatment of pancreatic cancer.

The primary treatment of pancreatic cancer was the topic of the 3rd St. Gallen Conference 2016. A multidisciplinary panel reviewed the current evidence and discussed controversial issues in a moderated consensus session. Here we report on the key expert recommendations. It was generally accepted that radical surgical resection followed by adjuvant chemotherapy offers the only evidence-based treatment with a chance for cure. Initial staging should classify localised tumours as resectable or unresectable (i.e. locally advanced pancreatic cancer) although there remains a large grey-zone of potentially resectable disease between these two categories which has recently been named as borderline resectable, a concept which was generally accepted by the panel members. However, the definition of these borderline-resectable (BR) tumours varies between classifications due to their focus on either (i) technical hurdles (e.g. the feasibility of vascular resection) or (ii) oncological outcome (e.g. predicting the risk of a R1 resection and/or occult metastases). The resulting expert discussion focussed on imaging standards as well as the value of pretherapeutic laparoscopy. Indications for biliary drainage were seen especially before neoadjuvant therapy. Following standard resection, the panel unanimously voted for the use of adjuvant chemotherapy after R0 resection and considered it as a reasonable standard of care after R1 resection, even though the optimal pathologic evaluation and the definition of R0/R1 was the issue of an ongoing debate. The general concept of BR tumours was considered as a good basis to select patients for preoperative therapy, albeit its current impact on the therapeutic strategy was far less clear. Main focus of the conference was to discuss the limits of surgical resection and to identify ways to standardise procedures and to improve curative outcome, including adjuvant and perioperative treatment.

Second St. Gallen European Organisation for Research and Treatment of Cancer Gastrointestinal Cancer Conference: consensus recommendations on controversial issues in the primary treatment of rectal cancer.

Primary treatment of rectal cancer was the focus of the second St. Gallen European Organisation for Research and Treatment of Cancer (EORTC) Gastrointestinal Cancer Conference. In the context of the conference, a multidisciplinary international expert panel discussed and voted on controversial issues which could not be easily answered using published evidence. Main topics included optimal pretherapeutic imaging, indication and type of neoadjuvant treatment, and the treatment strategies in advanced tumours. Here we report the key recommendations and summarise the related evidence. The treatment strategy for localised rectal cancer varies from local excision in early tumours to neoadjuvant radiochemotherapy (RCT) in combination with extended surgery in locally advanced disease. Optimal pretherapeutic staging is a key to any treatment decision. The panel recommended magnetic resonance imaging (MRI) or MRI + endoscopic ultrasonography (EUS) as mandatory staging modalities, except for early T1 cancers with an option for local excision, where EUS in addition to MRI was considered to be most important because of its superior near-field resolution. Primary surgery with total mesorectal excision was recommended by most panellists for some early tumours with limited risk of recurrence (i.e. cT1-2 or cT3a N0 with clear mesorectal fascia on MRI and clearly above the levator muscles), whereas all other stages were considered for multimodal treatment. The consensus panel recommended long-course RCT over short-course radiotherapy for most clinical situations where neoadjuvant treatment is indicated, with the exception of T3a/b N0 tumours where short-course radiotherapy or even no neoadjuvant therapy were regarded to be an option. In patients with potentially resectable tumours and synchronous liver metastases, most panel members did not see an indication to start with classical fluoropyrimidine-based RCT but rather favoured preoperative short-course radiotherapy with systemic combination chemotherapy or alternatively a liver-first resection approach in resectable metastases, which both allow optimal systemic therapy for the metastatic disease. In general, proper patient selection and discussion in an experienced multidisciplinary team was considered as crucial component of care.

Impact of lifestyle modification on left ventricular function and cardiopulmonary exercise capacity in patients with heart failure with normal ejection fraction and cardiometabolic syndrome: a prospective interventional study.

BACKGROUND: Heart failure with normal left ventricular ejection fraction (HFNEF) accounts for about one third of all heart failure patients with considerable mortality. The metabolic syndrome (MS) is a risk factor for diastolic dysfunction and HFNEF. We hypothesized that modifying metabolic burden by exercise training and weight loss might improve left ventricular diastolic function, heart failure symptoms and rehospitalization rate. METHODS AND RESULTS: Forty patients with HFNEF, MS and prediabetes were enrolled in this prospective study. Echocardiography and cardiopulmonary exercise testing (CPET) were done at baseline and after 3 months lifestyle modification (LSM). NT-pro BNP and adiponectin were determined at baseline as both peptidehormones play a crucial role in MS and heart failure. After discharge a 3-month LSM program with the aim of weight reduction by diet and exercise was started. After the intervention period a weight reduction of ≥ 2% was defined as successful LSM (group A = 23 patients), while a weight reduction < 2% was classified as unsuccessful LSM (group B = 17 patients). At baseline NT-pro BNP (424 ± 381 versus 121 ± 99 pg/ml, P < 0.01) and adiponectin (10.1 ± 6.2 versus 4.6-2.0 µg/ml, P < 0.01) were higher in group A than in group B. After 3 months of LSM, CPET showed a significant improve- ment of VO2 peak (P < 0.01), EqCO2 (P < 0.001), O2-pulse (P = 0.02) and VE / VCO2 slope (P = 0.01) in group A. After one year of follow-up a modest but significant reduction of left atrial size and mitral flow to mitral annulus velocity ratio E/E' was seen in group A. LSM resulted in significant improvement of NYHA status (P = 0.03) and higher freedom of rehospitalization (P = 0.04) in group A. CONCLUSION: Successful lifestyle modification in obese, prediabetic patients with HFNEF improves diastolic left ventricular function and cardiopulmonary exercise capacity. As these measures result in improved NYHA status and less hospitalization, LSM might be a promising approach to prevent chronic diastolic heart failure.

The transcriptional co-repressor TLE3 regulates development of trophoblast giant cells lining maternal blood spaces in the mouse placenta.

TLE3 is a transcriptional co-repressor that interacts with several DNA-binding repressors, including downstream effectors of the Notch signaling pathway. We generated Tle3-deficient mice and found that they die in utero and their death is associated with abnormal development of the placenta with major defects in the maternal vasculature. In the normal placenta, maternal blood spaces are lined, not as usual in the mammalian circulation by endothelial cells, but rather by specialized embryo-derived cells of the trophoblast cell lineage named trophoblast giant cells (TGC). Tle3 mRNA is expressed in those specialized TGC and Tle3 mutants show severe defects in differentiation of TGC-lined channels and lacunar spaces that take blood out of the labyrinth zone of the placenta and into the uterine veins. The mutants also show somewhat milder defects on the arterial-side of the maternal vascular circuit in spiral arteries and canals that take blood into the labyrinth. Notch2 and Tle3 expression patterns overlap in several TGC subtypes and we found that Tle3 and Notch2 mutants have some overlapping features. However, they also show differences implying that TLE3 may mediate some but not all of the effects of Notch2 signaling during placenta development. Therefore, formation of the different types of maternal blood spaces by different TGC subtypes is regulated by distinct molecular mechanisms.

Highlights of the EORTC St. Gallen International Expert Consensus on the primary therapy of gastric, gastroesophageal and oesophageal cancer - differential treatment strategies for subtypes of early gastroesophageal cancer.

The 1st St. Gallen EORTC Gastrointestinal Cancer Conference 2012 Expert Panel clearly differentiated treatment and staging recommendations for the various gastroesophageal cancers. For locally advanced gastric cancer (≥T3N+), the preferred treatment modality was pre- and postoperative chemotherapy. The majority of panel members would also treat T2N+ or even T2N0 tumours with a similar approach mainly because pretherapeutic staging was considered highly unreliable. It was agreed that adenocarcinoma of the gastroesophageal junction (AEG) is classified best according to Siewert et al. Preoperative radiochemotherapy (RCT) is the preferred treatment for AEG type I and II tumours. For AEG type III, i.e. tumours which may be considered as gastric cancer, perioperative chemotherapy is the majority approach. For resectable squamous cell cancer of the oesophagus a clear majority recommended radiochemotherapy followed by surgery as optimal approach, irrespective of tumour size. In contrast, definitive RCT was judged appropriate for advanced tumours with extended lymph node involvement (N2) or for cancers of the upper oesophagus. Additional recommendations are presented on the use of endosonography, PET-CT scan and laparoscopy for staging and on the preferred approach to surgery.

The transcriptional co-repressor Grg3/Tle3 promotes pancreatic endocrine progenitor delamination and ß-cell differentiation.

Pancreatic ß-cells arise from Ngn3(+) endocrine progenitors within the trunk epithelium of the embryonic pancreas. The emergence of endocrine cells requires E-cadherin downregulation, but the crucial steps that elicit such are not clear, yet probably important for ultimately being able to efficiently generate ß-cells de novo from stem cells. Grg3 (groucho-related gene 3, also known as Tle3), encodes a member of the Groucho/TLE family of co-repressors and its function in various cell contexts is mediated by recruitment to target genes by different transcription factors. Grg proteins broadly regulate the progression of progenitor cells to differentiated cell types, but specific developmental mechanisms have not been clear. We find that Grg3 is expressed in most ß-cells and a subset of other endocrine cell types in the pancreas. Grg3 is highly expressed in Ngn3(+) endocrine progenitor descendants just after transient Ngn3 expression. Grg3-null embryos die at E14.5, which is associated with placental defects, so we explanted E12.5 pancreata to allow endocrine differentiation to occur in culture. Grg3 knockout explants displayed a drastic decrease in the differentiation of all endocrine cell types owing to defects in the delamination of early endocrine progenitors from the trunk epithelium. We find that Grg3 normally suppresses E-cadherin gene expression, thereby allowing delamination of endocrine cells from the trunk epithelium and revealing how this transcriptional co-repressor modulates this crucial step of ß-cell development.

The antihormonal preventive therapy of breast cancer and prostate cancer.

With the continuing increase of median life expectancy of important segments of the world's population, cancer incidence, as well as cancer related morbidity and mortality, are constantly increasing, especially for developing countries and for breast and prostate cancer, the predominant gender-associated cancer types. In addition to continuing, with more and more expensive efforts to develop new and more effective cancer treatments, it is health-politically and medico-professionally important to realise that only successful approaches to primary cancer prevention of major and frequent cancer types will be able to change this socially and economically unfavourably outlook. It is therefore encouraging to see that primary (or pharmacologic, interventional) cancer prevention programs have been successfully developed over the past decade for individuals at elevated risk for breast and prostate cancer on the basis of several scientifically well-conducted, prospective chemoprevention trials, mainly with synthetic anti-hormones (anti-estrogens and anti-androgens) in the USA, in Europe and Australia. This paper summarises the presently published results and design of several completed and some currently running primary cancer prevention trials in breast cancer and prostate cancer, and also points to the important obstacles for their conduct and translation into general practice in the broader populations at risk outside of clinical prevention research.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep.

BACKGROUND: Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation. Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation. Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures. PURPOSE: The aim of this study was to compare sinus augmentation with rhBMP-2 on a poly-d, l-lactic-co-glycolic acid gelatine (PLPG) sponge with sinus augmentation with autologous pelvic cancellous bone in the maxillary sinus during the placement of different dental implants. MATERIALS AND METHODS: Nine adult female sheep were submitted to bilateral sinus-floor elevation. In one side (test group) the sinus lift was performed with rhBMP-2 on a PLPG-sponge, while the contralateral side served as the control by using cancellous bone from the iliac crest. Three different implants (Bränemark(®), 3i(®) and Straumann(®)) were inserted either simultaneously with the sinus augmentation or as a two staged procedure 6 weeks later. The animals were sacrificed at 6 and 12 weeks for histological and histomorphometrical evaluations during which bone-to-implant contact (BIC) and bone density (BD) were evaluated. RESULTS: BD and BIC were significantly higher at 12 weeks in the test group if the implants were placed at the time of the sinus lift (p<0.05). No difference was observed between the different implant systems or positions. CONCLUSIONS: The use of rhBMP-2 with PLPG-sponge increased BIC as well as BD in the augmented sinuses if compared to autologous bone. Different implant systems and positions of the implants had no effect on BIC or BD.

Reversal of nonlocal vortex motion in the regime of strong nonequilibrium.

We investigate nonlocal vortex motion in weakly pinning a-NbGe nanostructures, which is driven by a transport current I and remotely detected as a nonlocal voltage V{nl}. At a high I of a given polarity, V{nl} changes sign dramatically. This is followed by V{nl} becoming even in I, with the opposite sign at low and high temperatures T. These findings can be explained by a Nernst-like effect resulting from local electron overheating (low T), and a magnetization enhancement due to a nonequilibrium quasiparticle distribution that leads to a gap enhancement near the vortex core (high T).

EGFR and HER2 expression in advanced biliary tract cancer.

AIM: To analyze the pathogenetic role and potential clinical usefulness of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor 2 (HER2) in patients with advanced biliary tract cancer (BTC). METHODS: EGFR and HER2 expression was studied in biopsy samples from 124 patients (51% women; median age 64.8 years), with advanced BTC diagnosed between 1997 and 2004. Five micrometers sections of paraffin embedded tissue were examined by standard, FDA approved immunohistochemistry. Tumors with scores of 2+ or 3+ for HER2 expression on immunochemistry were additionally tested for HER2 gene amplification by fluorescence in situ hybridisation (FISH). RESULTS: 34/124 patients (27.4%) had gallbladder cancer, 47 (37.9%) had intrahepatic BTC and 43 (34.7%) had extrahepatic or perihilar BTC. EGFR expression was examined in a subset of 56 samples. EGFR expression was absent in 22/56 tumors (39.3%). Of the remaining samples expression was scored as 1+ in 12 (21.5%), 2+ in 13 (23.2%) and 3+ in 9 (16%), respectively. HER2 expression was as follows: score 0 73/124 (58.8%), score 1+ 27/124 (21.8%), score 2+ 21/124 (17%) and score 3+ 4/124 (3.2%). HER2 gene amplification was present in 6/124, resulting in an overall amplification rate of 5%. CONCLUSION: Our data suggest that routine testing and therapeutic targeting of HER2 does not seem to be useful in patients with BTC, while targeting EGFR may be promising.

Aspirin and non-steroidal anti-inflammatory drugs for cancer prevention: an international consensus statement.

Evidence clearly shows a chemopreventive effect for aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) on colorectal cancer and probably other cancer types; however, data on the risk-benefit profile for cancer prevention are insufficient and no definitive recommendations can be made. Aspirin has emerged as the most likely NSAID for use in chemoprevention because of its known cardiovascular benefit and available safety and efficacy data. Other traditional NSAIDs, particularly sulindac, and selective COX-2 inhibitors are now given to patients at high risk of colorectal cancer, although these drugs do not provide cardioprotection. More studies of aspirin and cancer prevention are needed to define the lowest effective dose, the age at which to initiate therapy, the optimum treatment duration, and the subpopulations for which the benefits of chemoprevention outweigh the risks of adverse side-effects. Although it might be possible to answer some of these questions with longer follow-up of existing clinical trials, randomised controlled trials with new study designs will be needed. Future projects should investigate the effects of aspirin treatment on multiple organ systems. Cancers of interest are colorectal, breast, prostate, lung, stomach, and oesophageal. The main side-effect of aspirin is peptic ulcers; therefore coadministration of aspirin with a proton-pump inhibitor is an attractive option and is under investigation in the AspECT trial.

Expression of Runx2 transcription factor in non-skeletal tissues, sperm and brain.

Runx2 is a master transcription factor for chondrocyte and osteoblast differentiation and bone formation. However expression of Runx2 (by RT-PCR), has been reported in non-skeletal tissues such as breast, T cells and testis. To better define Runx2 activity in non-skeletal tissues, we examined transgenic (Tg) mice expressing LacZ gene under control of 3.0 kb (3 kb Tg) or 1.0 kb (1 kb Tg) of the Runx2 distal (P1) promoter, Runx2 LacZ knock-in (Runx2(+/LacZ)) and Runx2/P1 LacZ knock-in (Runx2/P1(+/LacZ)). In the Runx2 3 kb Tg mouse, beta-galactosidase (beta-gal) expression appeared in various non-skeletal tissues including testis, skin, adrenal gland and brain. beta-gal expression from both 3 kb and 1 kb Tg, reflecting activity of the Runx2 promoter, was readily detectable in seminiferous tubules of the testis and the epididymis. At the single cell level, beta-gal was detected in spermatids and mature sperms not in sertoli or Leydig cells. We also detected a positive signal from the Runx2(+/LacZ) and Runx2/P1(+/LacZ) mice. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and Western blot analysis. Runx2, however, was not related to sex determination and sperm motility. Runx2 mediated beta-gal activity is also found robustly in the hippocampus and frontal lobe of the brain in Runx2(+/LacZ). Collectively, these results indicate that Runx2 is expressed in several non-skeletal tissues particularly sperms of testis and hippocampus of brain. It suggests that Runx2 may play an important role in male reproductive organ testis and brain.