Coiled-coil-mediated dimerization of Atg16 is required for binding to the PROPPIN Atg21.

PROPPINs/WIPIs are ß-propeller proteins that bind phosphoinositides and contribute to the recruitment of protein complexes involved in membrane remodelling processes such as autophagosome formation and endosomal trafficking. Yeast Atg21 and mammalian WIPI2 interact with Atg16/ATG16L1 to mediate recruitment of the lipidation machinery to the autophagosomal membrane. Here, we used the reverse double two-hybrid method (RD2H) to identify residues in Atg21 and Atg16 critical for protein-protein binding. Although our results are generally consistent with the crystal structure of the Atg21-Atg16 complex reported previously, they also reveal that dimerization of the Atg16 coiled-coil domain is required for Atg21 binding. Furthermore, most of the residues identified in Atg21 are conserved in WIPI2 and we showed that these residues also mediate ATG16L1 binding. Strikingly, these residues occupy the same position in the ß-propeller structure as residues in PROPPINs/WIPIs Hsv2 and WIPI4 that mediate Atg2/ATG2A binding, supporting the idea that these proteins use different amino acids at the same position to interact with different autophagic proteins. Finally, our findings demonstrate the effectiveness of the RD2H system to identify critical residues for protein-protein interactions and the utility of this method to generate combinatory mutants with a complete loss of binding capacity.

A burning question from the first international BPAN symposium: is restoration of autophagy a promising therapeutic strategy for BPAN?

Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurodegenerative disease associated with severe cognitive and motor deficits. BPAN pathophysiology and phenotypic spectrum are still emerging due to the fact that mutations in the WDR45 (WD repeat domain 45) gene, a regulator of macroautophagy/autophagy, were only identified a decade ago. In the first international symposium dedicated to BPAN, which was held in Lyon, France, a panel of international speakers, including several researchers from the autophagy community, presented their work on human patients, cellular and animal models, carrying WDR45 mutations and their homologs. Autophagy researchers found an opportunity to explore the defective function of autophagy mechanisms associated with WDR45 mutations, which underlie neuronal dysfunction and early death. Importantly, BPAN is one of the few human monogenic neurological diseases targeting a regulator of autophagy, which raises the possibility that it is a relevant model to directly assess the roles of autophagy in neurodegeneration and to develop autophagy restorative therapeutic strategies for more common disorders.Abbreviations: ATG: autophagy related; BPAN: beta-propeller protein-associated neurodegeneration; ER: endoplasmic reticulum; KO: knockout; NBIA: neurodegeneration with brain iron accumulation; PtdIns3P: phosphatidylinositol-3-phosphate; ULK1: unc-51 like autophagy activating kinase 1; WDR45: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting.

Diverticular Inflammation and Complication Assessment classification, CODA score and fecal calprotectin in clinical assessment of patients with diverticular disease: A decision curve analysis.

BACKGROUND AND AIMS: The Diverticular Inflammation and Complication Assessment (DICA) classification and the Combined Overview on Diverticular Assessment (CODA) were found to be effective in predicting the outcomes of Diverticular Disease (DD). We ascertain whether fecal calprotectin (FC) can further aid in improving risk stratification. METHODS: A three-year international, multicentre, prospective cohort study was conducted involving 43 Gastroenterology and Endoscopy centres. Survival methods for censored observations were used to estimate the risk of acute diverticulitis (AD) in newly diagnosed DD patients according to basal FC, DICA, and CODA. The net benefit of management strategies based on DICA, CODA and FC in addition to CODA was assessed with decision curve analysis, which incorporates the harms and benefits of using a prognostic model for clinical decisions. RESULTS: At the first diagnosis of diverticulosis/DD, 871 participants underwent FC measurement. FC was associated with the risk of AD at 3 years (HR per each base 10 logarithm increase: 3.29; 95% confidence interval, 2.13-5.10) and showed moderate discrimination (c-statistic: 0.685; 0.614-0.756). DICA and CODA were more accurate predictors of AD than FC. However, FC showed high discrimination capacity to predict AD at 3 months, which was not maintained at longer follow-up times. The decision curve analysis comparing the combination of FC and CODA with CODA alone did not clearly indicate a larger net benefit of one strategy over the other. CONCLUSIONS: FC measurement could be used as a complementary tool to assess the immediate risk of AD. In all other cases, treatment strategies based on the CODA score alone should be recommended.

Prevalence and Natural History of Segmental Colitis Associated With Diverticulosis.

INTRODUCTION: We assessed the prevalence and clinical outcomes of segmental colitis associated with diverticulosis (SCAD) in patients with newly diagnosed diverticulosis. METHODS: A 3-year international, multicenter, prospective cohort study was conducted involving 2,215 patients. RESULTS: SCAD diagnosis was posed in 44 patients (30 male patients; median age: 64.5 years; prevalence of 1.99%, 95% confidence interval, 1.45%-2.66%). Patients with SCAD types D and B showed worse symptoms, higher fecal calprotectin values, needed more steroids, and reached less likely complete remission. DISCUSSION: Although SCAD generally had a benign outcome, types B and D were associated with more severe symptoms and worse clinical course.

The association of lipid transfer protein VPS13A with endosomes is mediated by sorting nexin SNX5.

Human VPS13 proteins are implicated in severe neurological diseases. These proteins play an important role in lipid transport at membrane contact sites between different organelles. Identification of adaptors that regulate the subcellular localization of these proteins at specific membrane contact sites is essential to understand their function and role in disease. We have identified the sorting nexin SNX5 as an interactor of VPS13A that mediates its association with endosomal subdomains. As for the yeast sorting nexin and Vps13 endosomal adaptor Ypt35, this association involves the VPS13 adaptor-binding (VAB) domain in VPS13A and a PxP motif in SNX5. Notably, this interaction is impaired by mutation of a conserved asparagine residue in the VAB domain, which is also required for Vps13-adaptor binding in yeast and is pathogenic in VPS13D. VPS13A fragments containing the VAB domain co-localize with SNX5, whereas the more C-terminal part of VPS13A directs its localization to the mitochondria. Overall, our results suggest that a fraction of VPS13A localizes to junctions between the endoplasmic reticulum, mitochondria, and SNX5-containing endosomes.

Prognostic performance of the 'DICA' endoscopic classification and the 'CODA' score in predicting clinical outcomes of diverticular disease: an international, multicentre, prospective cohort study.

OBJECTIVE: To investigate the predictive value of the Diverticular Inflammation and Complication Assessment (DICA) classification and to develop and validate a combined endoscopic-clinical score predicting clinical outcomes of diverticulosis, named Combined Overview on Diverticular Assessment (CODA). DESIGN: A multicentre, prospective, international cohort study. SETTING: 43 gastroenterology and endoscopy centres located in Europe and South America. PARTICIPANTS: 2215 patients (2198 completing the study) at the first diagnosis of diverticulosis/diverticular disease were enrolled. Patients were scored according to DICA classifications. INTERVENTIONS: A 3-year follow-up was performed. MAIN OUTCOME MEASURES: To predict the acute diverticulitis and the surgery according to DICA classification. Survival methods for censored observation were used to develop and validate a novel combined endoscopic-clinical score for predicting diverticulitis and surgery (CODA score). RESULTS: The 3-year cumulative probability of diverticulitis and surgery was of 3.3% (95% CI 2.5% to 4.5%) in DICA 1, 11.6% (95% CI 9.2% to 14.5%) in DICA 2 and 22.0% (95% CI 17.2% to 28.0%) in DICA 3 (p<0.001), and 0.15% (95% CI 0.04% to 0.59%) in DICA 1, 3.0% (95% CI 1.9% to 4.7%) in DICA 2 and 11.0% (95% CI 7.5% to 16.0%) in DICA 3 (p<0.001), respectively. The 3-year cumulative probability of diverticulitis and surgery was ≤4%, and ≤0.7% in CODA A; <10% and <2.5% in CODA B; >10% and >2.5% in CODA C, respectively. The CODA score showed optimal discrimination capacity in predicting the risk of surgery in the development (c-statistic: 0.829; 95% CI 0.811 to 0.846) and validation cohort (c-statistic: 0.943; 95% CI 0.905 to 0.981). CONCLUSIONS: DICA classification has a significant role in predicting the risk of diverticulitis and surgery in patients with diverticulosis, which is significantly enhanced by the CODA score. TRIAL REGISTRATION NUMBER: NCT02758860.

A Dictyostelium model for BPAN disease reveals a functional relationship between the WDR45/WIPI4 homolog Wdr45l and Vmp1 in the regulation of autophagy-associated PtdIns3P and ER stress.

PROPPINs are conserved PtdIns3P-binding proteins required for autophagosome biogenesis that fold into a characteristic group of seven-bladed beta-propellers. Mutations in WDR45/WIPI4, a human member of this family, lead to BPAN, a rare form of neurodegeneration. We have generated mutants for the two PROPPIN proteins present in the model system Dictyostelium discoideum (Atg18 and Wdr45l) and characterized their function. Lack of Wdr45l greatly impairs autophagy, while Atg18 only causes subtle defects in the maturation of autolysosomes. The strong phenotype of the Wdr45l mutant is strikingly similar to that observed in Dictyostelium cells lacking Vmp1, an ER protein required for omegasome formation. Common phenotypes include impaired growth in axenic medium, lack of aggregation, and local enrichment of PtdIns3P as determined by the use of lipid reporters. In addition, Vmp1 and Wdr45l mutants show a chronically active response to ER stress. For both mutants, this altered PtdIns3P localization can be prevented by the additional mutation of the upstream regulator Atg1, which also leads to recovery of axenic growth and reduction of ER stress. We propose that, in addition to an autophagy defect, local autophagy-associated PtdIns3P accumulation might contribute to the pathogenesis of BPAN by disrupting ER homeostasis. The introduction of BPAN-associated mutations in Dictyostelium Wdr45l reveals the impact of pathogenic residues on the function and localization of the protein.

The WIPI Gene Family and Neurodegenerative Diseases: Insights From Yeast and Dictyostelium Models.

WIPIs are a conserved family of proteins with a characteristic 7-bladed ß-propeller structure. They play a prominent role in autophagy, but also in other membrane trafficking processes. Mutations in human WIPI4 cause several neurodegenerative diseases. One of them is BPAN, a rare disease characterized by developmental delay, motor disorders, and seizures. Autophagy dysfunction is thought to play an important role in this disease but the precise pathological consequences of the mutations are not well established. The use of simple models such as the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum provides valuable information on the molecular and cellular function of these proteins, but also sheds light on possible pathways that may be relevant in the search for potential therapies. Here, we review the function of WIPIs as well as disease-causing mutations with a special focus on the information provided by these simple models.

Proteins in Autophagic Machinery.

Autophagy is a conserved self-degradation process that is activated under a wide variety of stresses and physiological conditions [...].

A conserved ATG2 binding site in WIPI4 and yeast Hsv2 is disrupted by mutations causing ß-propeller protein-associated neurodegeneration.

PROPPINs are phosphoinositide-binding ß-propeller proteins that mediate membrane recruitment of other proteins and are involved in different membrane remodeling processes. The main role of PROPPINs is their function in autophagy, where they act at different steps in phagophore formation. The human PROPPIN WIPI4 (WDR45) forms a complex with ATG2 involved in phagophore elongation, and mutations in this gene cause ß-propeller protein-associated neurodegeneration (BPAN). The yeast functional counterpart of WIPI4 is Atg18, although its closest sequence homolog is another member of the PROPPIN family, Hsv2, whose function remains largely undefined. Here, we provide evidence that Hsv2, like WIPI4 and Atg18, interacts with Atg2. We show that Hsv2 and a pool of Atg2 colocalize on endosomes under basal conditions and at the pre-autophagosomal structure (PAS) upon autophagy induction. We further show that Hsv2 drives the recruitment of Atg2 to endosomes while Atg2 mediates Hsv2 recruitment to the PAS. HSV2 overexpression results in mis-sorting and secretion of carboxypeptidase CPY, suggesting that the endosomal function of this protein is related to the endosome-to-Golgi recycling pathway. Furthermore, we show that the Atg2 binding site is conserved in Hsv2 and WIPI4 but not in Atg18. Notably, two WIPI4 residues involved in ATG2 binding are mutated in patients with BPAN, and there is a correlation between the inhibitory effect of these mutations on ATG2 binding and the severity of the disease.

COVID-19 and the Global Impact on Colorectal Practice and Surgery.

BACKGROUND: The novel severe acute respiratory syndrome coronavirus 2 virus that emerged in December 2019 causing coronavirus disease 2019 (COVID-19) has led to the sudden national reorganization of health care systems and changes in the delivery of health care globally. The purpose of our study was to use a survey to assess the global effects of COVID-19 on colorectal practice and surgery. MATERIALS AND METHODS: A panel of International Society of University Colon and Rectal Surgeons (ISUCRS) selected 22 questions, which were included in the questionnaire. The questionnaire was distributed electronically to ISUCRS fellows and other surgeons included in the ISUCRS database and was advertised on social media sites. The questionnaire remained open from April 16 to 28, 2020. RESULTS: A total of 287 surgeons completed the survey. Of the 287 respondents, 90% were colorectal specialists or general surgeons with an interest in colorectal disease. COVID-19 had affected the practice of 96% of the surgeons, and 52% were now using telemedicine. Also, 66% reported that elective colorectal cancer surgery could proceed but with perioperative precautions. Of the 287 respondents, 19.5% reported that the use of personal protective equipment was the most important perioperative precaution. However, personal protective equipment was only provided by 9.1% of hospitals. In addition, 64% of surgeons were offering minimally invasive surgery. However, 44% reported that enough information was not available regarding the safety of the loss of intra-abdominal carbon dioxide gas during the COVID-19 pandemic. Finally, 61% of the surgeons were prepared to defer elective colorectal cancer surgery, with 29% willing to defer for ≤ 8 weeks. CONCLUSION: The results from our survey have demonstrated that, globally, COVID-19 has affected the ability of colorectal surgeons to offer care to their patients. We have also discussed suggestions for various practical adaptation strategies for use during the recovery period.

International consensus on natural orifice specimen extraction surgery (NOSES) for gastric cancer (2019).

At present, natural orifice specimen extraction surgery (NOSES) has attracted more and more attention worldwide, because of its great advantages including minimal cutaneous trauma and post-operative pain, fast post-operative recovery, short hospital stay, and positive psychological impact. However, NOSES for the treatment of gastric cancer (GC) is still in its infancy, and there is great potential to improve its theoretical system and clinical practice. Especially, several key points including oncological outcomes, bacteriological concerns, indication selection, and standardized surgical procedures are raised with this innovative technique. Therefore, it is necessary to achieve an international consensus to regulate the implementation of GC-NOSES, which is of great significance for healthy and orderly development of NOSES worldwide.

The Dictyostelium discoideum model system.

When we set out to organize this Special Issue, we faced the difficult task of gathering together a large variety of topics with the unique commonality of having been studied in a single model organism, Dictyostelium discoideum. This apparent setback turned into a wonderful opportunity to learn about an organism as a whole, which provides a more complete understanding of life processes, their natural meaning and their changes during evolution. From studies dedicated almost exclusively to cell motility, differentiation and patterning, the versatility of D. discoideum has allowed in recent years the expansion of our knowledge to other areas, including cell biology and many others related to human diseases. The present collection of papers can be considered as a journey throughout the mechanisms of life, where D. discoideum acts as a very special tourist guide.

Evidence for an evolutionary relationship between Vmp1 and bacterial DedA proteins.

VMP1 and DedA proteins are conserved families of transmembrane proteins in eukaryotes and prokaryotes respectively. Despite numerous reports involving these proteins in multiple cellular processes, their molecular function is still unknown. They share the domain of unknown function PF09335, suggesting a possible functional relationship between these protein families. Here we show that VMP1 from different species contain two short motifs conserved in the bacterial DedA proteins and the yeast protein Tvp38. The hallmark of one of these motifs is a glycine residue previously shown to be strictly conserved in all the DedA proteins. Substitution of this residue to leucine, glutamate or arginine in Dictyostelium Vmp1 inactivates the protein, as shown by the inability of the mutants to rescue the phenotypes associated with the lack of Vmp1 including development and lipid homeostasis. This is the first experimental approach that supports an evolutionary relationship between Vmp1 and DedA proteins and highlights the importance of the conserved glycine residue in the PF09335 domain.

VPS13A is closely associated with mitochondria and is required for efficient lysosomal degradation.

Members of the VPS13 family are associated with various human diseases. In particular, the loss of function of VPS13A leads to chorea-acanthocytosis (ChAc), a rare neurodegenerative disease without available curative treatments. Autophagy has been considered a promising therapeutic target because the absence of VPS13A causes a defective autophagy flux. However, the mechanistic details of this deficiency are unknown. Here, we identified Rab7A as an interactor of one of the VPS13 family members in Dictyostelium discoideum and showed that this interaction is conserved between the human homologs VPS13A and RAB7A in HeLa cells. As RAB7A is a key player in endosome trafficking, we addressed the possible function of VPS13A in endosome dynamics and lysosome degradation. Our results suggest that the decrease in autophagy observed in the absence of VPS13A may be the result of a more general defect in endocytic trafficking and lysosomal degradation. Unexpectedly, we found that VPS13A is closely localized to mitochondria, suggesting that the role of VPS13A in the endolysosomal pathway might be related to inter-organelle communication. We show that VPS13A localizes at the interface between mitochondria-endosomes and mitochondria-endoplasmic reticulum and that the presence of membrane contact sites is altered in the absence of VPS13A. Based on these findings, we propose that therapeutic strategies aimed at modulating the endolysosomal pathway could be beneficial in the treatment of ChAc.This article has an associated First Person interview with the first author of the paper.

International consensus on natural orifice specimen extraction surgery (NOSES) for colorectal cancer.

In recent years, natural orifice specimen extraction surgery (NOSES) in the treatment of colorectal cancer has attracted widespread attention. The potential benefits of NOSES including reduction in postoperative pain and wound complications, less use of postoperative analgesic, faster recovery of bowel function, shorter length of hospital stay, better cosmetic and psychological effect have been described in colorectal surgery. Despite significant decrease in surgical trauma of NOSES have been observed, the potential pitfalls of this technique have been demonstrated. Particularly, several issues including bacteriological concerns, oncological outcomes and patient selection are raised with this new technique. Therefore, it is urgent and necessary to reach a consensus as an industry guideline to standardize the implementation of NOSES in colorectal surgery. After three rounds of discussion by all members of the International Alliance of NOSES, the consensus is finally completed, which is also of great significance to the long-term progress of NOSES worldwide.

International Consensus on Diverticulosis and Diverticular Disease. Statements from the 3rd International Symposium on Diverticular Disease.

The statements produced by the Chairmen and Speakers of the 3rd International Symposium on Diverticular Disease, held in Madrid on April 11th-13th 2019, are reported. Topics such as current and evolving concepts on the pathogenesis, the course of the disease, the news in diagnosing, hot topics in medical and surgical treatments, and finally, critical issues on the disease were reviewed by the Chairmen who proposed 39 statements graded according to level of evidence and strength of recommendation. Each topic was explored focusing on the more relevant clinical questions. The vote was conducted on a 6-point scale and consensus was defined a priori as 67% agreement of the participants. The voting group consisted of 124 physicians from 18 countries, and agreement with all statements was provided. Comments were added explaining some controversial areas.

Hot Topics in Surgical Management of Acute Diverticulitiss.

In this session different issues for the surgical management of diverticular disease DD) were considered. The first session debated about the antibiotic treatment for acute uncomplicated diverticulitis (AUD), and supports their use selectively rather than routinely in patients with AUD. The second session discussed the best surgical treatment for those patients. Open approach is a valid choice especially in acute setting, while the laparoscopic approach should be individualised according to the level of skills of the surgeon and the risk factors of the patient (such as obesity and state of health at the time of the operation). The third session debated about the peritoneal lavage and drainage, which is still a safe surgical procedure. However, it requires longer follow-up and results of other trials to draw an adequate conclusion. The last session covers the current surgical certainties in managing complicated DD: 1. urgent colectomy has higher mortality in immune-compromised patients, while in elective surgery is comparable with other populations; 2. laparoscopic peritoneal lavage (LPL) should be the choice in young/fit patients; 3. elective resection is safer in an inflammation free interval; 4. laparoscopic resection shows advantages in several outcomes (such as post-operative morbidity and lower stoma and re-operation rate); 5. in Hinchey III/fecal peritonitis, primary sigmoid resection and anastomosis (open or laparoscopic) could be proposed in young/ fit patient; 6. in case of emergency surgery, Hartmann procedure (open or laparoscopic) must be considered in critically ill/unstable patient.

The DICA Endoscopic Classification for Diverticular Disease of the Colon Shows a Significant Interobserver Agreement among Community Endoscopists: an International Study.

BACKGROUND AND AIMS: The Diverticular Inflammation and Complication Assessment (DICA) endoscopic classification of diverticulosis and diverticular disease (DD) is currently available. It scores severity of the disease as DICA 1, DICA 2 and DICA 3. Our aim was to assess the agreement on this classification in an international endoscopists community setting. METHODS: A total of 96 doctors (82.9% endoscopists) independently scored a set of DD endoscopic videos. The percentages of overall agreement on DICA score and a free-marginal multirater kappa (κ) coefficient were reported as statistical measures of interrater agreement. RESULTS: Overall agreement in using DICA was 91.8% with a free-marginal kappa of 88% (95% CI 80-95). The overall agreement levels were: DICA 1, 85.2%; DICA 2, 96.5%; DICA 3, 99.5%. The free marginal κ was: DICA 1 = 0.753, DICA 2 = 0.958, DICA 3 = 0.919. The agreement about the main endoscopic items was 83.4% (k 67%) for diverticular extension, 62.6% (k 65%) for number of diverticula for each district, 86.8% (k 82%) for presence of inflammation, and 98.5 (k 98%) for presence of complications. CONCLUSIONS: The overall interrater agreement in this study ranges from good to very good. DICA score is a simple and reproducible endoscopic scoring system for diverticulosis and DD.

The Unfolded Protein Response Pathway in the Yeast Kluyveromyces lactis. A Comparative View among Yeast Species.

Eukaryotic cells have evolved signalling pathways that allow adaptation to harmful conditions that disrupt endoplasmic reticulum (ER) homeostasis. When the function of the ER is compromised in a condition known as ER stress, the cell triggers the unfolded protein response (UPR) in order to restore ER homeostasis. Accumulation of misfolded proteins due to stress conditions activates the UPR pathway. In mammalian cells, the UPR is composed of three branches, each containing an ER sensor (PERK, ATF6 and IRE1). However, in yeast species, the only sensor present is the inositol-requiring enzyme Ire1. To cope with unfolded protein accumulation, Ire1 triggers either a transcriptional response mediated by a transcriptional factor that belongs to the bZIP transcription factor family or an mRNA degradation process. In this review, we address the current knowledge of the UPR pathway in several yeast species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida glabrata, Cryptococcus neoformans, and Candida albicans. We also include unpublished data on the UPR pathway of the budding yeast Kluyveromyces lactis. We describe the basic components of the UPR pathway along with similarities and differences in the UPR mechanism that are present in these yeast species.