Protocol of the LATFIA trial (Laser Assisted Treatment of Fistula in Ano): a multicentre, prospective, randomized controlled trial comparing fistula-tract laser closure (FiLaC™) with rectal advancement flap for high trans-sphincteric fistulas.

AIM: Anal fistula is one of the most common anal diseases, affecting between 1 and 3 per 10 000 people per year. Symptoms have a potentially severe effect on a patient's quality of life. Surgery is the mainstay of treatment, aiming to cure the fistula and preserve anal sphincter function. Rectal advancement flap (RAF) is currently the gold standard treatment but has recurrence rates varying between 20% and 50% and might lead to disturbance of continence. The aim of the trial described in this work is to discover if the minimally invasive fistula tract laser closure (FiLaC™) technique could achieve higher healing rates and a better functional outcome than RAF. METHOD: We will perform a randomized prospective multicentre noninferiority study of the treatment of high trans-sphincteric perianal fistulas, comparing FiLaC™ with RAF in terms of fistula healing, recurrence rate, functional outcome and quality of life. Primary and secondary fistula healing will be evaluated at 26 and 52 weeks' follow-up. Quality of life will be evaluated using the SF-36 questionnaire, the Faecal Incontinence Quality of Life Scale questionnaire and the Vaizey score at 3, 6, 12 and 26 weeks postoperatively. CONCLUSION: High trans-sphincteric fistulas have a potentially severe effect on a patient's quality of life. Classical treatment with RAF is a time-consuming invasive procedure. The LATFIA trial aims to compare FiLaC™ with the gold standard treatment with RAF. In case of noninferiority, FiLaC™ treatment could be standardized as a first line treatment for high trans-sphincteric fistulas. Better conservation of the patient's anal sphincter function could possibly be obtained. Likewise, we will report on the postoperative quality of life when applying these two techniques.

N-glycosylation as a eukaryotic protective mechanism against protein aggregation.

The tendency for proteins to form aggregates is an inherent part of every proteome and arises from the self-assembly of short protein segments called aggregation-prone regions (APRs). While posttranslational modifications (PTMs) have been implicated in modulating protein aggregation, their direct role in APRs remains poorly understood. In this study, we used a combination of proteome-wide computational analyses and biophysical techniques to investigate the potential involvement of PTMs in aggregation regulation. Our findings reveal that while most PTM types are disfavored near APRs, N-glycosylation is enriched and evolutionarily selected, especially in proteins prone to misfolding. Experimentally, we show that N-glycosylation inhibits the aggregation of peptides in vitro through steric hindrance. Moreover, mining existing proteomics data, we find that the loss of N-glycans at the flanks of APRs leads to specific protein aggregation in Neuro2a cells. Our findings indicate that, among its many molecular functions, N-glycosylation directly prevents protein aggregation in higher eukaryotes.

Exploiting the aggregation propensity of beta-lactamases to design inhibitors that induce enzyme misfolding.

There is an arms race between beta-lactam antibiotics development and co-evolving beta-lactamases, which provide resistance by breaking down beta-lactam rings. We have observed that certain beta-lactamases tend to aggregate, which persists throughout their evolution under the selective pressure of antibiotics on their active sites. Interestingly, we find that existing beta-lactamase active site inhibitors can act as molecular chaperones, promoting the proper folding of these resistance factors. Therefore, we have created Pept-Ins, synthetic peptides designed to exploit the structural weaknesses of beta-lactamases by causing them to misfold into intracellular inclusion bodies. This approach restores sensitivity to a wide range of beta-lactam antibiotics in resistant clinical isolates, including those with Extended Spectrum variants that pose significant challenges in medical practice. Our findings suggest that targeted aggregation of resistance factors could offer a strategy for identifying molecules that aid in addressing the global antibiotic resistance crisis.

An observational study on lifestyle and environmental risk factors in patients with acute appendicitis.

Purpose: Acute appendicitis is a common abdominal emergency worldwide. This study aimed at characterizing environmental risk factors influencing the development and severity of acute appendicitis. Methods: Patients from a Belgian acute appendicitis cohort (n = 374) and healthy controls from the 500 functional genomics (500FG) cohort (n = 513) were compared. Individuals with a history of appendectomy (n = 1067) and without a history of appendectomy (n = 8656) were available from the Nijmegen Biomedical Study (NBS). Questionnaires on demographics, lifestyle and environment were available. Binary logistic regression was used for prediction models. Results: Fifteen risk factors for developing acute appendicitis were identified. Binary logistic regression showed that 7 were independent risk factors: family history of acute appendicitis, having grown up in a rural environment, having a lower education, probiotic use as well as antibiotic use increased the risk of developing appendicitis. Fruit and fiber-rich vegetable consumption decreased the risk. Findings on vegetable consumption, smoking and level of education were replicated in the NBS population. Independent risk factors for complicated appendicitis were being male, higher age, and a delay to diagnosis of more than 48 h. Conclusions: Environmental exposures influence the risk of developing appendicitis. Further research into these factors is needed.

Exploiting the intrinsic misfolding propensity of the KRAS oncoprotein.

Mutant KRAS is a major driver of oncogenesis in a multitude of cancers but remains a challenging target for classical small molecule drugs, motivating the exploration of alternative approaches. Here, we show that aggregation-prone regions (APRs) in the primary sequence of the oncoprotein constitute intrinsic vulnerabilities that can be exploited to misfold KRAS into protein aggregates. Conveniently, this propensity that is present in wild-type KRAS is increased in the common oncogenic mutations at positions 12 and 13. We show that synthetic peptides (Pept-ins™) derived from two distinct KRAS APRs could induce the misfolding and subsequent loss of function of oncogenic KRAS, both of recombinantly produced protein in solution, during cell-free translation and in cancer cells. The Pept-ins exerted antiproliferative activity against a range of mutant KRAS cell lines and abrogated tumor growth in a syngeneic lung adenocarcinoma mouse model driven by mutant KRAS G12V. These findings provide proof-of-concept that the intrinsic misfolding propensity of the KRAS oncoprotein can be exploited to cause its functional inactivation.

Investigating the Sequence Determinants of the Curling of Amyloid Fibrils Using Ovalbumin as a Case Study.

Highly ordered, straight amyloid fibrils readily lend themselves to structure determination techniques and have therefore been extensively characterized. However, the less ordered curly fibrils remain relatively understudied, and the structural organization underlying their specific characteristics remains poorly understood. We found that the exemplary curly fibril-forming protein ovalbumin contains multiple aggregation prone regions (APRs) that form straight fibrils when isolated as peptides or when excised from the full-length protein through hydrolysis. In the context of the intact full-length protein, however, the regions separating the APRs facilitate curly fibril formation. In fact, a meta-analysis of previously reported curly fibril-forming proteins shows that their inter-APRs are significantly longer and more hydrophobic when compared to straight fibril-forming proteins, suggesting that they may cause strain in the amyloid state. Hence, inter-APRs driving curly fibril formation may not only apply to our model protein but rather constitute a more general mechanism.

Impact of Human Genetic Variation on C-Reactive Protein Concentrations and Acute Appendicitis.

Background: Acute appendicitis is one of the most common abdominal emergencies worldwide. Both environmental and genetic factors contribute to the disease. C-reactive protein (CRP) is an important biomarker in the diagnosis of acute appendicitis. CRP concentrations are significantly affected by genetic variation. However, whether such genetic variation is causally related to appendicitis risk remains unclear. In this study, the causal relationship between single-nucleotide polymorphisms (SNPs) associated with circulating CRP concentrations and the risk and severity of acute appendicitis was investigated. Methods: CRP concentrations in serum of appendicitis patients (n = 325) were measured. Appendicitis was categorized as complicated/uncomplicated and gangrenous/non-gangrenous. Imputed SNP data (n = 287) were generated. A genome-wide association study (GWAS) on CRP concentrations and appendicitis severity was performed. Intersection and colocalization of the GWAS results were performed with appendicitis and CRP-associated loci from the Pan-UKBB cohort. A functional-genomics approach to prioritize genes was employed. Results: Thirteen percent of significant CRP quantitative trait loci (QTLs) that were previously identified in a large cohort of healthy individuals were replicated in our small patient cohort. Significant enrichment of CRP-QTLs in association with appendicitis was observed. Among these shared loci, the two top loci at chromosomes 1q41 and 8p23.1 were characterized. The top SNP at chromosome 1q41 is located within the promoter of H2.0 Like Homeobox (HLX) gene, which is involved in blood cell differentiation, and liver and gut organogeneses. The expression of HLX is increased in the appendix of appendicitis patients compared to controls. The locus at 8p23.1 contains multiple genes, including cathepsin B (CTSB), which is overexpressed in appendix tissue from appendicitis patients. The risk allele of the top SNP in this locus also increases CTSB expression in the sigmoid colon of healthy individuals. CTSB is involved in collagen degradation, MHC class II antigen presentation, and neutrophil degranulation. Conclusions: The results of this study prioritize HLX and CTSB as potential causal genes for appendicitis and suggest a shared genetic mechanism between appendicitis and CRP concentrations.

Protein structure and aggregation: a marriage of necessity ruled by aggregation gatekeepers.

Protein aggregation propensity is a pervasive and seemingly inescapable property of proteomes. Strikingly, a significant fraction of the proteome is supersaturated, meaning that, for these proteins, their native conformation is less stable than the aggregated state. Maintaining the integrity of a proteome under such conditions is precarious and requires energy-consuming proteostatic regulation. Why then is aggregation propensity maintained at such high levels over long evolutionary timescales? Here, we argue that the conformational stability of the native and aggregated states are correlated thermodynamically and that codon usage strengthens this correlation. As a result, the folding of stable proteins requires kinetic control to avoid aggregation, provided by aggregation gatekeepers. These unique residues are evolutionarily selected to kinetically favor native folding, either on their own or by coopting chaperones.

Comparison of the Clavien-Dindo and Comprehensive Complication Index systems for grading of surgical complications after colorectal resections.

INTRODUCTION: Postoperative complications are associated with prolonged hospital stay and a rise in costs of treatment. The Comprehensive Complication Index (CCI) was developed as a scoring system that does not only take the most severe complication into account but all complications after surgery. Our aim was to compare the Clavien-Dindo scoring system with the CCI in predicting length of hospital stay (LOHS) and in-hospital costs after colorectal resections. METHODS: Complications occurring after surgical procedures, performed between October 2012 and September 2013, were prospectively recorded. During this period 164 patients developed complication(s). Only patients that underwent a colorectal resection were included. Multivariable linear regression analysis was performed to find independent predictors of in-hospital costs and LOHS. RESULTS: 64 patients (age (range): 69 (10-91) years, M/F: 36/28) were retained. 46 (71.9%) patients had a Clavien-Dindo score ≥ IIIb. Median (IQR) CCI was 40 (30.2-53.9). Mean (±SD) in-hospitals costs for all patients were €12,920 ± €10,229. The adjusted difference (95% CI, p-value) in in-hospital costs for minor and major (Clavien-Dindo ≥ IIIb) complications was 10,021 (€4283 to €15,759, p = 0.001). A 10 point increase in CCI increased in-hospital costs by €2040. Multivariable analysis retained CCI > 40 as the only independent risk factor for increased in-hospital costs (Standard Beta Coeffic (p-value): 8063 (p = 0.022). CONCLUSION: CCI is a better predictor of in-hospital costs than Clavien-Dindo score to classify complications after colorectal resections, as it captures all complications. Further research is warranted to extrapolate our findings to other sub-specialities of surgery.

Heterotypic Amyloid ß interactions facilitate amyloid assembly and modify amyloid structure.

It is still unclear why pathological amyloid deposition initiates in specific brain regions or why some cells or tissues are more susceptible than others. Amyloid deposition is determined by the self-assembly of short protein segments called aggregation-prone regions (APRs) that favour cross-ß structure. Here, we investigated whether Aß amyloid assembly can be modified by heterotypic interactions between Aß APRs and short homologous segments in otherwise unrelated human proteins. Mining existing proteomics data of Aß plaques from AD patients revealed an enrichment in proteins that harbour such homologous sequences to the Aß APRs, suggesting heterotypic amyloid interactions may occur in patients. We identified homologous APRs from such proteins and show that they can modify Aß assembly kinetics, fibril morphology and deposition pattern in vitro. Moreover, we found three of these proteins upon transient expression in an Aß reporter cell line promote Aß amyloid aggregation. Strikingly, we did not find a bias towards heterotypic interactions in plaques from AD mouse models where Aß self-aggregation is observed. Based on these data, we propose that heterotypic APR interactions may play a hitherto unrealized role in amyloid-deposition diseases.

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation.

While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

Gene Erosion Can Lead to Gain-of-Function Alleles That Contribute to Bacterial Fitness.

Despite our extensive knowledge of the genetic regulation of heat shock proteins (HSPs), the evolutionary routes that allow bacteria to adaptively tune their HSP levels and corresponding proteostatic robustness have been explored less. In this report, directed evolution experiments using the Escherichia coli model system unexpectedly revealed that seemingly random single mutations in its tnaA gene can confer significant heat resistance. Closer examination, however, indicated that these mutations create folding-deficient and aggregation-prone TnaA variants that in turn can endogenously and preemptively trigger HSP expression to cause heat resistance. These findings, importantly, demonstrate that even erosive mutations with disruptive effects on protein structure and functionality can still yield true gain-of-function alleles with a selective advantage in adaptive evolution.

Investigating the mechanism of action of aggregation-inducing antimicrobial Pept-ins.

Aggregation can be selectively induced by aggregation-prone regions (APRs) contained in the target proteins. Aggregation-inducing antimicrobial peptides (Pept-ins) contain sequences homologous to APRs of target proteins and exert their bactericidal effect by causing aggregation of a large number of proteins. To better understand the mechanism of action of Pept-ins and the resistance mechanisms, we analyzed the phenotypic, lipidomic, and transcriptomic as well as genotypic changes in laboratory-derived Pept-in-resistant E. coli mutator cells. The analysis showed that the Pept-in resistance mechanism is dominated by a decreased Pept-in uptake, in both laboratory-derived mutator cells and clinical isolates. Our data indicate that Pept-in uptake involves an electrostatic attraction between the Pept-in and the bacterial membrane and follows a complex mechanism potentially involving many transporters. Furthermore, it seems more challenging for bacteria to become resistant toward Pept-ins that are less dependent on electrostatic attraction for uptake, suggesting that future Pept-ins should be selected for this property.

Local Recurrence After Transanal Total Mesorectal Excision for Rectal Cancer: A Multicenter Cohort Study.

OBJECTIVE: This study aimed to determine local recurrence (LR) rate and pattern after transanal total mesorectal excision (TaTME) for rectal cancer. BACKGROUND: TaTME for mid- and low rectal cancer has known a rapid and worldwide adoption. Recently, concerns have been raised on the oncological safety in light of reported high LR rates with a multifocal pattern. METHODS: This was a multicenter observational cohort study in 6 tertiary referral centers. All consecutive TaTME cases for primary rectal adenocarcinoma from the first TaTME case in every center until December 2018 were included for analysis. Patients with benign tumors, malignancies other than adenocarcinoma and recurrent rectal cancer, as well as exenterative procedures, were excluded. The primary endpoint was 2-year LR rate. Secondary endpoints included patterns and treatment of LR and histopathological characteristics of the primary surgery. RESULTS: A total of 767 patients were identified and eligible for analysis. Resection margins were involved in 8% and optimal pathological outcome (clear margins, (nearly) complete specimen, no perforation) was achieved in 86% of patients. After a median follow-up of 25.5 months, 24 patients developed LR, with an actuarial cumulative 2-year LR rate of 3% (95% CI 2-5). In none of the patients, a multifocal pattern of LR was observed. Thirteen patients had isolated LR (without systemic disease) and 10/13 could be managed by salvage surgery of whom 8 were disease-free at the end of follow-up. CONCLUSIONS AND RELEVANCE: This study shows good loco regional control after TaTME in selected cases from tertiary referral centers and does not indicate an inherent oncological risk of the surgical technique.

An observational study of innate immune responses in patients with acute appendicitis.

Acute appendicitis is a common surgical emergency worldwide. Exaggerated immune responses could be associated with appendicitis. This study aimed at characterizing immune responses towards a large variety of gut commensals and pathogens, and pattern recognition receptor (PRR) ligands, and investigating the course of systemic inflammation in a prospective cohort of acute appendicitis patients. PBMC responses of 23 patients of the cohort and 23 healthy controls were characterized more than 8 months post-surgery. Serum cytokine levels were measured in 23 patients at the time of appendicitis and after one month. CRP, WBC and percentage of neutrophils were analyzed in the total cohort of 325 patients. No differences in PBMC responses were found between patients and controls. Stronger IL-10 responses were found following complicated appendicitis. A trend towards lower IL-8 responses was shown following gangrenous appendicitis. Serum IL-10 and IL-6 were significantly elevated at presentation, and IL-6, IL-8 and TNF-α levels were higher in complicated appendicitis. Routine biomarkers could predict severity of appendicitis with high specificities, but low sensitivities. Cytokine responses in patients following acute appendicitis did not differ from healthy controls. Higher serum cytokine levels were found in acute complicated and gangrenous cases. Further research into discriminative biomarkers is warranted.

Reverse engineering synthetic antiviral amyloids.

Human amyloids have been shown to interact with viruses and interfere with viral replication. Based on this observation, we employed a synthetic biology approach in which we engineered virus-specific amyloids against influenza A and Zika proteins. Each amyloid shares a homologous aggregation-prone fragment with a specific viral target protein. For influenza we demonstrate that a designer amyloid against PB2 accumulates in influenza A-infected tissue in vivo. Moreover, this amyloid acts specifically against influenza A and its common PB2 polymorphisms, but not influenza B, which lacks the homologous fragment. Our model amyloid demonstrates that the sequence specificity of amyloid interactions has the capacity to tune amyloid-virus interactions while allowing for the flexibility to maintain activity on evolutionary diverging variants.

Autonomous aggregation suppression by acidic residues explains why chaperones favour basic residues.

Many chaperones favour binding to hydrophobic sequences that are flanked by basic residues while disfavouring acidic residues. However, the origin of this bias in protein quality control remains poorly understood. Here, we show that while acidic residues are the most efficient aggregation inhibitors, they are also less compatible with globular protein structure than basic amino acids. As a result, while acidic residues allow for chaperone-independent control of aggregation, their use is structurally limited. Conversely, we find that, while being more compatible with globular structure, basic residues are not sufficient to autonomously suppress protein aggregation. Using Hsp70, we show that chaperones with a bias towards basic residues are structurally adapted to prioritize aggregating sequences whose structural context forced the use of the less effective basic residues. The hypothesis that emerges from our analysis is that the bias of many chaperones for basic residues results from fundamental thermodynamic and kinetic constraints of globular structure. This also suggests the co-evolution of basic residues and chaperones allowed for an expansion of structural variety in the protein universe.