Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling.

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.

MALT-1 mediates IL-17 neural signaling to regulate C. elegans behavior, immunity and longevity.

Besides pro-inflammatory roles, the ancient cytokine interleukin-17 (IL-17) modulates neural circuit function. We investigate IL-17 signaling in neurons, and the extent it can alter organismal phenotypes. We combine immunoprecipitation and mass spectrometry to biochemically characterize endogenous signaling complexes that function downstream of IL-17 receptors in C. elegans neurons. We identify the paracaspase MALT-1 as a critical output of the pathway. MALT1 mediates signaling from many immune receptors in mammals, but was not previously implicated in IL-17 signaling or nervous system function. C. elegans MALT-1 forms a complex with homologs of Act1 and IRAK and appears to function both as a scaffold and a protease. MALT-1 is expressed broadly in the C. elegans nervous system, and neuronal IL-17-MALT-1 signaling regulates multiple phenotypes, including escape behavior, associative learning, immunity and longevity. Our data suggest MALT1 has an ancient role modulating neural circuit function downstream of IL-17 to remodel physiology and behavior.

Factors influencing the type, timing and severity of symptomatic responses to dietary gluten in patients with biopsy-proven coeliac disease.

BACKGROUND & AIM: There is a paucity of data reflecting the symptomatic responses to dietary gluten (SRDG) in patients with Coeliac Disease (CD). We aimed to determine the type, timing and severity of SRDG with reference to a range of disease-related factors. METHODS: Postal survey of 224 biopsy-proven patients including gluten-free diet (GFD) adherence, symptom checklist, ROME II criteria and The Hospital Anxiety & Depression Scale. Case-note review was also conducted. RESULTS: 26% of respondents were male. Full GFD adherence: n=159 (70%). Irritable bowel syndrome (IBS): n=50 (22%). Anxiety: n=30 (13%); Depression: n=33 (14%); Anxiety & Depression: n=72 (32%). Pruritus, fatigue and bloating were a more common SRDG in the partial/none GFD adherent group (p=ns). Co-existing IBS was associated with a greater prevalence of nausea and fatigue in response to gluten (p=<0.05). Fully GFD adherent patients are more likely to have SRDG <1hr than partial/none adherent (OR 4.8; p=0.004), as are a third of patients with co-existing IBS (OR 1.5; p=0.027) and those patients at risk of both anxiety and depression (OR 1.9; p=0.04). Inadvertent exposure to dietary gluten in the fully GFD adherent group is more likely to result in a severe SRDG in comparison to symptoms arising prior to consistent GFD adherence (OR 2.3; p=0.01). IBS sufferers are also more likely to rate their SRDG as severe in nature (OR 1.4; p=0.038). CONCLUSION: Patients with consistent GFD adherence experience a SRDG faster and more severe in comparison to prior gluten exposure possibly demonstrating an adept immunological response. Anxiety and depression also enhance the speed of symptom onset and co-existing visceral hypersensitivity is a risk factor for severe reactions to dietary gluten.

Quality of life in Coeliac Disease is determined by perceived degree of difficulty adhering to a gluten-free diet, not the level of dietary adherence ultimately achieved.

BACKGROUND: Coeliac Disease (CD) is an increasingly common autoimmune condition, the treatment of which is a gluten-free diet (GFD). Previous studies fail to reach consensus of the impact this restrictive diet has on an individual's quality of life (QoL). Furthermore, how patient characteristics, such as demographic and educational background, may predict GFD adherence is poorly understood. We aimed to assess which factors had an impact on Qol in patients with CD. METHODS: Case-control postal survey (n=573). Biopsy-proven CD patients (n=225; mean disease duration 8yrs; range 0.5-52yrs; male 26%) and age and sex matched controls (n=348; male 36%) completed The Short-Form 36-Item (SF-36) QoL measure, The Hospital Anxiety & Depression Scale (HADS), GFD assessment, and demographic questionnaire. RESULTS: We found a high proportion of GFD adherence: 'Full Adherence' 65%, 'Partial Adherence' 31%, 'None Adherence' 4%, accompanied however, by 80% of patients reporting difficulty adhering to the GFD: 'Impossible' 5%, 'Mostly difficult' 14%, 'Sometimes difficult' 61%, 'No difficulty' 20%. Negligible differences in QoL scores were observed when comparing full versus partial/none GFD patients (P=>0.05), however, stepwise reductions in QoL and increasing likelihood of anxiety/depression were found in association with increasing degree of difficulty adhering to the GFD (P=<0.0001). Demographic assessment suggests that an affluent background and a university education promote greater GFD adherence. CONCLUSIONS: Most coeliac patients adhere to a GFD but encounter difficulty doing so (potentially influenced by social and educational background). The degree of GFD difficulty is associated with reductions in patient wellbeing and psychological distress that the dietician is critically placed to address.

Reflux and irritable bowel syndrome are negative predictors of quality of life in coeliac disease and inflammatory bowel disease.

BACKGROUND AND AIM: An increased prevalence of reflux and irritable bowel syndrome (IBS) symptoms is associated with coeliac disease and inflammatory bowel disease (IBD). We aimed to determine the prevalence of reflux and IBS symptoms in a cohort of patients with coeliac disease and IBD and their relationship with quality of life (QoL) and psychological distress. METHODS: Histologically proven coeliac disease (n=225), ulcerative colitis (UC) (n=228), Crohn's disease (CD) (n=230) patients and age/sex-matched controls (n=348) completed the Short-Form 36 (SF-36)-Item Health Survey, Hospital Anxiety and Depression Scale (HADS), reflux screen and Rome II criteria. RESULTS: UC patients report higher SF-36 (QoL) scores than coeliac disease; CD fairing worse overall (P≤0.0001). Reflux prevalence: coeliac disease 66%; UC 62%; CD 72%; controls 50%. Patients report reflux of a greater severity: coeliac disease odds ratio=6.8, 95% confidence interval=3.6-12.7, P≤0.001; IBD odds ratio=2.2, 95% confidence interval=1.6-3.2, P≤0.0001. Stepwise reductions in SF-36 scores in association with increasing reflux severity were found (P≤0.0001). IBS prevalence: coeliac disease 22%; UC 16%; CD 24%; controls 6%. Concomitant IBS was associated with reduced SF-36 scores in patients (P≤0.0001). CONCLUSION: Reflux and IBS are more prevalent in coeliac disease and IBD in comparison with age-matched and sex-matched controls. These additional symptoms are associated with reduced QoL and increasing likelihood of anxiety and depression. QoL may be improved if coeliac disease and IBD patients were assessed for reflux and IBS.

Multimodal analgesia and intravenous nutrition preserves total body protein following major upper gastrointestinal surgery.

BACKGROUND AND OBJECTIVES: This study examined whether perioperative multimodal analgesia (MMA) improves the effectiveness of intravenous nutrition (IVN) as a means of preventing protein wasting following major upper abdominal surgery (UAS). The MMA regimen utilized combined epidural opioid/local anesthetic and the systemic nonsteroidal anti-inflammatory drug (NSAID) ketorolac for 48 hours. METHODS: In a prospective, randomized, nonblinded study, 47 patients scheduled for major UAS were allocated to receive the following: MMA +/- intravenous lipid-based nutrition (IVN) or patient-controlled analgesia with opioids (PCA) +/- IVN. Pain scores, nitrogen balance, total body protein (TBP), arterial blood gases, and various hormones were measured. RESULTS: Pain control was significantly better in the MMA patients at rest and coughing. Only the MMA + IVN group maintained TBP, mean (+/-95% confidence interval) preoperative day 1, 10.5 (+/-1.0) kg; day 14, 10.7 (+/-1.2) kg, whereas TBP decreased in the other groups (P =.04). Nitrogen balance was significantly greater in patients receiving IVN on day 7 (P =.01), but there was no effect related to the analgetic regimen. Decreased PaO(2) seen on postoperative day 2 was not prevented by MMA. The hormonal response to surgery was not influenced by treatment modality, apart from a return to postprandial insulin levels on postoperative day 7 in those patients receiving IVN (P =.002). CONCLUSIONS: In conclusion, we have shown that the combination of MMA and IVN prevents protein loss and improves pain control after major UAS. Our results suggest that after UAS, MMA significantly reduced pain and, in combination with IVN, preserves total body protein and fat. This is the first direct evidence of such effects associated with a commonly used multimodal regimen.