Potential of damage associated molecular patterns in synergising radiation and the immune response in oesophageal cancer.

BACKGROUND: There is an intimate crosstalk between cancer formation, dissemination, treatment response and the host immune system, with inducing tumour cell death the ultimate therapeutic goal for most anti-cancer treatments. However, inducing a purposeful synergistic response between conventional therapies and the immune system remains evasive. The release of damage associated molecular patterns (DAMPs) is indicative of immunogenic cell death and propagation of established immune responses. However, there is a gap in the literature regarding the importance of DAMP expression in oesophageal adenocarcinoma (OAC) or by immune cells themselves. AIM: To investigate the effects of conventional therapies on DAMP expression and to determine whether OAC is an immunogenic cancer. METHODS: We investigated the levels of immunogenic cell death-associated DAMPs, calreticulin (CRT) and HMGB1 using an OAC isogenic model of radioresistance. DAMP expression was also assessed directly using ex vivo cancer patient T cells (n = 10) and within tumour biopsies (n = 9) both pre and post-treatment with clinically relevant chemo(radio)therapeutics. RESULTS: Hypoxia in combination with nutrient deprivation significantly reduces DAMP expression by OAC cells in vitro. Significantly increased frequencies of T cell DAMP expression in OAC patients were observed following chemo(radio)therapy, which was significantly higher in tumour tissue compared with peripheral blood. Patients with high expression of HMGB1 had a significantly better tumour regression grade (TRG 1-2) compared to low expressors. CONCLUSION: In conclusion, OAC expresses an immunogenic phenotype with two distinct subgroups of high and low DAMP expressors, which correlated with tumour regression grade and lymphatic invasion. It also identifies DAMPs namely CRT and HMGB1 as potential promising biomarkers in predicting good pathological responses to conventional chemo(radio)therapies currently used in the multimodal management of locally advanced disease.

Metformin is a metabolic modulator and radiosensitiser in rectal cancer.

Resistance to neoadjuvant chemoradiation therapy, is a major challenge in the management of rectal cancer. Increasing evidence supports a role for altered energy metabolism in the resistance of tumours to anti-cancer therapy, suggesting that targeting tumour metabolism may have potential as a novel therapeutic strategy to boost treatment response. In this study, the impact of metformin on the radiosensitivity of colorectal cancer cells, and the potential mechanisms of action of metformin-mediated radiosensitisation were investigated. Metformin treatment was demonstrated to significantly radiosensitise both radiosensitive and radioresistant colorectal cancer cells in vitro. Transcriptomic and functional analysis demonstrated metformin-mediated alterations to energy metabolism, mitochondrial function, cell cycle distribution and progression, cell death and antioxidant levels in colorectal cancer cells. Using ex vivo models, metformin treatment significantly inhibited oxidative phosphorylation and glycolysis in treatment naïve rectal cancer biopsies, without affecting the real-time metabolic profile of non-cancer rectal tissue. Importantly, metformin treatment differentially altered the protein secretome of rectal cancer tissue when compared to non-cancer rectal tissue. Together these data highlight the potential utility of metformin as an anti-metabolic radiosensitiser in rectal cancer.

Application of PREVENA (Surgical Incision Protection System) in reducing surgical site infections following reversal of ileostomy or colostomy: the PRIC study protocol.

AIM: There is a current lack of evidence in the literature to support the routine use of negative pressure wound therapy (NPWT) to reduce the risk of surgical site infections (SSI) in the setting of ileostomy or colostomy reversal. The aim of this study is to examine whether routine NPWT confers a lower rate of SSI than conventional dressings following reversal of ileostomy or colostomy. METHODS: The PRIC study is a randomized, controlled, open-label, multi-centre superiority trial to assess whether routine NPWT following wound closure confers a lower rate of SSI following reversal of ileostomy or colostomy when compared to conventional dressings. Participants will be consecutively identified and recruited. Eligible participants will be randomized in a 1:1 allocation ratio, to receive either the NPWT (PREVENA) dressings or conventional dressings which will be applied immediately upon completion of surgery. PREVENA dressings will remain applied for a duration of 7 days. Surgical wounds will then be examined on post-operative day seven as well as during follow-up appointments in OPD for any evidence of SSI. In the interim, public health nurses (PHN) will provide out-patient support services incorporating wound assessment and care as part of a routine basis. Study investigators will liaise with PHN to gather the relevant data in relation to the time to wound healing. Our primary endpoint is the incidence of SSI within 30 days of stoma reversal. Secondary endpoints include measuring time to wound healing, evaluating wound healing and aesthetics and assessing patient satisfaction. CONCLUSION: The PRIC study will assess whether routine NPWT following wound closure is superior to conventional dressings in the reduction of SSI following reversal of ileostomy or colostomy and ascertain whether routine NPWT should be considered the new standard of care.

Prostate radiotherapy and the risk of secondary rectal cancer-a meta-analysis.

PURPOSE: Radiotherapy is being used increasingly in the treatment of prostate cancer. However, ionising radiation may confer a small risk of a radiation-induced secondary malignancy. We aim to assess the risk of rectal cancer following pelvic radiotherapy for prostate cancer. METHODS: A search was conducted of the PubMed/MEDLINE, EMBASE and Web of Science databases identifying studies reporting on the risk of rectal cancer following prostatic radiotherapy. Studies must have included an appropriate control group of non-irradiated prostate cancer patients. A meta-analysis was performed to assess the risk of prostatic radiotherapy on subsequent rectal cancer diagnosis. RESULTS: In total, 4757 articles were screened with eight studies meeting the predetermined criteria. A total of 796,386 patients were included in this meta-analysis which showed an increased odds ratio (OR) for subsequent rectal cancer in prostate cancer patients treated with radiotherapy compared to those treated by non-radiotherapy means (OR 1.45, 1.07-1.97, p = 0.02). CONCLUSION: These findings confirm that prostate radiotherapy significantly increases the risk of subsequent rectal cancer. This risk has implications for treatment selection, surveillance and patient counselling. However, it is crucial that this information is presented in a rational and comprehensible manner that does not disproportionately frighten or deter patients from what might be their most suitable treatment modality.

Opposing Immune-Metabolic Signature in Visceral Versus Subcutaneous Adipose Tissue in Patients with Adenocarcinoma of the Oesophagus and the Oesophagogastric Junction.

Oesophageal adenocarcinoma (OAC) is an exemplar model of obesity-associated cancer. Previous work in our group has demonstrated that overweight/obese OAC patients have better responses to neoadjuvant therapy, but the underlying mechanisms are unknown. Unravelling the immune-metabolic signatures of adipose tissue may provide insight for this observation. We hypothesised that different metabolic pathways predominate in visceral (VAT) and subcutaneous adipose tissue (SAT) and inflammatory secretions will differ between the fat depots. Real-time ex vivo metabolic profiles of VAT and SAT from 12 OAC patients were analysed. These samples were screened for the secretion of 54 inflammatory mediators, and data were correlated with patient body composition. Oxidative phosphorylation (OXPHOS) was significantly higher in VAT when compared to SAT. OXPHOS was significantly higher in the SAT of patients receiving neoadjuvant treatment. VEGF-A, VEGF-C, P1GF, Flt-1, bFGF, IL-15, IL-16, IL-17A, CRP, SAA, ICAM-1, VCAM-1, IL-2, IL-13, IFN-γ, and MIP-1ß secretions were significantly higher from VAT than SAT. Higher levels of bFGF, Eotaxin-3, and TNF-α were secreted from the VAT of obese patients, while higher levels of IL-23 and TARC were secreted from the SAT of obese patients. The angiogenic factors, bFGF and VEGF-C, correlated with visceral fat area. Levels of OXPHOS are higher in VAT than SAT. Angiogenic, vascular injury and inflammatory cytokines are elevated in VAT versus SAT, indicating that VAT may promote inflammation, linked to regulating treatment response.

Life-Threatening Intraparenchymal Hemorrhage of Steroid-Induced Hepatic Adenomas in a Healthy Man.

Hepatocellular adenomas are uncommon benign epithelial tumors of the liver that are associated with several risk factors such as anabolic androgens and oral contraceptive pills. They may present as incidental findings, with abdominal pain or hemorrhage. This case report details the presentation and management of a life-threatening hepatocellular adenomas hemorrhage in a seemingly healthy 28-year-old man. After initial conservative management, a clinical deterioration prompted urgent reevaluation and successful embolization of the liver through transarterial embolization. As oral contraceptive pills use and anabolic steroid abuse have become more prevalent in recent decades, we may begin to see more of these presentations.

Obesity and anastomotic leak rates in colorectal cancer: a meta-analysis.

PURPOSE: Anastomotic leak (AL) following colorectal cancer resection is associated with considerable morbidity and mortality with an impact on recurrence rates and survival. The impact of obesity on AL rates is debated. This meta-analysis aims to investigate the relationship between obesity and AL. METHODS: A search was conducted of the PubMed/MEDLINE, and Web of Science databases and included studies were split into Western and Asian groups based on population-specific body mass index (BMI) ranges for obesity. A meta-analysis was performed to assess the impact of obesity on AL rate following colorectal cancer resection. RESULTS: Two thousand three hundred and four articles were initially screened. Thirty-one studies totaling 32,953 patients were included. Patients with obesity had a statistically significant increase in AL rate in all Western and Asian study groups. However, this increase was only clinically significant in the rectal anastomotic subgroups-Western: 10.8% vs 8.4%, OR 1.57 (1.01-2.44) and Asian: 9.4% vs 7.4%, OR 1.58 (1.07-2.32). CONCLUSIONS: The findings of this analysis confirm that obesity is a significant risk factor for anastomotic leak, particularly in rectal anastomoses. This effect is thought to be primarily mediated via technical difficulties of surgery although metabolic and immunological factors may also play a role. Obesity in patients undergoing restorative CRC resection should be discussed and considered as part of the pre-operative counselling.

The role of intraoperative radiotherapy in advanced rectal cancer: a meta-analysis.

AIM: Patients with locally advanced and locally recurrent rectal cancer (LARC/LRRC) experience higher rates of local recurrence (LR) and poorer overall survival than patients with primary rectal cancer restricted to the mesorectum despite improved neoadjuvant treatment regimens and radical surgical procedures. Intraoperative radiotherapy (IORT) has been suggested as an adjunctive tool in the surgical management of these challenging cases. However, clear evidence regarding the oncological benefit of IORT is sparse. The aim of this review was to update this evidence in the era of standardized neoadjuvant radiotherapy administration. METHOD: A systematic review of patients who received IORT as part of multimodal treatment for advanced rectal cancer from 2000 to 2020 and an analysis of IORT and surgery/external beam radiotherapy (EBRT) groups was performed. The primary endpoint was the rate of LR between the two groups. RESULTS: Seven papers met the predefined criteria. LR was reduced by the addition of IORT when compared with the surgery/EBRT alone group (14.7% vs. 21.4%; OR 0.55, 95% CI 0.27-1.14; p = 0.11). There was no increase in reported genitourinary morbidity, wound issues, pelvic collections or anastomotic leak in those patients who received IORT. Notably, there was no survival difference between the two groups. CONCLUSION: The addition of IORT to current treatment strategies in the management of patients with LARC/LRRC is associated with a lower rate of locoregional recurrence without increased morbidity. However, this marks a highly selective group of patients, with heterogeneity regarding indications, prior neoadjuvant treatments and/or IORT dosing.

Management of chyle leaks following esophageal resection: a systematic review.

BACKGROUND: Chyle leakage is an uncommon but potentially life-threatening complication following esophageal resections. The optimal management strategy is not clear, with a limited evidence base. METHODS: Searches were conducted up to 31 December 2020 on MEDLINE, Embase, and Web of Science for randomized trials or retrospective studies that evaluated the management of chyle leakage following esophageal resection. Two authors independently screened studies, extracted data, and assessed for bias. The protocol was prospectively registered on PROSPERO (CRD: 42021224895) and reported in accordance with preferred reporting items for systematic reviews and meta-analyses guidelines. RESULTS: A total of 530 citations were reviewed. Twenty-five studies, totaling 1016 patients met the inclusion criteria, including two low-quality clinical trials and 23 retrospective case series. Heterogeneity of study design and outcomes prevented meta-analysis. The overall incidence of chyle leak/fistula was 3.2%. Eighteen studies describe management of chyle leaks conservatively, 17 by surgical ligation of the thoracic duct, 5 by pleurodesis, and 6 described percutaneous lymphangiography with thoracic duct embolization or disruption. CONCLUSIONS: The evidence base for optimal management of chyle leakage postesophagectomy is lacking, which may be related to its low incidence. There is a paucity of high-quality prospective studies directly comparing treatment modalities, but there is some low-certainty evidence that percutaneous approaches have reduced morbidity but lower efficacy compared with surgery. Further high-quality, prospective studies that compare interventions at different levels of severity are needed to determine the optimal approach to treatment.

The Protein Secretome Is Altered in Rectal Cancer Tissue Compared to Normal Rectal Tissue, and Alterations in the Secretome Induce Enhanced Innate Immune Responses.

Locally advanced rectal cancer is treated with neoadjuvant-chemoradiotherapy; however, only ~22% of patients achieve a complete response, and resistance mechanisms are poorly understood. The role of inflammation and immune cell biology in this setting is under-investigated. In this study, we profiled the inflammatory protein secretome of normal (non-cancer) (n = 8) and malignant rectal tissue (n = 12) pre- and post-radiation in human ex vivo explant models and examined the influence of these untreated and treated secretomes on dendritic cell biology (n = 8 for cancer and normal). These resultant profiles were correlated with patient clinical characteristics. Nineteen factors were secreted at significantly higher levels from the rectal cancer secretome when compared to the normal rectal secretome; Flt-1, P1GF, IFN-γ, IL-6, IL-10, CCL20, CCL26, CCL22, CCL3, CCL4, CCL17, GM-CSF, IL-12/IL-23p40, IL-17A, IL-1α, IL-17A/F, IL-1RA, TSLP and CXCL10 (p < 0.05). Radiation was found to have differential effects on normal rectal tissue and rectal cancer tissue with increased IL-15 and CCL22 secretion following radiation from normal rectal tissue explants (p < 0.05), while no significant alterations were observed in the irradiated rectal cancer tissue. Interestingly, however, the irradiated rectal cancer secretome induced the most potent effect on dendritic cell maturation via upregulation of CD80 and PD-L1. Patient's visceral fat area correlated with secreted factors including CCL20, suggesting that obesity status may alter the tumour microenvironment (TME). These results suggest that radiation does not have a negative effect on the ability of the rectal cancer TME to induce an immune response. Understanding these responses may unveil potential therapeutic targets to enhance radiation response and mitigate normal tissue injury. Tumour irradiation in this cohort enhances innate immune responses, which may be harnessed to improve patient treatment outcome.

Fibromatosis of the breast: a 10-year multi-institutional experience and review of the literature.

BACKGROUND: Breast fibromatosis is a rare clinical entity, but poses significant diagnostic and therapeutic challenges. In light of recent changes in management practices, the aim was to review our institutional experience of breast fibromatosis and provide a review of current available literature on such management. METHODS: A search of pathological databases within two tertiary institutions for all patients diagnosed with fibromatosis of the breast over a 10-year period (2007-2016) was performed. Clinicopathological characteristics and modes of treatment were recorded for each patient. Concurrently a comprehensive literature search was performed and studies relating to breast fibromatosis and its management were identified and reviewed. RESULTS: Sixteen patients were identified. Median age at diagnosis was 42 (range 21-70) and all patients were diagnosed with core biopsy. The most useful imaging modality in diagnosis was ultrasonography and magnetic resonance imaging. 13/16 were treated surgically whilst 3/16 were treated using a watch-and-wait approach. 6/13 (46%) required re-excision of margins and 2/13 (15%) had recurrence after surgery. On review of the literature, there is no dedicated guideline in place for the management of breast fibromatosis. Currently a 'watch and wait' approach is favoured over surgical intervention due to high levels of recurrence and associated surgical morbidity. All cases should be discussed at a sarcoma multidisciplinary team meeting and tyrosine kinase inhibitors should be considered in advanced cases. CONCLUSIONS: Breast fibromatosis is rare but affects young patients. Active surveillance is now favoured over surgical resection due to high recurrence rates and extensive morbidity. Dedicated guidelines are required to ensure best outcomes.

Current opinions and practices for the management of acute appendicitis: an international survey.

BACKGROUND: In recent decades the management of acute appendicitis has evolved significantly. Improved access to early imaging and better clinical scoring algorithms have resulted in less negative appendicectomy rates. In addition, non-operative management has become increasingly utilized. The aim of this study was to assess the variability of management of acute appendicitis globally. METHODS: This was a multi-national targeted survey of general surgeons across 39 countries. A structured set of questions was utilized to delineate nuances between management styles of consultants and trainees. Opinions on the pathological diagnosis of appendicitis, acceptable negative appendicectomy rates, and the role of non-operative treatment of appendicitis (NOTA) were surveyed. RESULTS: A total of 304 general surgeons responded to this survey, 42% of which were consultants/attendings. Sixty-nine percent advocated that a histologically normal appendix was the most appropriate definition of a negative appendicectomy, while 29% felt that anything other than inflammation, necrosis, gangrene, or perforation was more appropriate. Forty-three percent felt that negative appendicectomy rates should be less than 10%, with 41% reporting that their own negative appendicectomy rate was < 5%. Interestingly, only 17% reported routinely using NOTA for uncomplicated appendicitis, with one-fifth stating that they would undergo NOTA if they themselves had uncomplicated appendicitis. CONCLUSION: This study represents the largest sampling of management strategies for acute appendicitis. It shows substantial global heterogeneity between clinicians regarding what constitutes a negative appendicectomy as well as the appropriateness of non-operative management.

Radiation-induced Bystander Effect (RIBE) alters mitochondrial metabolism using a human rectal cancer ex vivo explant model.

Locally advanced rectal cancer is treated with neoadjuvant-chemoradiotherapy, however only 22% of patients achieve a complete response. Resistance mechanisms are poorly understood. Radiation-induced Bystander Effect (RIBE) describes the effect of radiation on neighbouring unirradiated cells. We investigated the effects of ex vivo RIBE-induction from normal and rectal cancer tissue on bystander cell metabolism, mitochondrial function and metabolomic profiling. We correlated bystander events to patient clinical characteristics. Ex vivo RIBE-induction caused metabolic alterations in bystander cells, specifically reductions in OXPHOS following RIBE-induction in normal (p = 0.01) and cancer tissue (p = 0.03) and reduced glycolysis following RIBE-induction in cancer tissue (p = 0.01). Visceral fat area correlated with glycolysis (p = 0.02) and ATP production (p = 0.03) following exposure of cells to TCM from irradiated cancer biopsies. Leucine levels were reduced in the irradiated cancer compared to the irradiated normal secretome (p = 0.04). ROS levels were higher in cells exposed to the cancer compared to the normal secretome (p = 0.04). RIBE-induction ex vivo causes alterations in the metabolome in normal and malignant rectal tissue along with metabolic alterations in bystander cellular metabolism. This may offer greater understanding of the effects of RIBE on metabolism, mitochondrial function and the secreted metabolome.

Linking Circulating Serum Proteins with Clinical Outcomes in Esophageal Adenocarcinoma-An Emerging Role for Chemokines.

Esophageal adenocarcinoma (EAC) is an aggressive cancer with poor prognosis and incidence is increasing rapidly in the Western world. Multi-modal treatment has improved survival outcomes but only for a minority of patients. Currently no markers have been identified to predict treatment response. This study investigated the association between clinical outcomes and pre-treatment levels of 54 serum proteins in n = 80 patients with EAC. Low tumor regression grade (TRG), corresponding to a favorable treatment response, was linked to prolonged overall survival (OS). CCL4 was higher in patients with a favorable treatment response, while Tie2 and CRP were higher in poor responders. Elevated CCL22 and CCL26 was associated with improved OS, while elevated IL-10 showed a negative association. CCL3, CCL4, IL-1α and IL-12/IL23p40 were highest in individuals with no adverse features of tumor biology, whereas levels of Tie2 and VEGF were lowest in this cohort. CCL4 was also elevated in patients with high tumor lymphocyte infiltration. Comparison of matched pre- and post-treatment serum (n = 28) showed a large reduction in VEGFC, and a concomitant increase in other cytokines, including CCL4. These data link several serum markers with clinical outcomes, highlighting an important role for immune cell trafficking in the EAC antitumor immune response.

Impact of progesterone receptor status on response to neoadjuvant chemotherapy in estrogen receptor-positive breast cancer patients.

INTRODUCTION: Breast cancer patients respond differently to neoadjuvant chemotherapy(NAC) based on receptor subtype. The aim of this study was to assess the impact of progesterone receptor (PgR) status on response to neoadjuvant chemotherapy (NAC) in estrogen receptor (ER)+, human epidermal growth factor receptor (HER)- breast cancer patients. METHODS: ER+ and HER- patients receiving NAC over a 7-year period (2011-2017) were identified. The primary outcome was breast complete pathological response (pCR) rate. Secondary outcomes included axillary pCR, axillary/breast pCR and complete radiological response (cRR). RESULTS: A total of 203 patients were identified (149 in the ER+, PgR+, and HER- group and 54 in the ER+, PgR-, and HER- group). Compared with the PgR+ group, PgR- patients were significantly associated with breast pCR (31.5% vs 7.4%; χ² test; P < .01). In multivariable analysis, PgR- status (odds ratio [OR], 4.58; 95% confidence interval [CI]: 1.58,13.28; P = .005), radiological size >50 mm (OR, 5.38; 95% CI: 1.07,27.04; P = .04) and grade (OR, 3.52;95% CI: 1.21,10.23;P = .02) were significant predictors of breast pCR. Only PgR- status was a significant predictor of cRR (OR, 6.234; 95% CI: 2.531, 15.355; P < .001). In node positive patients, PgR negativity was associated with a trend towards breast/axillary nodal pCR (22% vs 12.7%; χ² test; P = .055). CONCLUSION: Over 30% of ER+, PgR-, and HER- patients will have a breast pCR after NAC. PgR- is the only significant predictor of breast pCR/cRR in this tumor subtype. ER+, PgR-, and HER- patients should be considered for NAC.

Factors influencing surgical management of acute appendicitis in a large university hospital without a dedicated emergency theatre.

BACKGROUND: Acute appendicitis is the most common surgical emergency. Its management reflects the efficacy of acute care surgery. Limited theatre space is an escalating issue, especially without dedicated emergency theatre access. Pre-operative delays are associated with longer length of stay, higher costs and post-operative complications. AIMS: Calculate time to theatre (TTT) from admission to appendicectomy and investigate factors impacting TTT. METHODS: A retrospective review of all emergency appendicectomies from June 2017 to October 2018. Demographic, clinico-pathological and radiological data were extracted from electronic patient record. RESULTS: One hundred forty-eight patients underwent emergency appendicectomy during the study period. Fifty-six percent (n = 84) were male, and the median (range) age was 30.5 (17-76) years. Sixty-one percent had pre-operative imaging. The median (range) TTT was 18.37 (2-114) h; 7.5% (n = 11) waited > 48 h, 29.7% (n = 44) were operated on after 8 p.m. and 26% (n = 38) were done on elective lists. Male gender, admission CRP > 100 and admission before 12 p.m. significantly shortened TTT (p = 0.030, p = 0.004 and p = 0.001, respectively). However, pre-operative ultrasound, previous acute appendicitis and surgery on an elective list significantly prolonged TTT (p = 0.015 and p = 0.024, respectively). The median (range) LOS was 3 (1-24) nights. Ten percent (n = 15) had post-operative complications; however, longer TTT was not associated with higher complication rates (p = 0.196). CONCLUSIONS: This review highlights the impact of limited theatre access for on-call emergency admissions, with a significant portion of appendicectomies being done on elective lists or out-of-hours.

Improving data transparency in clinical trials using blockchain smart contracts.

The scientific credibility of findings from clinical trials can be undermined by a range of problems including missing data, endpoint switching, data dredging, and selective publication. Together, these issues have contributed to systematically distorted perceptions regarding the benefits and risks of treatments. While these issues have been well documented and widely discussed within the profession, legislative intervention has seen limited success. Recently, a method was described for using a blockchain to prove the existence of documents describing pre-specified endpoints in clinical trials. Here, we extend the idea by using smart contracts - code, and data, that resides at a specific address in a blockchain, and whose execution is cryptographically validated by the network - to demonstrate how trust in clinical trials can be enforced and data manipulation eliminated. We show that blockchain smart contracts provide a novel technological solution to the data manipulation problem, by acting as trusted administrators and providing an immutable record of trial history.

De novo membrane protein structure prediction.

Recent advances in identifying residue-residue contacts from large multiple sequence alignments have enabled impressive gains to be made in the field of protein structure prediction. In this chapter, we discuss these advances and provide a step-by-step guide to applying the latest tools to the de novo modelling of alpha-helical transmembrane proteins. As a practical example, we demonstrate the process of building an accurate 3D model of a G protein-coupled receptor, correctly orientated in the membrane, using only its primary protein sequence.

Evaluation of predictions in the CASP10 model refinement category.

Here we report on the assessment results of the third experiment to evaluate the state of the art in protein model refinement, where participants were invited to improve the accuracy of initial protein models for 27 targets. Using an array of complementary evaluation measures, we find that five groups performed better than the naïve (null) method-a marked improvement over CASP9, although only three were significantly better. The leading groups also demonstrated the ability to consistently improve both backbone and side chain positioning, while other groups reliably enhanced other aspects of protein physicality. The top-ranked group succeeded in improving the backbone conformation in almost 90% of targets, suggesting a strategy that for the first time in CASP refinement is successful in a clear majority of cases. A number of issues remain unsolved: the majority of groups still fail to improve the quality of the starting models; even successful groups are only able to make modest improvements; and no prediction is more similar to the native structure than to the starting model. Successful refinement attempts also often go unrecognized, as suggested by the relatively larger improvements when predictions not submitted as model 1 are also considered.

Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1.

O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functional importance of PL3 and PL5, with the former shown to require overall cationic properties. Essential Arg residues in the RX10G motifs of PL3 and PL5 were found to be conserved in putative homologues of WzyPa, as was the overall sequence homology between these two periplasmic loops in each protein. Amino acid substitutions in CL6 were found to alter Wzz-mediated O-antigen modality, with evidence suggesting that these changes may perturb the C-terminal WzyPa tertiary structure. Together, these data suggest that the catch-and-release mechanism of O-Ag polymerization is widespread among bacteria and that regulation of polymer length is affected by interaction of Wzz with Wzy.