A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

BACKGROUND: Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. RESULTS: Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. CONCLUSIONS: The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production

Exploring novel diagnostic and therapeutic strategies for disorders of anorectal function

Constipation and faecal incontinence (FI) are common gastrointestinal disorders, affecting 8-18% of the Western population. These disorders severely impact patients’ quality of life and are associated with a substantial financial burden on health systems. The diagnosis of both of these conditions relies on careful history taking, physical examination, including digital rectal examination, and anorectal physiological testing. When conservative management fails, anorectal biofeedback (BF) treatment is usually the treatment of choice. The aims of this work were to (1) improve the diagnostic algorithms currently used to diagnose and classify patients with chronic constipation and/or FI, and (2) refine and expand the treatment options, mainly BF, for these patients. Following a comprehensive literature review, data from anorectal physiology testing, namely anorectal manometry and balloon expulsion testing, are examined in both patients and in healthy subjects. The novel findings in this Section inform and guide the optimum methods and metrics for these tests, which are then used to suggest a novel diagnostic phenotype. In the following Section, the long-term efficacy of BF, and the predictors for successful BF treatment - key to improving patient selection and improving access to this effective yet limited resource - are explored. The effect of BF is found to be durable, and factors predicting successful long term treatment outcomes are determined. Next, the role of BF in 'difficult-to-treat' patients, such as patients with spinal cord injury are addressed, and the benefits of treatment in these patient groups, are quantified. Lastly, novel modifications to the BF treatment protocol by either abbreviating or augmenting it with peripheral neuromodulation are shown to be possible alternatives to standard BF treatment. In summary, the experimental studies undertaken and described in this thesis provide novel insights into the diagnostic and therapeutic aspects of constipation and FI. This work makes a significant contribution to improving patient care and quality of life in these patients whose lives are often severely impacted by these disorders. These studies also provide a firm basis for further research in these areas

Efficacy of anorectal biofeedback in scleroderma patients with fecal incontinence : a case-control study

Objective: To determine whether anorectal biofeedback therapy can improve the symptoms of fecal incontinence (FI) in patients with scleroderma when compared to patients with functional FI, and also whether there is any effect on anorectal physiology or quality of life (QOL). FI in patients with scleroderma is highly prevalent and is associated with significant loss of QOL. Biofeedback has been proven to be an effective treatment for functional FI, but there are no data to support its use in scleroderma. Materials and methods: 13 consecutive female patients (median age 59, IQR 47–65 years) with scleroderma, and 26 age- and parity-matched female patients with functional FI (disease controls, 2:1), underwent biofeedback therapy for management of FI. Fecal incontinence severity index (FISI), anorectal physiology, feeling of control and QOL were collected before and after 6 weeks of biofeedback therapy, with additional scoring repeated at 6-month follow-up. Results: After biofeedback treatment FISI, feeling of control and QOL significantly improved in both groups (p < 0.005). There was no difference in the degree in improvement in physiology, FISI or QOL between scleroderma patients and functional FI patients. Long-term improvement in FISI and control were seen in both groups and for QOL only in the scleroderma cohort (p < 0.05). Conclusions: Patients with scleroderma benefit from biofeedback therapy to the same extent as that achieved in patients with functional FI. There are significant improvements in symptoms, physiology and QOL. Biofeedback is an effective, low-risk treatment option in this patient group.6 page(s

Novel insights into fecal incontinence in men

Fecal incontinence (FI) in men is common, yet data on sex differences in clinical features, physiology, and treatment are scarce. Our aim was to provide insights into FI in males compared with females. Prospectively collected data from 73 men and 596 women with FI in a tertiary referral center were analyzed. Anorectal physiology, clinical characteristics, and outcome of instrumented biofeedback (BF) were recorded. Thirty-one men with FI proceeded to BF and were matched with 62 age-matched women with FI who underwent BF. Men with FI had higher resting, squeeze, and cough anal sphincter pressures (P < 0.001) and were more able to hold a sustained squeeze compared with women (P = 0.04). Men with FI had higher rectal pressure and less inadequate rectal pressure on strain and higher sensory thresholds (P < 0.05). Men, but not women, with isolated soiling had higher anal resting and squeeze pressures compared with those with overt FI (P < 0.05). Men were less likely to undergo BF when offered compared with women. Baseline symptom severity did not differ between the groups. In men, the absence of an organic cause for the FI and the presence of overt FI, but not isolated soiling, were correlated with improvement in patient satisfaction following BF. The outcomes of 50% reduction in FI episodes, physician assessment, symptoms, and quality of life scores after BF all significantly improved in men similarly to women. We conclude that men, compared with women, with FI have unique clinical features and physiology and are less likely to have investigations and treatment despite successful outcome with BF. Future studies to customize treatment in males and determine barriers to therapy are warranted. NEW & NOTEWORTHY Fecal incontinence in men is common, yet data on sex differences in clinical features, physiology, and treatment are scarce. We provide evidence that men, compared with women, with fecal incontinence have unique clinical features and physiology and are less likely to have investigations and treatment despite successful outcome with anorectal biofeedback therapy.6 page(s