Investigation of the gut microbiome, bile acid composition and host immunoinflammatory response in a model of azoxymethane-induced colon cancer at discrete timepoints.

BACKGROUND: Distinct sets of microbes contribute to colorectal cancer (CRC) initiation and progression. Some occur due to the evolving intestinal environment but may not contribute to disease. In contrast, others may play an important role at particular times during the tumorigenic process. Here, we describe changes in the microbiota and host over the course of azoxymethane (AOM)-induced tumorigenesis. METHODS: Mice were administered AOM or PBS and were euthanised 8, 12, 24 and 48 weeks later. Samples were analysed using 16S rRNA gene sequencing, UPLC-MS and qRT-PCR. RESULTS: The microbiota and bile acid profile showed distinct changes at each timepoint. The inflammatory response became apparent at weeks 12 and 24. Moreover, significant correlations between individual taxa, cytokines and bile acids were detected. One co-abundance group (CAG) differed significantly between PBS- and AOM-treated mice at week 24. Correlation analysis also revealed significant associations between CAGs, bile acids and the bile acid transporter, ASBT. Aberrant crypt foci and adenomas were first detectable at weeks 24 and 48, respectively. CONCLUSION: The observed changes precede host hyperplastic transformation and may represent early therapeutic targets for the prevention or management of CRC at specific timepoints in the tumorigenic process.

The Association of Coloproctology of Great Britain and Ireland consensus guidelines in surgery for inflammatory bowel disease.

AIM: There is a requirement of an expansive and up to date review of surgical management of inflammatory bowel disease (IBD) that can dovetail with the medical guidelines produced by the British Society of Gastroenterology. METHODS: Surgeons who are members of the ACPGBI with a recognised interest in IBD were invited to contribute various sections of the guidelines. They were directed to produce a procedure based document using literature searches that were systematic, comprehensible, transparent and reproducible. Levels of evidence were graded. An editorial board was convened to ensure consistency of style, presentation and quality. Each author was asked to provide a set of recommendations which were evidence based and unambiguous. These recommendations were submitted to the whole guideline group and scored. They were then refined and submitted to a second vote. Only those that achieved >80% consensus at level 5 (strongly agree) or level 4 (agree) after 2 votes were included in the guidelines. RESULTS: All aspects of surgical care for IBD have been included along with 157 recommendations for management. CONCLUSION: These guidelines provide an up to date and evidence based summary of the current surgical knowledge in the management of IBD and will serve as a useful practical text for clinicians performing this type of surgery.

Exacerbation risk in severe asthma is stratified by inflammatory phenotype using longitudinal measures of sputum eosinophils.

BACKGROUND: Airway inflammatory phenotyping is increasingly applied to subjects with asthma. However, its relationship to clinical outcomes in difficult asthma is incompletely elucidated. OBJECTIVE: The goal of our study was to determine the relationship between exacerbation rates and phenotypes of difficult asthma based on the longitudinal measures of sputum eosinophils and neutrophils. METHODS: Subjects in the longitudinal observational study from two tertiary care centres that completed 1 year of observation and provided at least three sputum samples were classified by inflammatory phenotypes using previously established thresholds. Kaplan-Meier curves and univariable and multivariable Cox proportional hazard models were used to determine the association between inflammatory phenotypes and exacerbation rate. RESULTS: During the study, 115 exacerbations occurred in 73 severe asthmatic subjects. Subjects with the persistently eosinophilic phenotype had a significantly shorter time to first exacerbation and greater risk of exacerbation over a 1-year period than those with the non-eosinophilic phenotype based on the univariable and multivariable Cox proportional hazard model (hazard ratio [HR], 3.24; 95% confidence interval [CI], 1.35-7.72; adjusted HR, 3.90; 95% CI, 1.34-11.36). No significant differences in time to first exacerbation or exacerbation risk over a 1-year period were observed among the neutrophilic phenotypes. CONCLUSIONS: The persistent eosinophilic phenotype is associated with increased exacerbation risk compared with the non-eosinophilic phenotype in severe asthma. No differences in time to first exacerbation or exacerbation risk over a 1-year period were detected among neutrophilic phenotypes.

Phenotyping of difficult asthma using longitudinal physiological and biomarker measurements reveals significant differences in stability between clusters.

BACKGROUND: Although the heterogeneous nature of asthma has prompted asthma phenotyping with physiological or biomarker data, these studies have been mostly cross-sectional. Longitudinal studies that assess the stability of phenotypes based on a combination of physiological, clinical and biomarker data are currently lacking. Our objective was to assess the longitudinal stability of clusters derived from repeated measures of airway and physiological data over a 1-year period in moderate and severe asthmatics. METHODS: A total of 125 subjects, 48 with moderate asthma (MA) and 77 with severe asthma (SA) were evaluated every 3 months and monthly, respectively, over a 1-year period. At each 3-month time point, subjects were grouped into 4 asthma clusters (A, B, C, D) based on a combination of clinical (duration of asthma), physiological (FEV1 and BMI) and biomarker (sputum eosinophil count) variables, using k-means clustering. RESULTS: Majority of subjects in clusters A and C had severe asthma (93 % of subjects in cluster A and 79.5 % of subjects in cluster C at baseline). Overall, a total of 59 subjects (47 %) had stable cluster membership, remaining in clusters with the same subjects at each evaluation time. Cluster A was the least stable (21 % stability) and cluster B was the most stable cluster (71 % stability). Cluster stability was not influenced by changes in the dosage of inhaled corticosteroids. CONCLUSION: Asthma phenotyping based on clinical, physiologic and biomarker data identified clusters with significant differences in longitudinal stability over a 1-year period. This finding indicates that the majority of patients within stable clusters can be phenotyped with reasonable accuracy after a single measurement of lung function and sputum eosinophilia, while patients in unstable clusters will require more frequent evaluation of these variables to be properly characterized.

Preoperative mapping of fistula-in-ano: a new three-dimensional MRI-based modelling technique.

AIM: We aimed to develop an intuitive, interactive, three-dimensional (3D) MRI modelling technique to produce a 3D image of fistula-in-ano. METHOD: The 3D model was created from standard two-dimensional (2D) MRI sequences to produce an image that is anatomically correct. Individual muscle and soft-tissue layers were extracted from T1-weighted sequences and fistula pathology from short TI inversion recovery (STIR) sequences, to produce two separate volumes. These were then fused using postprocessing software (Vitrea Workstation version 6.3) to generate a 3D model. RESULTS: The final 3D model was incorporated into a PDF file that has an integrated computer aided design (CAD) viewer, allowing the surgeon to rotate it in any direction during preoperative planning or whilst in theatre. CONCLUSION: As an adjunct to 2D MRI images and the associated radiology report, this model communicates the fistula anatomy to the clinician more clearly and should be particularly useful in complex cases.

Reading a preoperative CT scan to guide complex abdominal wall reconstructive surgery.

Computed tomography (CT) scanning is the imaging modality of choice when planning the overall management and operative approach to complex abdominal wall hernias. Despite its availability and well-recognised benefits there are no guidelines or recommendations regarding how best to read or report such scans for this application. In this paper we aim to outline an approach to interpreting preoperative CT scans in abdominal wall reconstruction (AWR). This approach breaks up the interpretive process into 4 steps-concentrating on the hernia or hernias, any complicating features of the hernia(s), the surrounding soft tissues and the abdominopelvic cavity as a whole-and was developed as a distillation of the authors' collective experience. We describe the key features that should be looked for at each of the four steps and the rationale for their inclusion.

Anti-inflammatory therapy enables robot-actuated regeneration of aged muscle.

Robot-actuated mechanical loading (ML)-based therapies ("mechanotherapies") can promote regeneration after severe skeletal muscle injury, but the effectiveness of such approaches during aging is unknown and may be influenced by age-associated decline in the healing capacity of skeletal muscle. To address this knowledge gap, this work used a noninvasive, load-controlled robotic device to impose highly defined tissue stresses to evaluate the age dependence of ML on muscle repair after injury. The response of injured muscle to robot-actuated cyclic compressive loading was found to be age sensitive, revealing not only a lack of reparative benefit of ML on injured aged muscles but also exacerbation of tissue inflammation. ML alone also disrupted the normal regenerative processes of aged muscle stem cells. However, these negative effects could be reversed by introducing anti-inflammatory therapy alongside ML application, leading to enhanced skeletal muscle regeneration even in aged mice.

Corticosterone levels of Atlantic puffins vary with breeding stage and sex but are not elevated in poor foraging years.

Corticosterone (CORT) levels in seabirds fluctuate across breeding stages and in different foraging conditions. Here we use a ten-year data set to examine whether CORT levels in Atlantic puffins differ in years with high or low availability of capelin, the preferred forage species. Female puffins had higher CORT levels than males, possibly related to cumulative costs of egg production and higher parental investment. Puffins had higher CORT levels and body mass during pre-breeding than during chick rearing. Yearly mean chick growth rates were higher in years when adults had higher body mass and in years where adults brought chicks a lower percentage of non-fish (invertebrates/larval fish) food. Unlike most results from seabird species with shorter chick-rearing periods, higher CORT levels in puffins were not associated with lower capelin abundance. Puffins may suppress CORT levels to conserve energy in case foraging conditions improve later in the prolonged chick-rearing period. Alternatively, CORT levels may be lowest both when food is very abundant (years not in our sample) or very scarce (e.g., 2009 in this study), and increase when extra foraging effort will increase foraging efficiency (most years in this study). If these data primarily represent years with medium to poor foraging, it is possible that CORT responses to variation in foraging conditions are similar for puffins and other seabirds.

Separating the components of an abdominal wall fellowship.

Complex abdominal wall reconstruction is an emerging subspecialty yet, despite the abundance of abdominal wall hernias requiring treatment and the increasing complexity of this type of surgery, there are few opportunities for surgeons to gain subspecialist training in this field. In this paper we discuss the need for focused training in complex abdominal wall reconstruction, outline some of the problems that may be hindering the availability of such opportunities and propose potential solutions to these issues.

Comprehensive multi-cohort transcriptional meta-analysis of muscle diseases identifies a signature of disease severity.

Muscle diseases share common pathological features suggesting common underlying mechanisms. We hypothesized there is a common set of genes dysregulated across muscle diseases compared to healthy muscle and that these genes correlate with severity of muscle disease. We performed meta-analysis of transcriptional profiles of muscle biopsies from human muscle diseases and healthy controls. Studies obtained from public microarray repositories fulfilling quality criteria were divided into six categories: (i) immobility, (ii) inflammatory myopathies, (iii) intensive care unit (ICU) acquired weakness (ICUAW), (iv) congenital muscle diseases, (v) chronic systemic diseases, (vi) motor neuron disease. Patient cohorts were separated in discovery and validation cohorts retaining roughly equal proportions of samples for the disease categories. To remove bias towards a specific muscle disease category we repeated the meta-analysis five times by removing data sets corresponding to one muscle disease class at a time in a "leave-one-disease-out" analysis. We used 636 muscle tissue samples from 30 independent cohorts to identify a 52 gene signature (36 up-regulated and 16 down-regulated genes). We validated the discriminatory power of this signature in 657 muscle biopsies from 12 additional patient cohorts encompassing five categories of muscle diseases with an area under the receiver operating characteristic curve of 0.91, 83% sensitivity, and 85.3% specificity. The expression score of the gene signature inversely correlated with quadriceps muscle mass (r = -0.50, p-value = 0.011) in ICUAW and shoulder abduction strength (r = -0.77, p-value = 0.014) in amyotrophic lateral sclerosis (ALS). The signature also positively correlated with histologic assessment of muscle atrophy in ALS (r = 0.88, p-value = 1.62 × 10-3) and fibrosis in muscular dystrophy (Jonckheere trend test p-value = 4.45 × 10-9). Our results identify a conserved transcriptional signature associated with clinical and histologic muscle disease severity. Several genes in this conserved signature have not been previously associated with muscle disease severity.

Physical activity recommendations pre and post abdominal wall reconstruction: a scoping review of the evidence.

PURPOSE: There are no universally agreed guidelines regarding which types of physical activity are safe and/or recommended in the perioperative period for patients undergoing ventral hernia repair or abdominal wall reconstruction (AWR). This study is intended to identify and summarise the literature on this topic. METHODS: Database searches of PubMed, CINAHL, Allied & Complementary medicine database, PEDro and Web of Science were performed followed by a snowballing search using two papers identified by the database search and four hand-selected papers of the authors' choosing. Inclusion-cohort studies, randomized controlled trials, prospective or retrospective. Studies concerning complex incisional hernia repairs and AWRs including a "prehabilitation" and/or "rehabilitation" program targeting the abdominal wall muscles in which the interventions were of a physical exercise nature. RoB2 and Robins-I were used to assess risk of bias. Prospero CRD42021236745. No external funding. Data from the included studies were extracted using a table based on the Cochrane Consumers and Communication Review Group's data extraction template. RESULTS: The database search yielded 5423 records. After screening two titles were selected for inclusion in our study. The snowballing search identified 49 records. After screening one title was selected for inclusion in our study. Three total papers were included-two randomised studies and one cohort study (combined 423 patients). All three studies subjected their patients to varying types of physical activity preoperatively, one study also prescribed these activities postoperatively. The outcomes differed between the studies therefore meta-analysis was impossible-two studies measured hernia recurrence, one measured peak torque. All three studies showed improved outcomes in their study groups compared to controls however significant methodological flaws and confounding factors existed in all three studies. No adverse events were reported. CONCLUSIONS: The literature supporting the advice given to patients regarding recommended physical activity levels in the perioperative period for AWR patients is sparse. Further research is urgently required on this subject.

Ileostomy formation does not prolong hospital length of stay after open anterior resection when performed within an enhanced recovery programme.

AIM: Previous studies have implicated stoma formation as an independent factor prolonging length of stay (LOS) after colorectal surgery. We examined whether loop ileostomy (LI) formation during an open anterior resection affected LOS within a newly developed enhanced recovery programme (ERP). This involved reorganization of services, including stoma care, to optimize pre- and perioperative patient education, training and expectation, while applying the standard scientific principles of enhanced recovery. METHOD: Data were collected prospectively on 72 consecutive open anterior resections (33 with LI) to see whether LI affected LOS. Stomas were fashioned at the surgeon's discretion and patients were discharged according to agreed criteria. RESULTS: Thirty-three of the 72 patients had a covering LI performed. The overall age range was 32-85 years (median 68), with 40 patients being men. LOS for all 72 patients ranged from 3 to 34 days. The median and mean LOS were the same for both groups (median 6 days, mean 8 days). The complication rate was 44% (32/72) with a leak rate of 7% (5/72) and a mortality of 1.4% (1/72). Between the two groups (no ileostomy vs ileostomy), there was no statistically significant difference in complications (41%vs 48%), leakage (8%vs 6%) and readmission rates (12%vs 5%). CONCLUSION: A covering loop ileostomy need not prolong hospital stay after open anterior resection.

Theatre ventilation.

INTRODUCTION: Owing to the COVID-19 pandemic, there has been significant disruption to all surgical specialties. In the UK, units have cancelled elective surgery and a decrease in aerosol generating procedures (AGPs) was favoured. Centres around the world advocate the use of negative pressure environments for AGPs in reducing the spread of infectious airborne particles. We present an overview of operating theatre ventilation systems and the respective evidence with relation to surgical site infection (SSI) and airborne pathogen transmission in light of COVID-19. METHODS: A literature search was conducted using the PubMed, Cochrane Library and MEDLINE databases. Search terms included "COVID-19", "theatre ventilation", "laminar", "turbulent" and "negative pressure". FINDINGS: Evidence for laminar flow ventilation in reducing the rate of SSI in orthopaedic surgery is widely documented. There is little evidence to support its use in general surgery. Following previous viral outbreaks, some centres have introduced negative pressure ventilation in an attempt to decrease exposure of airborne pathogens to staff and surrounding areas. This has again been suggested during the COVID-19 pandemic. A limited number of studies show some positive results for the use of negative pressure ventilation systems and reduction in spread of pathogens; however, cost, accessibility and duration of conversion remain an unexplored issue. Overall, there is insufficient evidence to advocate large scale conversion at this time. Nevertheless, it may be useful for each centre to have its own negative pressure room available for AGPs and high risk patients.

Individualization of exosuit assistance based on measured muscle dynamics during versatile walking.

Variability in human walking depends on individual physiology, environment, and walking task. Consequently, in the field of wearable robotics, there is a clear need for customizing assistance to the user and task. Here, we developed a muscle-based assistance (MBA) strategy wherein exosuit assistance was derived from direct measurements of individuals' muscle dynamics during specific tasks. We recorded individuals' soleus muscle dynamics using ultrasonographic imaging during multiple walking speeds and inclines. From these prerecorded images, we estimated the force produced by the soleus through inefficient concentric contraction and designed the exosuit assistance profile to be proportional to that estimated force. We evaluated this approach with a bilateral ankle exosuit at each measured walking task. Compared with not wearing a device, the MBA ankle exosuit significantly reduced metabolic demand by an average of 15.9, 9.7, and 8.9% for level walking at 1.25, 1.5, and 1.75 meters second−1, respectively, and 7.8% at 1.25 meters second−1 at 5.71° incline while applying lower assistance levels than in existing literature. In an additional study (n = 2), we showed for multiple walking tasks that the MBA profile outperforms other bioinspired strategies and the average profile from a previous optimization study. Last, we show the feasibility of online assistance generation in a mobile version for overground outdoor walking. This muscle-based approach enables relatively rapid (~10 seconds) generation of individualized low-force assistance profiles that provide metabolic benefit. This approach may help support the adoption of wearable robotics in real-world, dynamic locomotor tasks by enabling comfortable, tailored, and adaptive assistance.

mRNAs for alpha- and beta-tubulin and flagellar calmodulin are among those coordinately regulated when Naegleria gruberi amebae differentiate into flagellates.

Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361) have been identified as coding for flagellar proteins. The products of these mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kb RNA (class II) all selected mRNA that was translated into two alpha-tubulins. The principal in vitro product, alpha-1, comigrated with a cytoplasmic alpha-tubulin, while the minor product with a more acidic pI, alpha-2, comigrated with flagellar alpha-tubulin. While Naegleria flagellar alpha-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that alpha-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second alpha-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected beta-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the alpha- and beta-subunits of tubulin, that Naegleria alpha-tubulin migrated faster than beta-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.

Nucleolus-like morphology produced during the in vitro reassociation of nucleolar components.

Nucleoli, the sites of rRNA synthesis, rRNA processing, and the assembly of ribosomes, are dynamic organelles that, in most cells, disperse and reform during mitosis. The mechanisms that regulate nucleolar formation are unknown as is the relationship between nucleolar morphology and the pathway of ribosome biogenesis. In this report we describe the in vitro formation of nucleolus-like particles (NLPs) from soluble extracts of nucleoli. NLPs, which reached sizes comparable to nucleoli (1-3 microns), were found to contain 40% of the nucleolar DNA, RNA, and protein. The ultrastructure of NLPs resembled that of a number of in vivo structures including compact nucleoli, prenucleolar bodies, and pseudonucleoli. The particles were composed of two morphologically distinct regions. The core resembled the dense fibrillar component (DFC) of nucleoli while the cortex resembled the granular component (GC) of nucleoli. The cortex of NLPs contained numerous 15-20 nm osmophilic granules that resembled the preribosomes found in the GC of nucleoli. The distribution of nucleolar proteins in NLPs also resembled that in nucleoli. BN46/51, a component of the GC of nucleoli, was restricted to the GC-like cortex of NLPs. A mAb that bound to the DFC of nucleoli, bound only to the DFC-like core of NLPs while a second mAb that bound to both the DFC and GC of nucleoli, bound to both the core and cortex of NLPs. Thus solubilized components of nucleoli can reassociate in vitro to produce particles that resemble nucleoli in their size, ultrastructure, and protein distribution.

New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates.

We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. Cell Biol., 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these differentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis. Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation (Lai, E.Y., C. Walsh, D. Wardell, and C. Fulton, 1979, Cell, 17:867-878).