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Background and Objective: Inflammatory bowel disease (IBD) fatigue aetiology is poorly understood. This study quantified body composition and physical function alongside proton magnetic resonance imaging (1H MRI) and spectroscopy (31P MRS) measures of organ structure and function in quiescent Crohn's disease patients (CD) and healthy volunteers (HVs), to identify a physiological basis for IBD fatigue., Methods: Body composition was determined using dual-energy X-ray absorptiometry and 1H MRI. Knee extensor isometric strength and isokinetic fatigue were measured using dynamometry. 1H MRI was used to quantify cardiac output, cerebral blood flow (gmCBF), and brain oxygen extraction fraction (OEF) at rest, and during supine, steady-state exercise, and recovery. 31P MRS was used to quantify post-exercise muscle phosphocreatine (PCr) resynthesis., Results: Sixteen CD and 12 HV (age, sex, and BMI matched) were recruited. Fatigue perception was greater (13.9 ± 1 vs 8.3 ± 0.9, P = .001), and daily step count was less (5482 ± 684 vs 8168 ± 1123, P = .04) in CD. During steady-state exercise, gmCBF was less in CD (653 ± 30 vs 823 ± 40 mL/min, P = .003). Cardiac output and brain OEF were no different. Post-exercise PCr resynthesis was less in CD (17.2 ± 2.0 vs 25.3 ± 2.4 mM·min-1, P = .02). Body composition, isometric strength, and isokinetic fatigability were no different., Conclusions: CD self-reported increased fatigue perception and exhibited a slower rate of post-exercise PCr resynthesis compared to HV. This occurred independently of changes in body composition, muscle strength, and fatigability. IBD fatigue may be linked to peripheral muscle deconditioning and lower gmCBF during submaximal exercise., (© The Author(s) 2025. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Background: Imaging is used to monitor disease activity in small bowel Crohn's disease (CD). Magnetic Resonance Enterography is often employed as a first modality in the United Kingdom for assessment and monitoring; however, waiting times, cost, patient burden and limited access are significant. It is as yet uncertain if small bowel intestinal ultrasound (IUS) may be a quicker, more acceptable, and cheaper alternative for monitoring patients with CD., Methods: A clinical service evaluation of imaging pathways was undertaken at a single NHS site in England, United Kingdom. Data were collected about patients who were referred and underwent an imaging analysis for their IBD. Only patients who underwent a therapy change were included in the analysis. Data were collected from care episodes between 01 January 2021-30 March 2022., Results: A combined total of 193 patient care episodes were reviewed, 107 from the IUS pathway and 86 from the MRE pathway. Estimated costs per patient in the IUS pathway was £78.86, and £375.35 per patient in the MRE pathway. The MRE pathway had an average time from referral to treatment initiation of 91 days (SD= ±61) with patients in the IUS pathway waiting an average of 46 days (SD= ±17)., Conclusions: Findings from this work indicate that IUS is a potential cost-saving option when compared to MRE when used in the management of CD. This is in addition to the cost difference of the radiological modalities. A large, multicentre, prospective study is needed to validate these initial findings.

Background: Alterations in resting state functional connectivity (rs-FC) in Crohn's Disease (CD) have been documented in default mode network (DMN) and frontal parietal network (FPN) areas, visual, cerebellar, salience and attention resting-state-networks (RSNs), constituting a CD specific neural phenotype. To date, most studies are in patients in remission, with limited studies in active disease., Methods: Twenty five active CD cases and 25 age-, BMI- and gender-matched healthy controls (HC) were recruited to a resting-state-functional Magnetic Resonance Imaging (rs-fMRI) study. Active disease was defined as C-reactive protein>5 mg/dL, faecal calprotectin>250 μg/g, or through ileocolonoscopy or MRE. rs-fMRI data were analysed using independent component analysis (ICA) and dual regression. Differences in RSNs between HCs and active CD were assessed, and rs-FC was associated with disease duration and abdominal pain., Results: Increased connectivity in the FPN (fusiform gyrus, thalamus, caudate, posterior cingulate cortex, postcentral gyrus) and visual RSN (orbital frontal cortex) were observed in CD versus HC. Decreased activity was observed in the salience network (cerebellum, postcentral gyrus), DMN (parahippocampal gyrus, cerebellum), and cerebellar network (occipital fusiform gyrus, cerebellum) in CD versus HCs. Greater abdominal pain scores were associated with lower connectivity in the precuneus (visual network) and parietal operculum (salience network), and higher connectivity in the cerebellum (frontal network). Greater disease duration was associated with greater connectivity in the middle temporal gyrus and planum temporale (visual network)., Conclusion: Alterations in rs-FC in active CD in RSNs implicated in cognition, attention, emotion, and pain may represent neural correlates of chronic systemic inflammation, abdominal pain, disease duration, and severity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Thapaliya, Eldeghaidy, Radford, Francis and Moran.)

Background: Infliximab is a monoclonal antibody that binds and neutralises tumour necrosis factor-alpha (TNF-α), which is present in high levels in the blood serum, mucosa and stool of people with Crohn's disease., Objectives: To evaluate the benefits and harms of infliximab alone or in combination with another agent for induction of remission in Crohn's disease compared to placebo or active medical therapies., Search Methods: On 31 August 2021 and 4 March 2023, we searched CENTRAL, MEDLINE, Embase, ClinicalTrials.gov and World Health Organization ICTRP., Selection Criteria: Randomised control trials (RCTs) comparing infliximab alone or in combination with another agent to placebo or another active comparator in adults with active Crohn's disease., Data Collection and Analysis: Pairs of review authors independently selected studies and conducted data extraction and risk of bias assessment. We expressed outcomes as risk ratios (RR) and mean differences (MD) with 95% confidence intervals (CI). We assessed the certainty of the evidence using GRADE. Our primary outcomes were clinical remission, clinical response and withdrawals due to adverse events. Our secondary outcomes were endoscopic remission, histological remission, endoscopic response, and serious and total adverse events., Main Results: The search identified 10 RCTs with 1101 participants. They were conducted between 1999 and 2019, and 7/10 RCTs included biologically naive participants. All but one RCT, which did not provide information, were multicentre and funded by pharmaceutical companies, and their authors declared conflicts. The age of the participants ranged from 26 to 65 years. Results were based on one study unless otherwise stated. Infliximab 5 mg/kg to 10 mg/kg may be more effective than placebo at week four for clinical remission (30/55 versus 3/25; RR 4.55, 95% CI 1.53 to 13.50; number needed to treat for an additional beneficial outcome (NNTB) 3) and response (36/55 versus 4/25; RR 4.09, 95% CI 1.63 to 10.25, NNTB 3). The evidence was low certainty. The study did not report withdrawals due to adverse events. We could not draw conclusions on the effects of infliximab 5 mg/kg to 10 mg/kg compared to placebo for fistulating participants for clinical remission (29/63 versus 4/31; RR 3.57, 95% CI 1.38 to 9.25; NNTB 4), response (48/106 versus 15/75; RR 1.94, 95% CI 1.10 to 3.41; NNTB 6; 2 studies) or withdrawals due to adverse events (2/63 versus 0/31; RR 2.50, 95% CI 0.12 to 50.54). The evidence was very low certainty. Infliximab used in combination with purine analogues is probably more effective than purine analogues alone for clinical remission at weeks 24 to 26 (182/301 versus 95/302; RR 1.92, 95% CI 1.59 to 2.32, NNTB 4; 4 studies; moderate-certainty evidence) and clinical response at week 26 (107/177 versus 66/178; RR 1.64, 95% CI 1.31 to 2.05; NNTB 5; 2 studies; moderate-certainty evidence). There may be little or no difference in withdrawals due to adverse events at week 26 (62/302 versus 53/301; RR 0.87, 95% CI 0.63 to 1.21; 4 studies; low-certainty evidence). Infliximab alone may be more effective than purine analogues alone at week 26 for clinical remission (85/177 versus 57/178; RR 1.50, 95% CI 1.15 to 1.95; NNTB 7; 2 studies) and response (94/177 versus 66/178; RR 1.44, 95% CI 1.13 to 1.82; NNTB 7; 2 studies). There may be little or no difference in withdrawals due to adverse events (30/177 versus 43/178; RR 0.70, 95% CI 0.46 to 1.06; 4 studies). The evidence was low certainty. We could not draw any conclusions on the effects of infliximab 5 mg/kg compared to 10 mg/kg for clinical remission (19/27 versus 11/28; RR 1.79, 95% CI 1.06 to 3.02) and response (22/27 versus 24/28; RR 1.63, 95% CI 1.08 to 2.46). The evidence was very low certainty. Withdrawals due to adverse events were not reported. We could not draw any conclusions on the effects of infliximab 5 mg/kg compared to 10 mg/kg in an exclusively fistulating population for clinical remission (17/31 versus 12/32; RR 1.46, 95% CI 0.84 to 2.53), response (21/31 versus 18/32; RR 1.20, 95% CI 0.82 to 1.78), or withdrawals due to adverse events (1/31 versus 1/32; RR 1.03, 95% CI 0.07 to 15.79). The evidence was very low certainty. We could not draw any conclusions on the effects of infliximab 5 mg/kg compared to 20 mg/kg for clinical remission (19/27 versus 11/28; RR 1.79, 95% CI 1.06 to 3.02) or response (22/27 versus 18/28; RR 1.27, 95% CI 0.91 to 1.76). The evidence was very low certainty. Withdrawals due to adverse events were not reported. We could not draw any conclusions on the effects of infliximab 10 mg/kg compared to 20 mg/kg for clinical remission (11/28 versus 11/28; RR 1.00, 95% CI 0.52 to 1.92) or response (14/28 versus 18/28; RR 0.78, 95% CI 0.49 to 1.23). The evidence was very low certainty. Withdrawals due to adverse events were not reported. There may be little or no difference between infliximab and a CT-P13 biosimilar at week six for clinical remission (47/109 versus 49/111; RR 0.98, 95% CI 0.72 to 1.32), response (67/109 versus 70/111; RR 0.97, 95% CI 0.79 to 1.20) and withdrawals due to adverse events (21/109 versus 17/111; RR 1.26, 95% CI 0.70 to 2.25). The evidence was low certainty., Authors' Conclusions: Infliximab in combination with purine analogues is probably more effective than purine analogues alone in inducing clinical remission and clinical response. Infliximab alone may be more effective in inducing clinical remission and response than purine analogues alone or placebo. Infliximab may be similar in efficacy to a CT-P13 biosimilar and there may be little or no difference in withdrawals due to adverse events. We were unable to draw meaningful conclusions as to whether infliximab alone is effective when used for exclusively fistulating populations. There was evidence that there may be little or no difference in withdrawal due to adverse events between infliximab plus purines compared with purines alone, as well as infliximab alone compared with purines alone. Meaningful conclusions cannot be drawn on all other outcomes related to adverse events due to very low certainty evidence., (Copyright © 2023 The Authors. Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.)

In this second part of an introduction to research for gastroenterology and hepatology nurses, we aim to build on the first article that introduced the significance and structure of the National Institute for Health and Care Research clinical research landscape in the UK and the importance of nurse engagement. This article introduces possible career pathways available in the profession and specialty. Practical information on how to start research is provided, including an overview of the education, training and support required for a career in research delivery and academic research. Some of the potential barriers to nursing research careers are highlighted, and solutions to navigate a successful career in nursing research are proposed., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

This is the first of two articles from the joint British Society of Gastroenterology Nurses association and Research Committee working group. The group is dedicated to signposting and improving access to research for specialist nurses working in gastroenterology and hepatology. This article is an introduction to the significance and structure of the National Institute for Health Research clinical research landscape in the UK and the importance of encouraging nurse engagement in research. This paper describes and 'demystifies' the clinical trials infrastructure in the UK, which is one of the most organised in the world. Going forwards this working group will organise and conduct educational events encouraging specialist nurses to become more aware of and engage in clinical research in their area of practice., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Fatigue is a frequently reported symptom of Inflammatory Bowel Disease (IBD), having a negative impact on Health-Related Quality of Life (HRQoL). Patients' experiences of this have not been researched in IBD., Methods: Semi-structured interviews were conducted with adults with Crohn's Disease from out-patient clinics in the United Kingdom. Interviews were audio-recorded and transcribed verbatim, then analysed using thematic analysis., Results: Fourteen participant interviews were conducted. Three key themes were identified: 1) 'The new normal' established through adaptation and acceptance; 2) 'Energy as a resource' describing attempts to better manage fatigue through planning and prioritising tasks; 3) 'Keeping healthy' encompasses participants' beliefs that 'good health' allows better management of fatigue., Conclusion: Participants establish a 'new' normality, through maintaining the same or similar level of employment/education activities. However, this is often at the expense of social activities. Further research is required to explore patient led self-management interventions in IBD fatigue., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

The use of cross-sectional imaging and ultrasonography has long complemented endoscopic assessment of inflammatory bowel disease (IBD). Clinical symptoms alone are often not enough to assess disease activity, so a reliance on non-invasive techniques is essential. In this paper, we aim to examine the current use of radiological modalities in aiding the management of patients with IBD. We focus on the various sections of the gastrointestinal tract and how different modalities can aid in assessing current disease state and response to treatments. We also have a look at how newer sequences in cross-sectional imaging and ultrasonography can allow for better differentiation of disease activity (ie, fibrotic vs inflammatory) as well improve evaluation of small bowel, colonic and perianal disease. Furthermore, we examine how advanced image processing has the potential to allow radiology to be a surrogate for biomarkers. An example of this is explored when reviewing the ability of MR sequences to quantify visceral fat, which potentially plays a role in determining disease activity in Crohn's disease. Lastly, we look into the expected role for artificial intelligence to be used as an adjunct to radiology to better improve IBD evaluation., Competing Interests: Competing interests: GM receives funding from AztraZenca and Jansen and is a consultant for Alimentiv., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Small bowel ultrasound has very good diagnostic accuracy for disease extent, presence and activity in Crohn's Disease, is well tolerated by patients and is cheaper when compared with MRI. However, uptake of ultrasound in the UK is limited., Methods: An online survey to assess the current usage of ultrasound throughout the UK was undertaken by BSG IBD group members between 9/06/2021- 25/06/2021. Responses were anonymous., Results: 103 responses were included in the data analysis. Responses came from 66 different NHS trusts from 14 different regions of the UK. All respondents reported that they currently have an MRI service for Crohn's disease, whereas only 31 had an ultrasound service. Average time for results to be reported for MRI scans was reported as between 4- and 6 weeks, with a range of 2 days to 28 weeks. The average time for an ultrasound to be reported was stated as 1-4 weeks, with a range of 0-8 weeks. There was disparity between the reported confidence of clinicians making clinical decisions when using ultrasound compared to MRI. Of those respondents who did not have access to an ultrasound service, 72 stated that they would be interested in developing an ultrasound service., Conclusion: There is an appetite for the uptake of ultrasound in the UK for assessment of Crohn's disease, however, there remains a significant number of UK centres with little or no access to an ultrasound service. Further research is necessary to understand why this is the case., Competing Interests: Competing interests: GM receives research funding from Janssen, Arla foods and Astra Zeneca and is a consultant for Alimentiv. ST is a consultant to Alimentiv and has share options in Motilent., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Fatigue is the inability to achieve or maintain an expected work output resulting from central or peripheral mechanisms. The prevalence of inflammatory bowel disease (IBD) fatigue can reach 86% in active disease, persisting in 50%-52% of patients with mild to inactive disease. Fatigue is the commonest reason for work absence in IBD, and patients often report fatigue burden to be greater than that of primary disease symptoms. Relatively few evidence-based treatment options exist, and the aetiology is poorly understood., Aim: To review the available data and suggest a possible aetiology of IBD fatigue and to consider the efficacy of existing management strategies and highlight potential future interventions., Methods: We reviewed fatigue-related literature in IBD using PubMed database., Results: Disease related factors such as inflammation and pharmacological treatments negatively impact skeletal muscle and brain physiology, likely contributing to fatigue symptoms. Secondary factors such as malnutrition, anaemia, sleep disturbance and psychological comorbidity are potential determinants. Immune profile, faecal microbiota composition and physical fitness differ significantly between fatigued and non-fatigued patients, suggesting these may be aetiological factors. Solution-focused therapy, high-dosage thiamine supplementation and biological therapy may reduce fatigue perception in IBD. The effect of physical activity interventions is inconclusive., Conclusions: A multimodal approach is likely required to treat IBD fatigue. Established reversible factors like anaemia, micronutrient deficiencies and active disease should initially be resolved. Psychosocial intervention shows potential efficacy in reducing fatigue perception in quiescent disease. Restoring physical deconditioning by exercise training intervention may further improve fatigue burden., (© 2021 John Wiley & Sons Ltd.)

Background: Ultrasound (US) is an alternative to magnetic resonance enterography, and has the potential to significantly reduce waiting times, expedite clinical decision-making and improve patient experience. Point of care US is an advantage of the US imaging modality, where same day scanning, interpretation and treatment decisions can be made., Aim: To systematically scope the literature on point of care US use in small bowel Crohn's disease, generating a comprehensive list of factors relating to the current understanding of clinical utility of this imaging modality., Methods: Searches included MEDLINE, EMBASE, Cochrane Library, Cumulative Index to Nursing and Allied Health Literature, PsycINFO, clinicaltrial.gov,'TRIP' and Epistemonikos. Reference lists of included studies were hand searched. Search terms were searched for as both keywords and subject headings (MeSH) as appropriate. Searches were performed with the 'suggested search terms' and 'explode' selection, and restricted to 'human', 'adult' and 'English language' publications. No date limits were applied to be as inclusive as possible. Two investigators conducted abstract and full-text review. No formal quality appraisal process was undertaken; however, quality of sources was considered when reporting findings. A narrative synthesis was conducted., Results: The review included 42 sources from the UK, Europe, Japan, Canada and the USA. Small bowel ultrasound (SBUS) has been shown to be as accurate in detecting the presence of small bowel Crohn's disease, is quicker, safer and more acceptable to patients, compared with magnetic resonance enterography. SBUS is used widely in central Europe and Canada but has not been embraced in the UK. Further research considering economic evaluation, clinical decision-making and exploration of perceived barriers to future implementation of SBUSs is required., Competing Interests: Competing interests: SJR, BS, PL and CC—nil to declare GWM has received educational support from Abbvie, Janssen, NAPP, Takeda Pharmaceuticals, Merck Sharp & Dohme Ltd, Ferring and Dr Falk. He has received speaker honoraria from Merck Sharp & Dohme Ltd, Abbvie, Janssen, Ferring and Takeda Pharmaceuticals. He attended advisory boards for Abbvie, Celgene, Takeda Pharmaceuticals, Janssen, Medtronic, Phebra Pharmaceuticals, Servertus Associates Ltd and Dr Falk. Dr Moran is a consultant for Alimentiv. SAT is consultant to Alimentiv and has share options in Motilent., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Rab1A is a small GTPase known for its role in vesicular trafficking. Recent evidence indicates that Rab1A is essential for amino acids (aas) sensing and signaling to regulate mTORC1 in normal and cancer cells. However, Rab1A's in vivo function in mammals is not known. Here, we report the generation of tamoxifen (TAM)-induced whole body Rab1A knockout (Rab1A -/- ) in adult mice. Rab1A -/- mice are viable but become hyperglycemic and glucose intolerant due to impaired insulin transcription and β-cell proliferation and maintenance. Mechanistically, Rab1A mediates AA-mTORC1 signaling, particularly branched chain amino acids (BCAA), to regulate the stability and localization of the insulin transcription factor Pdx1. Collectively, these results reveal a physiological role of aa-Rab1A-mTORC1 signaling in the control of whole-body glucose homeostasis in mammals. Intriguingly, Rab1A expression is reduced in β-cells of type 2 diabetes (T2D) patients, which is correlated with loss of insulin expression, suggesting that Rab1A downregulation contributes to T2D progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Deep eutectic solvents (DES) are one of the most promising green technologies to emerge in recent years given their combination of environmentally friendly credentials and useful functionalities. Considering the continued search for new DES - especially those that exemplify the aforementioned characteristics, we report the preparation of DES based on natural analogues of l-ascorbic acid for the first time. The onset of eutectic melting occurred at temperatures far below the melting point of the individual components and resulted in the generation of glass forming fluids with glass transition temperatures, viscosities and flow behavior that are comparable to similar systems. This work expands the current array of DES that can be produced using naturally occurring components, which given their potential to be bio-derived, interesting physicochemical properties (e. g. propensity to supercool and vitrify) and apparent antibacterial nature, may provide utility within a range of applications., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Background: Fatigue is frequently reported in inflammatory bowel disease (IBD) and impacts on health-related quality of life (HRQoL). HRQoL has not been systematically reviewed in IBD fatigue., Aim: To investigate what impact IBD fatigue has on HRQoL in adults with IBD., Methods: Systematic searches (CINAHL, EMBASE, PsychINFO, Medline) were conducted on 25 September 2018, restricted to 'human', 'adult', 'primary research' and 'English language'. Search terms encompassed concepts of 'fatigue', 'IBD' and 'HRQoL'. A 5-year time limit (2013-2018) was set to include the most relevant publications. Publications were screened, data extracted and quality appraised by two authors. A narrative synthesis was conducted., Results: Eleven studies were included, presenting data from 2823 participants. Fatigue experiences were significantly related to three HRQoL areas: symptom acceptance, psychosocial well-being and physical activity. Patients reporting high fatigue levels had low symptom acceptance. Psychosocial factors were strongly associated with both fatigue and HRQoL. Higher social support levels were associated with higher HRQoL. Physical activity was impaired by higher fatigue levels, lowering HRQoL, but it was also used as a means of reducing fatigue and improving HRQoL. Quality appraisal revealed methodological shortcomings in a number of studies. Notably, use of multiple measures, comparison without statistical adjustment and fatigue and HRQoL assessment using the same tool were some of the methodological shortcomings., Conclusion: Psychosocial factors, symptom management and acceptance and physical activity levels have significant impact on HRQoL. Results support application of psychosocial or exercise interventions for fatigue management. Further exploration of HRQoL factors in IBD fatigue is required, using validated fatigue and HRQoL measures., Prospero Registration Number: CRD42018110005., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Long term stabilization of water-in-oil (W/O) emulsions remains a particularly challenging problem in colloid science. Recent studies have shown that polyphenols act as Pickering stabilizers at the water-oil interface. In this work we propose a novel way to stabilize water droplets via interfacial complex formation. It was observed that polyphenol crystals (curcumin or quercetin) absorb at the interface and provide stabilization of water droplets for several days; however formation of a polyphenol- whey protein (WPI) complex at the water-oil interface revealed a pronounced improvement in the stabilization. The mechanism of complex formation was tested by subjecting the systems to different environmental conditions, such as ionic strength and temperature. The evidence suggests that the complex is probably stabilized by electrostatic attraction between the oppositely-charged polyphenol particles and protein at the interface, although hydrogen bonding between the two components may also contribute. The resulting stable water droplets have a Sauter mean diameter (D 3,2 ) of approximately ∼22 and ∼27 μm for curcumin and quercetin systems, respectively. Emulsions were more stable at pH 3 than at pH 7, due to either weaker complex formation at pH 7 and/or chemical degradation of the polyphenols at this more alkaline pH. Interfacial shear viscosity measurements confirmed that there was strong interfacial complex formation with aqueous WPI concentrations of ∼0.5 wt.%., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.

The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD, and the plus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster How multiple microtubule-bundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. We have found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. We propose that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces, resulting in the capacity to accurately segregate chromosomes., (Copyright © 2017 by the Genetics Society of America.)

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore-microtubule attachment in oocytes., (© 2015 Gluszek et al.)

In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation, but the nature and function of these interactions is not clear. We have examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes.

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Abscission is the last step of cytokinesis that physically separates the cytoplasm of sister cells. As the final stage of cell division, abscission is poorly characterized during animal development. Here, we show that Aurora B and Survivin regulate the number of germ cells in each Drosophila egg chamber by inhibiting abscission during differentiation. This inhibition is mediated by an Aurora B-dependent phosphorylation of Cyclin B, as a phosphomimic form of Cyclin B rescues premature abscission caused by a loss of function of Aurora B. We show that Cyclin B localizes at the cytokinesis bridge, where it promotes abscission. We propose that mutual inhibitions between Aurora B and Cyclin B regulate the duration of abscission and thereby the number of sister cells in each cyst. Finally, we show that inhibitions of Aurora B and Cyclin-dependent kinase 1 activity in vertebrate cells also have opposite effects on the timing of abscission, suggesting a possible conservation of these mechanisms., (Copyright © 2013 Elsevier Inc. All rights reserved.)

During meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, we focused on the chromosomal passenger complex (CPC). We have found that the CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, we show that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data we propose that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes.

During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. We show here that a member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila melanogaster oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation.

Meiotic recombination gives rise to crossovers, which are required in most organisms for the faithful segregation of homologous chromosomes during meiotic cell division. Characterization of crossover-defective mutants has contributed much to our understanding of the molecular mechanism of crossover formation. We report here a molecular analysis of recombination in a Drosophila melanogaster crossover-defective mutant, mei-9. In the absence of mei-9 activity, postmeiotic segregation associated with noncrossovers occurs at the expense of crossover products, suggesting that the underlying meiotic function for MEI-9 is in crossover formation rather than mismatch repair. In support of this, analysis of the arrangement of heteroduplex DNA in the postmeiotic segregation products reveals different patterns from those observed in Drosophila Msh6 mutants, which are mismatch-repair defective. This analysis also provides evidence that the double-strand break repair model applies to meiotic recombination in Drosophila. Our results support a model in which MEI-9 nicks Holliday junctions to generate crossovers during meiotic recombination, and, in the absence of MEI-9 activity, the double Holliday junction intermediate instead undergoes dissolution to generate noncrossover products in which heteroduplex is unrepaired.

Crossovers (COs) generated through meiotic recombination are important for the correct segregation of homologous chromosomes during meiosis. Several models describing the molecular mechanism of meiotic recombination have been proposed. These models differ in the arrangement of heteroduplex DNA (hDNA) in recombination intermediates. Heterologies in hDNA are usually repaired prior to the recovery of recombination products, thereby obscuring information about the arrangement of hDNA. To examine hDNA in meiotic recombination in Drosophila melanogaster, we sought to block hDNA repair by conducting recombination assays in a mutant defective in mismatch repair (MMR). We generated mutations in the MMR gene Msh6 and analyzed recombination between highly polymorphic homologous chromosomes. We found that hDNA often goes unrepaired during meiotic recombination in an Msh6 mutant, leading to high levels of postmeiotic segregation; however, hDNA and gene conversion tracts are frequently discontinuous, with multiple transitions between gene conversion, restoration, and unrepaired hDNA. We suggest that these discontinuities reflect the activity of a short-patch repair system that operates when canonical MMR is defective.

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2-7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.

In most eukaryotes, crossovers are not independently distributed along the length of a chromosome. Instead, they appear to avoid close proximity to one another--a phenomenon known as crossover interference. Previously, for three of the five Arabidopsis chromosomes, we measured the strength of interference and suggested a model wherein some crossovers experience interference while others do not. Here we show, using the same model, that the fraction of interference-insensitive crossovers is significantly smaller on the remaining two chromosomes. Since these two chromosomes bear the Arabidopsis NOR domains, the possibility that these chromosomal regions influence interference is discussed.

Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates.

We have investigated the combined effect of ionic calcium and ethanol on the visual creaming behavior and rheology of sodium caseinate-stabilized emulsions (4 wt% protein, 30 vol% oil, pH 6.8, mean droplet diameter 0.4 microm). A range of ionic calcium concentrations, expressed as a calcium/caseinate molar ratio R, was adjusted prior to homogenization and varying concentrations of ethanol were added shortly after homogenization. A stability map was produced on the basis of visual creaming behavior over a minimum period of 8 h for different calcium/caseinate/ethanol emulsion compositions. A single narrow stable (noncreaming) region was identified, indicating limited cooperation between calcium ions and ethanol. The shear-thinning behavior of the caseinate-stabilized emulsions is typical of systems undergoing depletion flocculation. Addition of calcium ions and/or ethanol was found to lead to a pronounced reduction in viscosity and the onset of Newtonian flow. The state of aggregation was correlated with emulsion microstructure from confocal laser scanning microscopy. Time-dependent rheology (18 h) with a density-matched oil phase (1-bromohexadecane) revealed that the visually stable emulsions were time-independent low-viscosity fluids. Surface coverage data showed that increasing amounts of caseinate were associated with the oil-water interface with increasing R and ethanol content. A decrease in free calcium ions in the aqueous phase with moderate increases in R and ethanol content was observed, which is consistent with greater calcium-caseinate binding (aggregation). Ostwald ripening occurred at the high-ethanol emulsion compositions that were stable to depletion flocculation. While the coarsening rate was low, this can account for the cream plug formation observed during gravity creaming experiments. The caseinate emulsion with no ionic calcium or ethanol exhibits depletion flocculation from excess nonadsorbed caseinate submicelles. Addition of calcium ions reduces the submicelle number density via specific calcium-binding in the aqueous phase (fewer, larger calcium-caseinate aggregates) and at the droplet surface (increased surface coverage). Nonspecific ethanol-induced (calcium-dependent) caseinate submicelle aggregation in the bulk phase and on the droplet surface (increased surface coverage) culminates in a reduction in the number density of caseinate submicelles. A narrow window of inhibition of depletion flocculation occurs in systems containing both calcium ions and ethanol, both species combining to aggregate the protein and so reduce the density of free submicelles.