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Cancer survivors consider nutrition to be highly important and are motivated to seek information about lifestyle changes, including nutrition, to improve their long-term health. Despite this, suboptimal dietary intake is still reported. Understanding cancer-specific barriers and facilitators to healthy eating among this population could help develop targeted interventions for this group. This study explored the barriers and facilitators to following a healthy diet among cancer survivors using the Theoretical Domains Framework (TDF) and COM-B model. Semi-structured focus groups with 20 cancer survivors were conducted between February and August 2021, which were transcribed verbatim. Seven key TDF domains accounted for 87% of all coded responses. These were (a) environmental context and resources; (b) knowledge; (c) behavioural regulation; (d) social/professional role and identity; (e) belief about consequences; (f) social influences; and (g) skills. Key barriers were lack of knowledge, non-specific or irrelevant information, environmental aspects, and family. Facilitators were awareness of the importance of nutrition, the health benefits of a plant-based diet, confidence in cooking skills, organisation, balance, family, time, and viewing themselves as someone who eats healthily and access to fresh produce. Enablement was the most prominently linked intervention function. This work can inform future interventions in this area and ensure they are end-user-centred.

In Ireland, the number of individuals living with and beyond cancer equates to almost 4% of the population, projected to increase. Little is known about how cancer changes views on food and nutrition among cancer survivors. A patient-centered perspective is imperative for developing future health interventions in this cohort. This study aimed to explore these perceptions and views amongst Irish cancer survivors. Participants living across Ireland and who had completed active cancer treatment at least six months ago were eligible for this study. Semi-structured focus groups were conducted online via Microsoft Teams. Data was analysed using Braun and Clarke's updated six-phase process for reflexive thematic analysis. Five focus group interviews were held with cancer survivors (n=20). The cohort was predominately female (n=17) with a mean age of 51.3 ± 11.9 years. Three core themes emerged (i) the role of nutrition, (ii) cancer as a catalyst for change and (iii) the overall lack of nutritional support in oncology. Findings suggest how a cancer diagnosis prompts a re-evaluation of health status and may prime survivors toward healthier dietary choices. The views of our participants indicate that survivors long for guidance from health professionals about their nutrition and the side effects of their cancer treatment and medication that impact dietary intake. This article is protected by copyright. All rights reserved.

BackgroundMalnutrition is a global emergency, creating an overlapping burden on individual, public and economic health. The double burden of malnutrition affects approximately 2.3 billion adults worldwide. Following 3 years of capacity building work in Kolkata, with assistance of local volunteers and organisations, we established an empowering nutrition education model in the form of a ‘mobile teaching kitchen (MTK)’ with the aim of creating culinary health educators from lay slum-dwelling women.AimsTo evaluate the piloting of a novel MTK nutrition education platform and its effects on the participants, alongside data collection feasibility.MethodsOver 6 months, marginalised (RG Kar and Chetla slums) women underwent nutrition training using the MTK supported by dietitians, doctors and volunteers. Preintervention and postintervention assessments of knowledge, attitudes and practices (KAP), as well as anthropometric and clinical nutritional status of both the women and their children were recorded. The education was delivered by a ‘See One, Do One, Teach One’ approach with a final assessment of teaching delivery performed in the final session.ResultsTwelve women were trained in total, six from each slum. Statistically significant improvements were noted in sections of KAP, with improvements in nutrition knowledge (+4.8) and practices (+0.8). In addition, statistically significant positive changes were seen in ‘understanding of healthy nutrition for their children’ (p=0.02), ‘sources of protein rich food’ (p=0.02) and ‘not skipping meals if a child is ill’ (p≤0.001).ConclusionThe MTK as a public health intervention managed to educate, empower and upskill two groups of lay marginalised women into MTK Champions from the urban slums of Kolkata, India. Improvements in their nutrition KAP demonstrate just some of the effects of this programme. By the provision of healthy meals and nutritional messages, the MTK Champions are key drivers nudging improvements in nutrition and health related awareness with a ripple effect across the communities that they serve. There is potential to upscale and adapt this programme to other settings, or developing into a microenterprise model, that can help future MTK Champions earn a stable income.

The present study aimed to describe the proportion of cancer patients and survivors who receive nutrition advice, the sources of that advice and to explore the nutrition advice this cohort would like to have received.A descriptive cross-sectional survey was completed by 211 cancer patients and survivors recruited through two hospitals in the North-West of Ireland. The survey consisted of open-ended and closed questions that aimed to explore the provision of and sources of nutrition advice and desired guidance in this cohort. Descriptive analysis was conducted using SPPS, while open-ended questions underwent thematic analysis.Respondents were mainly female (n = 133; 63%), aged 50-69 years old (n = 118; 56%), attending the outpatient department (n = 128; 60.7%) and5 years since diagnosis (n = 150; 71.7%). Breast (n = 69; 32.7%) was the most common cancer type. Respondents experienced a mean ± SD of 5 ± 3.1 nutrition impact symptoms. The most common was changes in taste/smell (n = 122; 57.8%). Although 53.6% (n = 113) reported being given advice, only 34.1% (n = 72) received this advice from a dietitian. The main sources of nutritional advice were friends and family. Respondents expressed their desire for nutrition advice from a dietitian and that this should be individualised, clear and practical. There was also a desire to avoid misinformation and uncertainty.The results of the present study can be used to help tailor nutrition support for this group. There is a need to ensure that patients and survivors consistently receive evidence-based advice tailored to their needs, cancer type and treatment stage.

Ascertaining cancer survivors' specific needs and preferences regarding nutrition information delivery is important in developing and designing future health interventions in oncology. The present study aimed to explore the nutrition information needs of cancer survivors and their preferences regarding intervention delivery.The present study is a mixed-methods study comprising a cross-sectional survey and focus groups. Participants were eligible for inclusion if they were aged ≥ 18 years, living in Ireland with a cancer diagnosis and had completed treatment at least 6 months previously. Cancer support centres promoted the online survey on social media. Focus groups were conducted with 20 individuals.The cohort (n = 56) was predominantly female (n = 50; 89.3%) breast cancer survivors (n = 42; 75%). Seven (12.5%) had received nutrition advice from a dietitian. Most were interested in receiving nutrition advice (n = 52; 92.9%), with variability in delivery mode. There was interest in improving sleep quality (n = 35; 62.5%), making positive lifestyle changes (n = 28; 50%) and accessing cancer-specific physical activity classes (n = 27; 48.2%). The best time to deliver information was throughout the cancer journey (n = 31; 55.4%). Time and motivation (n = 15; 26.8%) were the main barriers. Facilitators were keeping healthy (n = 42; 75%) and weight maintenance (n = 31; 55.4%). Four themes emerged from thematic analysis: lack of nutrition guidance, an abundance of misinformation, one size does not fit all and dietitians as the preferred source of advice.There is a desire for evidence-based nutrition advice and for dietitians to be integrated into cancer clinics and cancer survivorship care. This research guides future nutrition interventions to improve cancer survivorship outcomes.

The purpose of this research was to explore the nutrition practices among post-treatment cancer survivors across Ireland. Cancer survivors aged 18+ years living across Ireland, who were not palliative and had completed active cancer treatment at least six months previous, were recruited to complete an online survey assessing dietary quality, food choice and satisfaction with food-related life as well as clinical and nutrition status. It was circulated by cancer support networks and on social media. Descriptive statistics are presented. The cohort (n = 170) was predominantly female (85.9%) and had breast cancer (64.7%). Mean age was 51.5 ± 10.9 years and 42.7% of the cohort were >five years post-treatment. Only 20% and 12% of the cohort had been assessed by a dietitian during and post-treatment, respectively. The mean dietary quality score was 10.3 ± 1.7, which was measured by the Leeds short-form food frequency questionnaire (SFFFQ). Using a 5-point Likert scale, the median satisfaction with food-related life score was 19 (3.3), which evaluates cognitive judgements on the person’s food-related life. The food choice questionnaire (FCQ) assesses the relative importance of a range of factors related to dietary choice to individuals. The primary determinant of food choice in this cohort was the natural content (31.7%) followed by health (24.7%). Vitamin and mineral supplement use was reported by 69.8% of the cohort; the most consumed was Vitamin D. Four themes emerged from an optional open-ended question: awareness of nutritional importance; desire for specific nutritional advice and dietetic referral; cancer and treatment nutrition impacts were highlighted; as well as struggles with weight gain. This research provides useful insight into the nutrition practices of Irish cancer survivors. A desire and need for individualised and specific advice are evident.

Processing bodies (PBs) are ribonucleoprotein granules important for cytokine mRNA decay that are targeted for disassembly by many viruses. Kaposi’s sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposi’s sarcoma, and a PB-regulating virus. The virus encodes kaposin B (KapB), which induces actin stress fibers (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors, and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB. We propose that the viral protein KapB activates a mechanoresponsive signaling axis and links changes in cell shape and cytoskeletal structures to enhanced inflammatory molecule expression using PB disassembly. Our work implies that cytoskeletal changes in other pathologies may similarly impact the inflammatory environment.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121., The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.

Processing bodies (PBs) are ribonucleoprotein granules that suppress cytokine mRNA translation that are targeted for disassembly by many viruses. Kaposi’s sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposi’s sarcoma, and a PB-regulating virus. The virus encodes Kaposin B (KapB), which induces actin stress fibres (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB and mechanoresponsive PB disassembly also required YAP. Using the viral protein KapB, we identified a new consequence of the exposure of cells to mechanical forces that alter actin dynamics and activate YAP, namely the disassembly of PBs.ImportanceFor the first time, we demonstrate that processing bodies (PBs), cytoplasmic sites of RNA decay, are regulated by mechanical signaling events that alter actin dynamics and that this requires the mechanoresponsive transcription factor, YAP. Using the overexpression of a viral protein called KapB, known previously to mediate PB disassembly, we show that actin stress fibers (SFs) and the mechanoresponsive transcription factor, YAP, are required for PB loss. We also show that other established mechanical signals (shear stress or stiff extracellular matrix) that lead to the formation of SFs and activate YAP also cause PB disassembly. This is important because it means that KapB activates, from the inside out, a pathway that links cell shape to post-transcriptional gene regulation via cytoplasmic PBs.

Centrosomal protein of 192 kDa (CEP192) is a scaffolding protein that recruits the mitotic protein kinases Aurora A and PLK1 to the centrosome. Here we demonstrate that CEP192 also recruits the type one protein phosphatase (PP1) via a highly conserved KHVTF docking motif. The threonine of the KHVTF motif is phosphorylated during mitosis and protein kinase inhibition studies suggest this to be a PLK1-dependent process.

Up to 50% of the adult population fail to meet the recommended total water intake despite the adverse health outcomes associated with chronic low fluid intake. Total fluid intake plays an important role in the energy and nutrient intake of individuals. Dietitians’ role is to advise and guide the population toward a healthier diet. However, based on existing evidence, dietitians’ current knowledge and practices regarding healthy hydration is an area for improvement. The European Healthy Hydration Awareness Campaign (EuHHAC) is an initiative between the European Federation of the Associations of Dietitians (EFAD) and the Hydration and Health Department of Danone Research, which aims to increase knowledge and awareness of healthy hydration and provide dietitians with evidence-based resources to use in practice. Since 2016, a series of tasks were undertaken by the EuHHAC group targeting dietitians across Europe. These tasks included an online survey addressing gaps in hydration knowledge, a webinar providing information on the terminology and impact of hydration on health, an interactive session addressing facilitators and barriers to healthy hydration, and a tutorial summarizing key hydration information and take-home messages. Dietitians were actively engaged in each aspect. Based on the feedback received, awareness of hydration was increased among dietitians. To further enhance understanding, an online Hydration Resource Center, including the resources of EuHHAC, will be created and uploaded on the EFAD website. Once completed an evaluation study will be undertaken to assess the usefulness of the resources in practice. The deliverables will be cascaded across Europe and worldwide via social media, conferences, and publications.

Background The NNEdPro Core Team, Bhavishya Shakti Researchers and Inner Wheel Club of Greater Calcutta launched Mobile Teaching Kitchens as a nutritional education tool in two Indian slums in February 2018 to improve awareness of diet diversity and disease prevention by using locally sourced foods and cooking skills. Local volunteers, trained in healthy cooking, transferred core nutritional principals by cooking sustainable, nutritional and affordable meals. They followed a “See one, Do one and Teach one” model to transfer knowledge to their peers where markers of malnutrition were assessed before and after this programme. Objective To evaluate the efficacy of using mobile teaching kitchens to provide nutrition education through cooking, teaching healthy eating to a disadvantaged community in the urban slums of India. Study Design, Settings, Participants Longitudinal evaluation of mothers and children in RG Kar Canal Slum and Chetla Slum in Kolkata, India from 2018 to 2019. Measurable Outcome/Analysis Anthropometric and clinical signs of the children were assessed in person, alongside assessment of knowledge, attitudes and practice of the mothers through questionnaires. Results Preliminary measurements post-analysis from both slums (n = 14) showed that overall anthropometric measures of health in the mothers remained static. There was a mean change of 2.75cm growth in height in children. Other markers of nutritional status such as weight and mid-upper arm circumference did not significantly change. Statistically significant changes were seen in self-perceived nutritional knowledge about dietary protein sources, where the median response increased from 2 to 3 out of 5 (5 indicating excellent understanding) (P Conclusion Overall improvement in clinical status and nutritional knowledge was seen using Mobile Teaching Kitchens as an educational tool. Further evaluation of this particular teaching method is warranted due to the small sample size and heterogeneous results. Funding British Medical Association (BMA), Economic and Social Research Council (ESRC), Global Open Data for Agriculture and Nutrition (GODAN), The British Dietetics Association (BDA), University of Cambridge.

Aim. The aim of this study was to investigate dietitians’ knowledge, attitudes, and practices (KAP) regarding hydration and patient care. Methods. A cross-sectional online survey was administered to UK dietitians via the British Dietetic Association monthly newsletter and included 18 items on hydration knowledge (n=8), attitudes (n=4), and practices (n=6). KAP scores were calculated by adding the total number of correct knowledge responses and by ranking attitude and practice responses on a Likert scale. Results. 97 dietitians completed the online survey and displayed varying levels of KAP regarding hydration and patient care. The mean unweighted scores were knowledge 5.0 (±1.3) out of 8; attitude 13.9 (±1.3) out of 16; practice 14.9 (±2.6) out of 24. Dietitians appeared to be guided by clinical reasoning and priorities for nutrition care. Conclusions. There may be scope to further assess and potentially enhance the KAP of dietitians regarding hydration and patient care. Innovative approaches to hydration promotion are warranted and may include focusing on dietitians’ personal hydration status, increasing communication with other healthcare professionals, and partnering with patients to take a proactive role in hydration monitoring.

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis.

Staff play key roles in the prevention, detection, and treatment of hospital malnutrition. Understanding staff knowledge, attitudes, and practices (KAP) is important for developing and evaluating change management strategies.The More-2-Eat project improved nutrition care in 5 Canadian hospitals by implementing the Integrated Nutrition Pathway for Acute Care (INPAC). To understand staff views before (T1) and after 1 year of implementation (T2), a reliable KAP questionnaire, based on INPAC, was administered. T2 included questions about involvement in implementation. The mean difference between T2 and T1 responses was calculated, and t tests were used for comparisons.The questionnaire was completed at T1 (n = 189) and T2 (n = 147) (unpaired); 57 staff completed both questionnaires (paired). A significant increase in total score was seen in unpaired results at T2 (from 93.6/128 [range, 51-124] to 99.5/128 [range, 54-119]; t = 5.97, P.0001), with an increase in knowledge/attitudes (KA) (t = 2.4, P = .016) and practice (t = 3.57, P.0001) components. There were no statistically significant changes in paired responses. Seventy percent (n = 102/147) noticed positive changes in practices, 12% (n = 18) noticed positive/negative changes, 1% (n = 1) noticed negative change, and 17% (n = 25) noticed no change. Fifty-nine percent (n = 86) felt involved in the change, and these staff had higher KA and KAP scores than those who did not feel involved.Staff involvement is important in the implementation process for improving nutrition care.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G(2)/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (gamma-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of gamma-H2AX, the dissolution of IR-induced foci, and release from the G(2)/M checkpoint in vivo.

Recent studies have established that DNA-dependent protein kinase (DNA-PK) undergoes a series of autophosphorylation events that facilitate successful completion of nonhomologous DNA end joining. Autophosphorylation at sites in two distinct clusters regulates DNA end access to DNA end-processing factors and to other DNA repair pathways. Autophosphorylation within the kinase's activation loop regulates kinase activity. Additional autophosphorylation events (as yet undefined) occur that mediate kinase dissociation. Here we provide the first evidence that autophosphorylation within the two major clusters (regulating end access) occurs in trans. Further, both UV-induced and double-strand break (DSB)-induced phosphorylation in the two major clusters is predominately autophosphorylation. Finally, we show that while autophosphorylation in trans on one of two synapsed DNA-PK complexes facilitates appropriate end processing, this is not sufficient to promote efficient end joining. This suggests that end joining in living cells requires additional phosphorylation events that either occur in cis or that occur on both sides of the DNA-PK synapse. These data support an emerging consensus that, via a series of autophosphorylation events, DNA-PK undergoes a sequence of conformational changes that promote efficient and appropriate repair of DSBs.

The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

Pharmacists are one of the largest and most accessible community healthcare professions and can engage up to four times more with patients than general practitioners. They are ideally placed to pro...

Hydration is essential for life but clinical guidelines on hydration are lacking. Dietitians assess, diagnose and treat nutritional problems for individuals in the UK, ideally placing them to deliver on both acute clinical and preventative aspects of hydration care. The aim of this study was to identify key topics for a training intervention to promote incorporation of hydration care into dietetic practice. A review of literature identified key topics for a cross-sectional online survey for UK dietitians, containing 18 items on hydration knowledge (n8), attitudes (n4) and practices (n6). Ninety-seven dietitians completed the survey which prioritised topics for an educational intervention. Knowledge questions where the majority (蠅70%) of dietitians answered correctly were; impact of dehydration on performance tasks (94%), physical signs of dehydration (92%) and the definition of dehydration (76%). More than half of dietitians answered the following knowledge questions incorrectly; European Food Safety Auth...

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser(343)), SMC1 (Ser(957)), Chk1 (Ser(317) and Ser(345)), and Chk2 (Ser(33/35) and Thr(68)). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.

Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.

DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.

ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC(50) of approximately 100 nM. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme.

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.

Background One in four adults are estimated to be at medium to high risk of malnutrition when screened using the ‘Malnutrition Universal Screening Tool’ upon admission to hospital in the United Kingdom. The Need for Nutrition Education/Education Programme (NNEdPro) Group was developed to address this issue and the Nutrition Education and Leadership for Improved Clinical Outcomes (NELICO) is a project within this group. The objective of NELICO was to assess whether an intensive training intervention combining clinical and public health nutrition, organisational management and leadership strategies, could equip junior doctors to contribute to improvement in nutrition awareness among healthcare professionals in the National Health Service in England. Methods Three junior doctors were self-selected from the NNEdPro Group original training. Each junior doctor recruited three additional team members to attend an intensive training weekend incorporating nutrition, change management and leadership. This equipped them to run nutrition awareness weeks in their respective hospitals. Knowledge, attitudes and practices were evaluated at baseline as well as one and four months post-training as a quality assurance measure. The number and type of educational events held, pre-awareness week Online Hospital Survey results, attendance and qualitative feedback from training sessions, effectiveness of dissemination methods such as awareness stalls, Hospital Nutrition Attitude Survey results and overall feedback were also used to determine impact. Results When the weighted average score for knowledge, attitudes and practices at baseline was compared with four months post-intervention scores, there was a significant increase in the overall score (p = 0.03). All three hospital teams conducted an effective nutrition awareness week, as determined by qualitative data collected from interviews and feedback from educational sessions. Conclusion The NELICO project and its resulting nutrition awareness weeks were considered innovative in terms of concept and content. It was considered useful, both for the junior doctors who showed improvement in their nutrition knowledge and reported enthusiasm and for the hospital setting, increasing awareness of clinical and public health nutrition among healthcare professionals. The NELICO project is one innovative method to promote nutrition awareness in tomorrow’s doctors and shows they have the enthusiasm and drive to be nutrition champions.

Objectives To (1) assess the hydration knowledge, attitudes and practices (KAP) of doctors; (2) develop an evidence-based training package; and (3) evaluate the impact of the training package. Design Educational intervention with impact evaluation. Setting Cambridgeshire, UK. Participants General practitioners (GPs (primary care physicians)). Interventions Hydration and healthcare training. Main outcome measures Hydration KAP score before and immediately after the training session. Results Knowledge gaps of doctors identified before the teaching were the definition of dehydration, European Food Safety Authority water intake recommendations, water content of the human body and proportion of water from food and drink. A face-to-face teaching package was developed on findings from the KAP survey and literature search. 54 questionnaires were completed before and immediately after two training sessions with GPs. Following the training, total hydration KAP scores increased significantly (p

The low-activity, phosphorylated form of nitrate reductase (NR) became activated during purification from spinach (Spinacia oleracea) leaves harvested in the dark. This activation resulted from its separation from an approximately 110-kd nitrate reductase inhibitor protein (NIP). Readdition of NIP inactivated the purified phosphorylated NR, but not the active dephosphorylated form of NR, indicating that the inactivation of NR requires its interaction with NIP as well as phosphorylation. Consistent with this hypothesis, NR that had been inactivated in vitro in the presence of NR kinase, ATP-Mg, and NIP could be reactivated either by dephosphorylation with protein phosphatase 2A or by dissociation of NIP from NR.

Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5′-DNA kinase/3′-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.

How a cell chooses between nonhomologous end joining (NHEJ) and homologous recombination (HR) to repair a double-strand break (DSB) is a central and largely unanswered question. Although there is evidence of competition between HR and NHEJ, because of the DNA-dependent protein kinase (DNA-PK)'s cellular abundance, it seems that there must be more to the repair pathway choice than direct competition. Both a mutational approach and chemical inhibition were utilized to address how DNA-PK affects HR. We find that DNA-PK's ability to repress HR is both titratable and entirely dependent on its enzymatic activity. Still, although requisite, robust enzymatic activity is not sufficient to inhibit HR. Emerging data (including the data presented here) document the functional complexities of DNA-PK's extensive phosphorylations that likely occur on more than 40 sites. Even more, we show here that certain phosphorylations of the DNA-PK large catalytic subunit (DNA-PKcs) clearly promote HR while inhibiting NHEJ, and we conclude that the phosphorylation status of DNA-PK impacts how a cell chooses to repair a DSB.

Comment on: Douglas P, et al. Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and dephosphorylates {gamma}-H2AX. Mol Cell Biol. 2010 Jan 11. [Epub ahead of print].

NNEdPro aimed to conduct an educational needs analysis and pilot a non-vertical curriculum intervention through aninnovatively developed educational package in a national sample of tomorrow’s doctors.An intervention group of 100 senior/clinical medical students from 15 medical schools were recruited. A non-vertical educationalpackage was delivered over 2d, incorporating interactive lectures, role-play, simulated consultations, small group work and mini-PBL. Aspiral learning approach revisited topics in order to build upon consolidated basic concepts. Clinical nutrition knowledge, attitudes andpractices (KAP) were assessed before and after intervention, and at 3 months, using a construct-validated questionnaire. Questionnaireitems were randomised before and after intervention, to minimise recall bias. A student-reported evaluation of the educational interventionwas also conducted and yielded favourable results.

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to DNA double-strand breaks caused by ionizing radiation. Ionizing radiation induces the autophosphorylation of ATM on serine 1981; however, the precise mechanisms that regulate ATM autophosphorylation are not fully understood. By treating cells with okadaic acid, a cell-permeable protein phosphatase inhibitor, together with assays to quantify the activity of particular protein phosphatases, we have demonstrated that the autophosphorylation of ATM on serine 1981 is regulated by a protein phosphatase 2A-like activity. Here, we describe the series of experiments that employed protein phosphatase inhibitors to establish that ATM was regulated by a type-2A protein phosphatase.

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to DNA double-strand breaks caused by ionizing radiation. Ionizing radiation induces the autophosphorylation of ATM on serine 1981; however, the precise mechanisms that regulate ATM autophosphorylation are not fully understood. By treating cells with okadaic acid, a cell-permeable protein phosphatase inhibitor, together with assays to quantify the activity of particular protein phosphatases, we have demonstrated that the autophosphorylation of ATM on serine 1981 is regulated by a protein phosphatase 2A-like activity. Here, we describe the series of experiments that employed protein phosphatase inhibitors to establish that ATM was regulated by a type-2A protein phosphatase.

The Ku70/80 heterodimer is a major player in non-homologous end joining and the repair of DNA double-strand breaks. Studies suggest that once bound to a DNA double-strand break, Ku recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form the DNA-dependent protein kinase holoenzyme complex (DNA-PK). We previously identified four DNA-PK phosphorylation sites on the Ku70/80 heterodimer: serine 6 of Ku70, serine 577 and 580 and threonine 715 of Ku80. This raised the interesting possibility that DNA-PK-dependent phosphorylation of Ku could provide a mechanism for the regulation of non-homologous end joining. Here, using mass spectrometry and phosphospecific antibodies we confirm that these sites are phosphorylated in vitro by purified DNA-PK. However, we show that neither DNA-PK nor the related protein kinase ataxia-telangiectasia mutated (ATM) is required for phosphorylation of Ku at these sites in vivo. Furthermore, Ku containing serine/threonine to alanine mutations at these sites was fully able to complement the radiation sensitivity of Ku negative mammalian cells indicating that phosphorylation at these sites is not required for non-homologous end joining. Interestingly, both Ku70 and Ku80 were phosphorylated in cells treated with the protein phosphatase inhibitor okadaic acid under conditions known to inactivate protein phosphatase 2A-like protein phosphatases. Moreover, okadaic acid-induced phosphorylation of Ku80 was inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. These results suggest that the phosphorylation of Ku70 and Ku80 is regulated by a protein phosphatase 2A-like protein phosphatase and a staurosporine sensitive protein kinase in vivo, but that DNA-PK-mediated phosphorylation of Ku is not required for DNA double-strand break repair.

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.

Leach and colleagues’ article reiterates the issues that the Need for Nutrition Education Programme (NNEdPro; www.nnedpro.org.uk; comprising doctors, dietitians, and other healthcare professionals) has been working on for more than five years.1 We strongly agree that a greater emphasis on nutrition and hydration education is needed among healthcare professionals, particularly tomorrow’s doctors. As a multiprofessional group, we …