AMPK: a nutrient and energy sensor that maintains energy homeostasis.

AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor. Once activated by falling energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism while conserving ATP by switching off biosynthetic pathways. AMPK also regulates metabolic energy balance at the whole-body level. For example, it mediates the effects of agents acting on the hypothalamus that promote feeding and entrains circadian rhythms of metabolism and feeding behaviour. Finally, recent studies reveal that AMPK conserves ATP levels through the regulation of processes other than metabolism, such as the cell cycle and neuronal membrane excitability.

Frequent loss-of-function mutations in the AMPK-α2 catalytic subunit suggest a tumour suppressor role in human skin cancers.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status activated by increases in AMP or ADP relative to ATP. Once activated, it phosphorylates targets that promote ATP-generating catabolic pathways or inhibit ATP-consuming anabolic pathways, helping to restore cellular energy balance. Analysis of human cancer genome studies reveals that the PRKAA2 gene (encoding the α2 isoform of the catalytic subunit) is often subject to mis-sense mutations in cancer, particularly in melanoma and non-melanoma skin cancers, where up to 70 mis-sense mutations have been documented, often accompanied by loss of the tumour suppressor NF1. Recently it has been reported that knockout of PRKAA2 in NF1-deficient melanoma cells promoted anchorage-independent growth in vitro, as well as growth as xenografts in immunodeficient mice in vivo, suggesting that AMPK-α2 can act as a tumour suppressor in that context. However, very few of the mis-sense mutations in PRKAA2 that occur in human skin cancer and melanoma have been tested to see whether they cause loss-of-function. We have addressed this by making most of the reported mutations and testing their activity when expressed in AMPK knockout cells. Of 55 different mis-sense mutations (representing 75 cases), 9 (12%) appeared to cause a total loss of activity, 18 (24%) a partial loss, 11 (15%) an increase in phenformin-stimulated kinase activity, while just 37 (49%) had no clear effect on kinase activity. This supports the idea that AMPK-α2 acts as a tumour suppressor in the context of human skin cancer.

Intentional rounding: a realist evaluation using case studies in acute and care of older people hospital wards.

BACKGROUND: In response to concerns about high hospital mortality rates, patient and carer complaints, a Mid Staffordshire NHS Foundation Trust public inquiry was conducted at the request of the UK government. This inquiry found serious failures in the quality of basic care provided and as a consequence, recommended that patients should have more regular visits, organised at predictable times from nursing staff. Intentional rounding, also known as nursing ward rounds, was widely adopted to meet this need. OBJECTIVE: To test, refine or refute eight programme theories to understand what works, for whom, and in what circumstances. SETTING: Six wards (older people and acute wards) in three NHS trusts in England. PARTICIPANTS: Board level and senior nursing managers (N = 17), nursing ward staff (N = 33), allied health and medical professionals (N = 26), patients (N = 34) and relatives (N = 28) participated in an individual, in-depth interview using the realist method. In addition, ward-based nurses (N = 39) were shadowed whilst they conduced intentional rounds (240 rounds in total) and the direct care of patients (188 h of patient care in total) was observed. METHODS: The mixed methods design included: Phase (1) Theory development - A realist synthesis was undertaken to identify any programme theories which were tested, refined and/or refuted, using data from phases 2 and 3; Phase (2) A survey of all English NHS acute Trusts; Phase (3) Six case studies of wards involving realist interviews, shadowing and non-participant observations, analysis of ward outcome and cost data; and Phase (4) Synthesis of findings from phases 1, 2 and 3. RESULTS: The realist synthesis identified eight programme theories of intentional rounding: 'Consistency and comprehensiveness', 'Accountability', 'Visibility of nurses', 'Anticipation', 'Allocated time to care', 'Nurse-patient relationships', 'Multi-disciplinary teamwork and communication' and 'Patient empowerment'. Key findings showed that of the original eight programme theories of intentional rounding, only two partially explained how the intervention worked ('Consistency and comprehensiveness' and 'Accountability'). Of the remaining six programme theories, the evidence for two was inconclusive ('Visibility of nurses' and 'Anticipation') and there was no evidence for four ('Allocated time to care'; 'Nurse-patient relationships'; 'Multi-disciplinary teamwork and communication'; and 'Patient empowerment'). CONCLUSIONS: This first theory-informed evaluation of intentional rounding, demonstrates that the effectiveness of intentional rounding in the English healthcare context is very weak. Furthermore, the evidence collected in this study has challenged and refuted some of the underlying assumptions about how intentional rounding works. This study has demonstrated the crucial role context plays in determining the effectiveness of an intervention and how caution is needed when implementing interventions developed for the health system of one country into another.

BAY-3827 and SBI-0206965: Potent AMPK Inhibitors That Paradoxically Increase Thr172 Phosphorylation.

AMP-activated protein kinase (AMPK) is the central component of a signalling pathway that senses energy stress and triggers a metabolic switch away from anabolic processes and towards catabolic processes. There has been a prolonged focus in the pharmaceutical industry on the development of AMPK-activating drugs for the treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. However, recent findings suggest that AMPK inhibitors might be efficacious for treating certain cancers, especially lung adenocarcinomas, in which the PRKAA1 gene (encoding the α1 catalytic subunit isoform of AMPK) is often amplified. Here, we study two potent AMPK inhibitors, BAY-3827 and SBI-0206965. Despite not being closely related structurally, the treatment of cells with either drug unexpectedly caused increases in AMPK phosphorylation at the activating site, Thr172, even though the phosphorylation of several downstream targets in different subcellular compartments was completely inhibited. Surprisingly, the two inhibitors appear to promote Thr172 phosphorylation by different mechanisms: BAY-3827 primarily protects against Thr172 dephosphorylation, while SBI-0206965 also promotes phosphorylation by LKB1 at low concentrations, while increasing cellular AMP:ATP ratios at higher concentrations. Due to its greater potency and fewer off-target effects, BAY-3827 is now the inhibitor of choice for cell studies, although its low bioavailability may limit its use in vivo.

The role of faecal calprotectin in diagnosis and staging of colorectal neoplasia: a systematic review and meta-analysis.

INTRODUCTION: The presence of inflammation is a key hallmark of cancer and, plays an important role in disease progression and survival in colorectal cancer (CRC). Calprotectin detected in the faeces is a sensitive measure of colonic inflammation. The role of FC as a diagnostic test that may categorise patients by risk of neoplasia is poorly defined. This systematic review and meta-analysis aims to characterise the relationship between elevations of FC and colorectal neoplasia. METHODS: A systematic review was performed using the keywords (MESH terms) and a statistical and meta-analysis was performed. RESULTS: A total of 35 studies are included in this review. CRC patients are more likely than controls to have an elevated FC OR 5.19, 95% CI 3.12-8.62, p < 0.001 with a heterogeneity (I2 = 27%). No tumour characteristics significantly correlated with FC, only stage of CRC shows signs that it may potentially correlate with FC. CONCLUSION: FC levels are significantly higher in CRC, with high sensitivity. Its low specificity prevents it from being used to diagnose or screen for CRC.

The role of faecal calprotectin in the identification of colorectal neoplasia in patients attending for screening colonoscopy.

AIM: Although the relationship between colorectal neoplasia and inflammation is well described, the role of faecal calprotectin (FC) in clinical practice to diagnose or screen patients for colorectal neoplasia is less defined. This prospective study characterizes the relationship between FC and colorectal neoplasia in patients within the faecal occult blood testing (FOBT) positive patients in the Scottish Bowel Screening Programme. METHODS: All FOBT positive patients attending for colonoscopy between February 2016 and July 2017 were invited to participate. Patients provided a stool sample for FC before commencing bowel preparation. All demographics and endoscopic findings were collected prospectively. RESULTS: In all, 352 patients were included. 210 patients had FC > 50 µg. Colorectal cancer (CRC) patients had a higher median FC (138.5 µg/g, P < 0.05), in comparison to those without CRC, and 13/14 had an FC > 50 µg/g (93%). FC had a high sensitivity (92.8%) and negative predictive value (99.3%) for CRC, but with a low specificity (41.7%) and positive predictive value (6.2%). FC sensitivity increased sequentially as neoplasms progressed from non-advanced to malignant neoplasia (48.6% non-advanced adenoma vs. 92.9% CRC). However, no significant relationship was observed between FC and non-cancer neoplasia. CONCLUSION: In an FOBT positive screening population, FC was strongly associated with CRC (sensitivity 92.8%, specificity 41.7% for CRC, at 50 µg/g). However, although sensitive for the detection of CRC, FC failed to show sufficient sensitivity or specificity for the detection of non-cancer neoplasia. Based on these results we cannot recommend routine use of FC in a bowel screening population to detect cancer per se, but it is apparent that, with further optimization, faecal assessments including quantification of haemoglobin and inflammation could form part of a risk assessment tool aimed at refining the selection of patients for colonoscopy in both symptomatic and screening populations.

"Raising the curtain on the equality theatre": a study of recruitment to first healthcare job post-qualification in the UK National Health Service.

BACKGROUND: UK equality law and National Health Service (NHS) policy requires racial equality in job appointments and career opportunities. However, recent national workforce race equality standard (WRES) data show that nearly all NHS organisations in the UK are failing to appoint ethnically diverse candidates with equivalent training and qualifications as their white counterparts. This is problematic because workforce diversity is associated with improved patient outcomes and other benefits for staff and organisations. AIM: To better understand the reasons behind underrepresentation of ethnically diverse candidates in first NHS healthcare jobs post-qualification and to identify any structural or systemic barriers to employment for such groups. METHODS: The study was informed by critical theory and the authors' interdisciplinary perspectives as educators and researchers in the healthcare professions. Data collected from semi-structured face-to-face interviews with 12 nurse and physiotherapy recruiting managers from two NHS trusts in London were analysed using a healthcare workforce equity and diversity conceptual lens we developed from the literature. Using this lens, we devised questions to examine six dimensions of equity and diversity in the interview data from recruiting managers. RESULTS: Recruiting managers said they valued the benefits of an ethnically diverse workforce for patients and their unit/organisation. However, their adherence to organisational policies for recruitment and selection, which emphasise objectivity and standardisation, acted as constraints to recognising ethnicity as an important issue in recruitment and workforce diversity. Some recruiting managers sense that there are barriers for ethnically diverse candidates but lacked information about workforce diversity, systems for monitoring recruitment, or ways to engage with staff or candidates to talk about these issues. Without this information there was no apparent problem or reason to try alternative approaches. CONCLUSION: These accounts from 12 recruiting managers give a 'backstage' view into the reasons behind ethnic inequalities in recruitment to first healthcare job in the UK NHS. Adherence to recruitment and selection policies, which aim to support equality through standardisation and anonymisation, appear to be limiting workforce diversity and creating barriers for ethnically diverse candidates to attain the jobs that they are trained and qualified for. The Healthcare Workforce Equity + Diversity Lens we have developed can help to 'raise the curtain on the equality theatre' and inform more inclusive approaches to recruitment such as contextualised recruitment or effective allyship between employers and universities.

Constitutional and somatic rearrangement of chromosome 21 in acute lymphoblastic leukaemia.

Changes in gene dosage are a major driver of cancer, known to be caused by a finite, but increasingly well annotated, repertoire of mutational mechanisms. This can potentially generate correlated copy-number alterations across hundreds of linked genes, as exemplified by the 2% of childhood acute lymphoblastic leukaemia (ALL) with recurrent amplification of megabase regions of chromosome 21 (iAMP21). We used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. Here we show that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have approximately 2,700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by a chromothripsis event involving both sister chromatids of the Robertsonian chromosome, a novel mechanism for cancer predisposition. In sporadic iAMP21, breakage-fusion-bridge cycles are typically the initiating event, often followed by chromothripsis. In both sporadic and rob(15;21)c-associated iAMP21, the final stages frequently involve duplications of the entire abnormal chromosome. The end-product is a derivative of chromosome 21 or the rob(15;21)c chromosome with gene dosage optimized for leukaemic potential, showing constrained copy-number levels over multiple linked genes. Thus, dicentric chromosomes may be an important precipitant of chromothripsis, as we show rob(15;21)c to be constitutionally dicentric and breakage-fusion-bridge cycles generate dicentric chromosomes somatically. Furthermore, our data illustrate that several cancer-specific mutational processes, applied sequentially, can coordinate to fashion copy-number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.

Global nurse shortages-the facts, the impact and action for change.

INTRODUCTION: Nurses comprise half the global health workforce. A nine million shortage estimated in 2014 is predicted to decrease by two million by 2030 but disproportionality effect regions such as Africa. This scoping review investigated: what is known about current nurse workforces and shortages and what can be done to forestall such shortages? SOURCES OF DATA: Published documents from international organisations with remits for nursing workforces, published reviews with forward citation and key author searches. AREAS OF AGREEMENT: Addressing nurse shortages requires a data informed, country specific model of the routes of supply and demand. It requires evidence informed policy and resource allocation at national, subnational and organisation levels. AREAS OF CONTROVERSY: The definition in law, type of education, levels and scope of practice of nurses varies between countries raising questions of factors and evidence underpinning such variation. Most policy solutions proposed by international bodies draws on data and research about the medical workforce and applies that to nurses, despite the different demographic profile, the work, the career options, the remuneration and the status. GROWING POINTS: Demand for nurses is increasing in all countries. Better workforce planning in nursing is crucial to reduce health inequalities and ensure sustainable health systems. AREAS TIMELY FOR DEVELOPING RESEARCH: Research is needed on: the nursing workforce in low income countries and in rural and remote areas; on the impact of scope of practice and task-shifting changes; on the impact over time of implementing system wide policies as well as raising the profile of nursing.

Coexistent hyperdiploidy does not abrogate poor prognosis in myeloma with adverse cytogenetics and may precede IGH translocations.

The acquisition of the cytogenetic abnormalities hyperdiploidy or translocations into the immunoglobulin gene loci are considered as initiating events in the pathogenesis of myeloma and were often assumed to be mutually exclusive. These lesions have clinical significance; hyperdiploidy or the presence of the t(11;14) translocation is associated with a favorable outcome, whereas t(4;14), t(14;16), and t(14;20) are unfavorable. Poor outcomes are magnified when lesions occur in association with other high-risk features, del17p and +1q. Some patients have coexistence of both good and poor prognostic lesions, and there has been no consensus on their risk status. To address this, we have investigated their clinical impact using cases in the Myeloma IX study (ISRCTN68454111) and shown that the coexistence of hyperdiploidy or t(11;14) does not abrogate the poor prognosis associated with adverse molecular lesions, including translocations. We have also used single-cell analysis to study cases with coexistent translocations and hyperdiploidy to determine how these lesions cosegregate within the clonal substructure, and we have demonstrated that hyperdiploidy may precede IGH translocation in a proportion of patients. These findings have important clinical and biological implications, as we conclude patients with coexistence of adverse lesions and hyperdiploidy should be considered high risk and treated accordingly.

Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms.

The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ1, γ2 or γ3 isoform. When assayed at a physiological ATP concentration (5 mM), γ1- and γ2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr(172) phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ1 complexes, activation and Thr(172) phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKß)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr(172) dephosphorylation; with γ2 complexes, ADP had similar potency to AMP, but with γ1 and γ3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.

Examining the nature of interprofessional interventions designed to promote patient safety: a narrative review.

PURPOSE: This narrative review aimed to scope the patient safety literature to identify interprofessional intervention approaches, sources of evidence and reported outcomes. DATA SOURCES: Two major databases (MEDLINE and CINAHL) were searched from 2005 to 2015. STUDY SELECTION: A total of 1552 abstracts were initially identified. After screening these abstracts, 129 full papers were obtained. Further screening resulted in a total of 89 papers included in this review. DATA EXTRACTION: The following information was extracted from each included paper: details on the patient safety intervention, study methods employed and outcomes reported. RESULTS OF DATA SYNTHESIS: It was found that the bulk of the included studies was undertaken in a North American acute care context. Most often, studies involved qualified professionals from nursing and medicine collaborating in hospitals and medical centres. Nearly half the studies reported in this review employed educational interventions, such as TeamSTEPPS, aimed at enhancing practitioners' competence of delivering safe patient care. Nearly a third of studies involved practice-based interventions (e.g. checklists) aimed at improving the delivery of safe care. Most of the studies used a quasi-experimental design and typically gathered survey data. The majority reported outcomes related to changes in professionals' attitudes, knowledge and skills. There were, however, fewer studies reporting changes in practitioners' safety behaviours, organizational practices or patient benefit. CONCLUSION: The use of different interprofessional interventions are key activities involved in promoting safe patient care practices. However, further work is needed to strengthen these interventions and their evaluations.

AMP-activated protein kinase inhibits Kv 1.5 channel currents of pulmonary arterial myocytes in response to hypoxia and inhibition of mitochondrial oxidative phosphorylation.

KEY POINTS: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear. AMP-activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension. Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited Kv 1.5 channels in pulmonary arterial myocytes. AMPK activation by 5-aminoimidazole-4-carboxamide riboside, A769662 or C13 attenuated Kv 1.5 currents in pulmonary arterial myocytes, and this effect was non-additive with respect to Kv 1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human Kv 1.5 channels in cell-free assays, and inhibited K(+) currents when introduced into HEK 293 cells stably expressing Kv 1.5. These results suggest that AMPK is the primary mediator of reductions in Kv 1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. ABSTRACT: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP-activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1-dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited Kv currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte Kv currents were also markedly inhibited upon AMPK activation by A769662, 5-aminoimidazole-4-carboxamide riboside and C13 and by intracellular dialysis from a patch-pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2ß2γ1 or α1ß1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK-sensitive K(+) currents, which were also blocked by the selective Kv 1.5 channel inhibitor diphenyl phosphine oxide-1 but unaffected by the presence of the BKCa channel blocker paxilline. Moreover, recombinant human Kv 1.5 channels were phosphorylated by AMPK in cell-free assays, and K(+) currents carried by Kv 1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates Kv channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of Kv 1.5 and/or an associated protein.

The NF-κB1 transcription factor prevents the intrathymic development of CD8 T cells with memory properties.

The role of specific members of the NF-κB family of transcription factors in CD8 T-cell selection and development is largely unknown. Here, we show that mice lacking NF-κB1 develop a unique population of conventional CD8 single-positive (SP) thymocytes with memory T cell-like properties that populate peripheral immune organs. Development of this memory-like population is not due to PLZF(+) thymocytes and instead coincides with changes in CD8 T-cell selection. These include a reduction in the efficiency of negative selection and a dependence on MHC class Ia or Ib expressed by haematopoietic cells. These findings indicate that NF-κB1 regulates multiple events in the thymus that collectively inhibit the excess development of CD8(+) thymocytes with memory cell characteristics.

PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain.

AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two ß subunits (ß1/ß2) and one of three γ subunits (γ1/γ2/γ3). The ability to selectively activate specific isoforms would be a useful research tool and a promising strategy to combat diseases such as cancer and Type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 (unc-51-like autophagy-activating kinase 1) on Ser555, but not proposed AMPK-γ3 substrates such as Ser231 on TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 (TBC1D1) or Ser212 on acetyl-CoA carboxylase subunit 2 (ACC2), nor did it stimulate glucose transport. Surprisingly, however, in human embryonic kidney (HEK) 293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the kinase and auto-inhibitory domains (AIDs) of the α subunit. We show instead that PT-1 activates AMPK indirectly by inhibiting the respiratory chain and increasing cellular AMP:ATP and/or ADP:ATP ratios. Consistent with this mechanism, PT-1 failed to activate AMPK in HEK293 cells expressing an AMP-insensitive R299G mutant of AMPK-γ1. We propose that the failure of PT-1 to activate γ3-containing complexes in muscle is not an intrinsic feature of such complexes, but is because PT-1 does not increase cellular AMP:ATP ratios in the specific subcellular compartment(s) in which γ3 complexes are located.

A molecular diagnostic approach able to detect the recurrent genetic prognostic factors typical of presenting myeloma.

Risk stratification in myeloma requires an accurate assessment of the presence of a range of molecular abnormalities including the differing IGH translocations and the recurrent copy number abnormalities that can impact clinical behavior. Currently, interphase fluorescence in situ hybridization is used to detect these abnormalities. High failure rates, slow turnaround, cost, and labor intensiveness make it difficult and expensive to use in routine clinical practice. Multiplex ligation-dependent probe amplification (MLPA), a molecular approach based on a multiplex polymerase chain reaction method, offers an alternative for the assessment of copy number changes present in the myeloma genome. Here, we provide evidence showing that MLPA is a powerful tool for the efficient detection of copy number abnormalities and when combined with expression assays, MLPA can detect all of the prognostically relevant molecular events which characterize presenting myeloma. This approach opens the way for a molecular diagnostic strategy that is efficient, high throughput, and cost effective.

Characterization of IGH locus breakpoints in multiple myeloma indicates a subset of translocations appear to occur in pregerminal center B cells.

Translocations in myeloma are thought to occur solely in mature B cells in the germinal center through class switch recombination (CSR). We used a targeted captured technique followed by massively parallel sequencing to determine the exact breakpoints in both the immunoglobulin heavy chain (IGH) locus and the partner chromosome in 61 presentation multiple myeloma samples. The majority of samples (62%) have a breakpoint within the switch regions upstream of the IGH constant genes and are generated through CSR in a mature B cell. However, the proportion of CSR translocations is not consistent between cytogenetic subgroups. We find that 100% of t(4;14) are CSR-mediated; however, 21% of t(11;14) and 25% of t(14;20) are generated through DH-JH recombination activation gene-mediated mechanisms, indicating they occur earlier in B-cell development at the pro-B-cell stage in the bone marrow. These 2 groups also generate translocations through receptor revision, as determined by the breakpoints and mutation status of the segments used in 10% and 50% of t(11;14) and t(14;20) samples, respectively. The study indicates that in a significant number of cases the translocation-based etiological events underlying myeloma may arise at the pro-B-cell hematological progenitor cell level, much earlier in B-cell development than was previously thought.

Assessing the oral bioavailability of difluorosialic acid prodrugs, potent viral neuraminidase inhibitors, using a snapshot PK screening assay.

Difluorosialic acids (DFSAs) are potent inhibitors of viral neuraminidase that demonstrate activity against oseltamivir- and zanamivir-resistant strains of influenza. Unfortunately, low oral bioavailability precludes their development as second generation neuraminidase inhibitors for treating influenza as this leaves them unsuitable for use in an oral formulation. Herein is described the preparation of a series of DFSA prodrugs designed to increase oral bioavailability. These prodrugs were evaluated using a snapshot PK screen and stability tests, with successful candidates being further assessed with a full pharmacokinetic workup. These new prodrugs increased oral bioavailability by up to three times that seen for the parent DFSAs.

Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells.

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the 'ST loop' (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser487 phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr172 phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)-AMPK pathway, which would otherwise inhibit cell growth and proliferation.