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1.  International Myeloma Working Group Consensus Statement for the Management, Treatment, and Supportive Care of Patients With Myeloma Not Eligible for Standard Autologous Stem-Cell Transplantation 
Journal of Clinical Oncology  2014;32(6):587-600.
To provide an update on recent advances in the management of patients with multiple myeloma who are not eligible for autologous stem-cell transplantation.
A comprehensive review of the literature on diagnostic criteria is provided, and treatment options and management of adverse events are summarized.
Patients with symptomatic disease and organ damage (ie, hypercalcemia, renal failure, anemia, or bone lesions) require immediate treatment. The International Staging System and chromosomal abnormalities identify high- and standard-risk patients. Proteasome inhibitors, immunomodulatory drugs, corticosteroids, and alkylating agents are the most active agents. The presence of concomitant diseases, frailty, or disability should be assessed and, if present, treated with reduced-dose approaches. Bone disease, renal damage, hematologic toxicities, infections, thromboembolism, and peripheral neuropathy are the most frequent disabling events requiring prompt and active supportive care.
These recommendations will help clinicians ensure the most appropriate care for patients with myeloma in everyday clinical practice.
PMCID: PMC3918540  PMID: 24419113
2.  Global Myeloma Research Clusters, Output, and Citations: A Bibliometric Mapping and Clustering Analysis 
PLoS ONE  2015;10(1):e0116966.
International collaborative research is a mechanism for improving the development of disease-specific therapies and for improving health at the population level. However, limited data are available to assess the trends in research output related to orphan diseases.
Methods and Findings
We used bibliometric mapping and clustering methods to illustrate the level of fragmentation in myeloma research and the development of collaborative efforts. Publication data from Thomson Reuters Web of Science were retrieved for 2005–2009 and followed until 2013. We created a database of multiple myeloma publications, and we analysed impact and co-authorship density to identify scientific collaborations, developments, and international key players over time. The global annual publication volume for studies on multiple myeloma increased from 1,144 in 2005 to 1,628 in 2009, which represents a 43% increase. This increase is high compared to the 24% and 14% increases observed for lymphoma and leukaemia. The major proportion (>90% of publications) was from the US and EU over the study period. The output and impact in terms of citations, identified several successful groups with a large number of intra-cluster collaborations in the US and EU. The US-based myeloma clusters clearly stand out as the most productive and highly cited, and the European Myeloma Network members exhibited a doubling of collaborative publications from 2005 to 2009, still increasing up to 2013.
Conclusion and Perspective
Multiple myeloma research output has increased substantially in the past decade. The fragmented European myeloma research activities based on national or regional groups are progressing, but they require a broad range of targeted research investments to improve multiple myeloma health care.
PMCID: PMC4309532  PMID: 25629620
3.  Probiotics in the prevention of eczema: a randomised controlled trial 
Archives of Disease in Childhood  2014;99(11):1014-1019.
To evaluate a multistrain, high-dose probiotic in the prevention of eczema.
A randomised, double-blind, placebo-controlled, parallel group trial.
Antenatal clinics, research clinic, children at home.
Pregnant women and their infants.
Women from 36 weeks gestation and their infants to age 6 months received daily either the probiotic (Lactobacillus salivarius CUL61, Lactobacillus paracasei CUL08, Bifidobacterium animalis subspecies lactis CUL34 and Bifidobacterium bifidum CUL20; total of 1010 organisms/day) or matching placebo.
Main outcome measure
Diagnosed eczema at age 2 years. Infants were followed up by questionnaire. Clinical examination and skin prick tests to common allergens were done at 6 months and 2 years.
The cumulative frequency of diagnosed eczema at 2 years was similar in the probiotic (73/214, 34.1%) and placebo arms (72/222, 32.4%; OR 1.07, 95% CI 0.72 to 1.6). Among the secondary outcomes, the cumulative frequency of skin prick sensitivity at 2 years was reduced in the probiotic (18/171; 10.5%) compared with the placebo arm (32/173; 18.5%; OR 0.52, 95% CI 0.28 to 0.98). The statistically significant differences between the arms were mainly in sensitisation to cow's milk and hen's egg proteins at 6 months. Atopic eczema occurred in 9/171 (5.3%) children in the probiotic arm and 21/173 (12.1%) in the placebo arm (OR 0.40, 95% CI 0.18 to 0.91).
The study did not provide evidence that the probiotic either prevented eczema during the study or reduced its severity. However, the probiotic seemed to prevent atopic sensitisation to common food allergens and so reduce the incidence of atopic eczema in early childhood.
Trial registration Number
PMCID: PMC4215350  PMID: 24947281
Allergy; Microbiology; Dermatology
4.  Cancer-Selective Targeting of the NF-κB Survival Pathway with GADD45β/MKK7 Inhibitors 
Cancer Cell  2014;26(4):495-508.
Constitutive NF-κB signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-κB-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-κB-regulated antiapoptotic factor GADD45β and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45β/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-κB pathway is possible and, at least for myeloma patients, promises a profound benefit.
•GADD45β is a critical mediator of the NF-κB antiapoptotic function in MM•GADD45β binds to MKK7 and promotes MM cell survival by blocking MKK7/JNK signaling•GADD45β/MKK7 inhibitors display potent activity against MM, in vitro and in vivo•GADD45β/MKK7 inhibitors are far more cancer selective than IKK/NF-κB inhibitors
NF-κB is implicated in MM and other malignancies, but it is challenging to block NF-κB only in diseased cells. Tornatore et al. identify an interaction between MKK7 and NF-κB-regulated GADD45β as a therapeutic target and develop a peptide that disrupts the complex and selectively kills MM cells.
PMCID: PMC4197335  PMID: 25314077
5.  Intraclonal heterogeneity is a critical early event in the development of myeloma and precedes the development of clinical symptoms 
Leukemia  2013;28(2):384-390.
The mechanisms involved in progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia (PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole genome sequencing on a series of MGUS (n=4), high risk (HR)-SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors which drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.
PMCID: PMC3916874  PMID: 23817176
myeloma; smoldering; progression; genome; sequencing
6.  Risk of Progression and Survival in Multiple Myeloma Relapsing After Therapy with IMiDs and Bortezomib: A Multicenter International Myeloma Working Group Study 
Leukemia  2011;26(1):149-157.
Promising new drugs are being evaluated for treatment of multiple myeloma (MM), but their impact should be measured against the expected outcome in patients failing current therapies. However, the natural history of relapsed disease in the current era remains unclear. We studied 286 patients with relapsed MM, who were refractory to bortezomib and were relapsed, refractory, or ineligible, to an IMiD (Immunomodulatory Drug), with measurable disease and ECOG PS of 0, 1 or 2. The date patients satisfied the entry criteria was defined as time zero (T0). The median age at diagnosis was 58 years and time from diagnosis to T0 was 3.3 years. Following T0, 213 (74%) patients had a treatment recorded with one or more regimens (median=1; range 0-8). The first regimen contained bortezomib in 55 (26%) patients and an IMiD in 70 (33%). A minor response or better was seen to at least one therapy after T0 in 94 patients (51%) including >=partial response in 69 (38%). The median overall survival and event free survival from T0 were 9 and 5 months respectively. This study confirms the poor outcome once patients become refractory to current treatments. The results provide context for interpreting ongoing trials of new drugs.
PMCID: PMC4109061  PMID: 21799510
multiple Myeloma; relapse; natural history; survival
7.  A Novel Functional Role for MMSET in RNA Processing Based on the Link Between the REIIBP Isoform and Its Interaction with the SMN Complex 
PLoS ONE  2014;9(6):e99493.
The chromosomal translocation t(4;14) deregulates MMSET (WHSC1/NSD2) expression and is a poor prognostic factor in multiple myeloma (MM). MMSET encodes two major protein isoforms. We have characterized the role of the shorter isoform (REIIBP) in myeloma cells and identified a clear and novel interaction of REIIBP with members of the SMN (survival of motor neuron) complex that directly affects the assembly of the spliceosomal ribonucleic particles. Using RNA-seq we show that REIIBP influences the RNA splicing pattern of the cell. This new discovery provides novel insights into the understanding of MM pathology, and potential new leads for therapeutic targeting.
PMCID: PMC4055699  PMID: 24923560
8.  Faxing ECGs from peripheral hospitals to Tertiary Paediatric Cardiology Units- Is it Safe and Sustainable? 
The Ulster Medical Journal  2014;83(1):13-15.
Recent local involvement with the United Kingdom“Safe and Sustainable review of paediatric cardiology services” has highlighted the need for development of clinical networks and improvement of the communication infrastructure within and between teams.
One common communication between peripheral and tertiary hospitals is facsimile transfer of electrocardiograms. The quality of fax transmission can be variable, raising concerns regarding the quality of the received image, accuracy of the diagnosis and appropriateness of the resultant advice.
We performed a systematic quality evaluation of faxed ECGs to determine whether they should be replaced on the basis of patient safety and information governance.
A sample of 50 ECGs was selected from over 300 which had been faxed to our tertiary department. These were scored according to a structured system leading to a 10 point Likert scale, assessing technical quality and the ability to make a clinically relevant assessment of the information.
Only 1 from 50 faxed ECGs fulfilled all 9 objective criteria set. Heart rate and quadrant of the QRS axis were only identifiable in 10%. Comparing the faxed ECGs with the rating given to an original ECG confirmed a significant difference in the interpretability of faxed and original ECGs (p<0.05).
Our study suggests that faxed ECGs do not provide consistent, accurate diagnostic information. It suggests that this currently widespread practice should be considered as a potential patient safety issue within developing paediatric cardiology networks. We would recommend that faxing of ECGs be replaced with scanning of ECGs, transmitted via secure email.
PMCID: PMC3992088  PMID: 24757263
9.  Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α 
BMC Biochemistry  2014;15:3.
Endoplasmic reticulum stress, caused by the presence of misfolded proteins, activates the stress sensor inositol-requiring enzyme 1α (IRE1α). The resulting increase in IRE1α RNase activity causes sequence-specific cleavage of X-box binding protein 1 (XBP1) mRNA, resulting in upregulation of the unfolded protein response and cellular adaptation to stress. The precise mechanism of human IRE1α activation is currently unclear. The role of IRE1α kinase activity is disputed, as results from the generation of various kinase-inactivating mutations in either yeast or human cells are discordant. Kinase activity can also be made redundant by small molecules which bind the ATP binding site. We set out to uncover a role for IRE1α kinase activity using wild-type cytosolic protein constructs.
We show that concentration-dependent oligomerisation is sufficient to cause IRE1α cytosolic domain RNase activity in vitro. We demonstrate a role for the kinase activity by showing that autophosphorylation enhances RNase activity. Inclusion of the IRE1α linker domain in protein constructs allows hyperphosphorylation and further enhancement of RNase activity, highlighting the importance of kinase activity. We show that IRE1α phosphorylation status correlates with an increased propensity to form oligomeric complexes and that forced dimerisation causes great enhancement in RNase activity. In addition we demonstrate that even when IRE1α is forced to dimerise, by a GST-tag, phospho-enhancement of activity is still observed.
Taken together these experiments support the hypothesis that phosphorylation is important in modulating IRE1α RNase activity which is achieved by increasing the propensity of IRE1α to dimerise. This work supports the development of IRE1α kinase inhibitors for use in the treatment of secretory cancers.
PMCID: PMC3928614  PMID: 24524643
Endoplasmic reticulum stress; Enzyme mechanisms; ER stress; Mass spectrometry (MS); Multiple myeloma; Ribonuclease; Unfolded protein response; IRE1; Autophosphorylation
10.  A new strategy to identify potentially dangerous coronary arterial patterns before percutaneous pulmonary valve implantation 
Despite advances in surgical techniques, right ventricular outflow tract (RVOT) conduits are prone to fail over time. Percutaneous pulmonary valve implantation was introduced to expand the lifetime of these conduits and to decrease the number of open heart operations during a patient's lifetime. The procedure can be performed with excellent results; however, serious complications such as coronary arterial compression and conduit rupture have been reported. We present percutaneous treatment of a patient after Ross-Konno operation with RVOT conduit dysfunction and a potentially problematic course of the left anterior descending artery.
PMCID: PMC4252329  PMID: 25489326
percutaneous pulmonary valve implantation; coronary compression
11.  Proof of the Concept to Use a Malignant B Cell Line Drug Screen Strategy for Identification and Weight of Melphalan Resistance Genes in Multiple Myeloma 
PLoS ONE  2013;8(12):e83252.
In a conceptual study of drug resistance we have used a preclinical model of malignant B-cell lines by combining drug induced growth inhibition and gene expression profiling. In the current report a melphalan resistance profile of 19 genes were weighted by microarray data from the MRC Myeloma IX trial and time to progression following high dose melphalan, to generate an individual melphalan resistance index. The resistance index was subsequently validated in the HOVON65/GMMG-HD4 trial data set to prove the concept. Biologically, the assigned resistance indices were differentially distributed among translocations and cyclin D expression classes. Clinically, the 25% most melphalan resistant, the intermediate 50% and the 25% most sensitive patients had a median progression free survival of 18, 32 and 28 months, respectively (log-rank P-value  = 0.05). Furthermore, the median overall survival was 45 months for the resistant group and not reached for the intermediate and sensitive groups (log-rank P-value  = 0.003) following 38 months median observation. In a multivariate analysis, correcting for age, sex and ISS-staging, we found a high resistance index to be an independent variable associated with inferior progression free survival and overall survival. This study provides clinical proof of concept to use in vitro drug screen for identification of melphalan resistance gene signatures for future functional analysis.
PMCID: PMC3869769  PMID: 24376673
12.  A gene expression based predictor for myeloma patients at high risk of developing bone disease on bisphosphonate treatment 
Myeloma bone disease (BD) impairs quality of life and is associated with impaired survival. Even with effective bisphosphonate treatment a significant proportion of patients still develop skeletal-related events (SREs). Identifying such patients at presentation would allow treatment modification.
Experimental Design
In order to investigate the molecular basis of BD at presentation, and to develop a predictive signature for patients at high risk of developing SREs on bisphosphonates, 261 presenting myeloma samples were analyzed by global gene expression profiling. The derived “SRE gene signature” was complemented by the integration of associated clinical parameters to generate an optimal predictor.
Fifty genes were significantly associated with presenting BD including the WNT-signalling antagonist DKK1 and genes involved in growth factor signalling and apoptosis. Higher serum calcium (Ca) level, the presence of BD and hyperdiploidy at presentation were associated with high risk of SRE development. A gene signature derived from the fourteen genes overexpressed in the SRE group was able to identify patients at high risk of developing an SRE on treatment. These genes either belonged to the interferon-induced family or were involved in cell signalling and mitosis. Multivariate logistic model selection yielded an optimal SRE predictor comprising 7 genes and Ca level, which was validated as an effective predictor in a further set of patients.
The simple expression-based SRE predictor can effectively identify individuals at high risk of developing BD while being on bisphosphonates. This predictor could assist developing future trials on novel therapies aimed at reducing myeloma BD.
PMCID: PMC3866998  PMID: 21856767
13.  Hsp70 inhibition induces myeloma cell death via the intracellular accumulation of immunoglobulin and the generation of proteotoxic stress☆ 
Cancer Letters  2013;339(1):49-59.
Multiple myeloma (MM) cells rely on protein homeostatic mechanisms for survival. These mechanisms could be therapeutically targeted via modulation of the heat shock response. We studied the roles of Hsp72 and Hsc70, and show that the two major cytoplasmic Hsp70s play a key role in regulating protein homeostasis and controlling multiple oncogenic pathways in MM, and their inhibition can lead to myeloma cell death. Our study provides further evidence that targeting Hsp70 represents a novel therapeutic approach which may be effective in the treatment of MM.
PMCID: PMC3778988  PMID: 23887058
Haematology; Multiple myeloma; Protein folding; ER stress; Hsp70
14.  Volunteer Bias in Recruitment, Retention, and Blood Sample Donation in a Randomised Controlled Trial Involving Mothers and Their Children at Six Months and Two Years: A Longitudinal Analysis 
PLoS ONE  2013;8(7):e67912.
The vulnerability of clinical trials to volunteer bias is under-reported. Volunteer bias is systematic error due to differences between those who choose to participate in studies and those who do not.
Methods and Results
This paper extends the applications of the concept of volunteer bias by using data from a trial of probiotic supplementation for childhood atopy in healthy dyads to explore 1) differences between a) trial participants and aggregated data from publicly available databases b) participants and non-participants as the trial progressed 2) impact on trial findings of weighting data according to deprivation (Townsend) fifths in the sample and target populations. 1) a) Recruits (n = 454) were less deprived than the target population, matched for area of residence and delivery dates (n = 6,893) (mean [SD] deprivation scores 0.09[4.21] and 0.79[4.08], t = 3.44, df = 511, p<0.001). b) i)As the trial progressed, representation of the most deprived decreased. These participants and smokers were less likely to be retained at 6 months (n = 430[95%]) (OR 0.29,0.13–0.67 and 0.20,0.09–0.46), and 2 years (n = 380[84%]) (aOR 0.68,0.50–0.93 and 0.55,0.28–1.09), and consent to infant blood sample donation (n = 220[48%]) (aOR 0.72,0.57–0.92 and 0.43,0.22–0.83). ii)Mothers interested in probiotics or research or reporting infants’ adverse events or rashes were more likely to attend research clinics and consent to skin-prick testing. Mothers participating to help children were more likely to consent to infant blood sample donation. 2) In one trial outcome, atopic eczema, the intervention had a positive effect only in the over-represented, least deprived group. Here, data weighting attenuated risk reduction from 6.9%(0.9–13.1%) to 4.6%(−1.4–+10.5%), and OR from 0.40(0.18–0.91) to 0.56(0.26–1.21). Other findings were unchanged.
Potential for volunteer bias intensified during the trial, due to non-participation of the most deprived and smokers. However, these were not the only predictors of non-participation. Data weighting quantified volunteer bias and modified one important trial outcome.
Trial Registration
This randomised, double blind, parallel group, placebo controlled trial is registered with the International Standard Randomised Controlled Trials Register, Number (ISRCTN) 26287422. Registered title: Probiotics in the prevention of atopy in infants and children.
PMCID: PMC3706448  PMID: 23874465
15.  Characterization of a Novel Mouse Model of Multiple Myeloma and Its Use in Preclinical Therapeutic Assessment 
PLoS ONE  2013;8(2):e57641.
To aid preclinical development of novel therapeutics for myeloma, an in vivo model which recapitulates the human condition is required. An important feature of such a model is the interaction of myeloma cells with the bone marrow microenvironment, as this interaction modulates tumour activity and protects against drug-induced apoptosis. Therefore NOD/SCIDγcnull mice were injected intra-tibially with luciferase-tagged myeloma cells. Disease progression was monitored by weekly bioluminescent imaging (BLI) and measurement of paraprotein levels. Results were compared with magnetic resonance imaging (MRI) and histology. Assessment of model suitability for preclinical drug testing was investigated using bortezomib, melphalan and two novel agents. Cells engrafted at week 3, with a significant increase in BLI radiance occurring between weeks 5 and 7. This was accompanied by an increase in paraprotein secretion, MRI-derived tumour volume and CD138 positive cells within the bone marrow. Treatment with known anti-myeloma agents or novel agents significantly attenuated the increase in all disease markers. In addition, intra-tibial implantation of primary patient plasma cells resulted in development of myeloma within bone marrow. In conclusion, using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma by ensuring the key interaction of tumour cells with the microenvironment.
PMCID: PMC3578800  PMID: 23437401
16.  Proton NMR-Based Metabolite Analyses of Archived Serial Paired Serum and Urine Samples from Myeloma Patients at Different Stages of Disease Activity Identifies Acetylcarnitine as a Novel Marker of Active Disease 
PLoS ONE  2013;8(2):e56422.
Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an ‘ideal’ fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm.
We performed proton nuclear magnetic resonance spectroscopy-based metabolomics on blood serum and urine samples from 32 patients representative of a total cohort of 1970 multiple myeloma patients entered into the United Kingdom Medical Research Council Myeloma IX trial.
Using serial paired blood and urine samples we detected metabolite profiles that associated with diagnosis, post-treatment remission and disease progression. These studies identified carnitine and acetylcarnitine as novel potential biomarkers of active disease both at diagnosis and relapse and as a mediator of disease associated pathologies.
These findings show that samples conventionally processed and archived can provide useful metabolomic information that has important implications for understanding the biology of myeloma, discovering new therapies and identifying biomarkers potentially useful in deciding the choice and application of therapy.
PMCID: PMC3576408  PMID: 23431376
17.  Correction: A Modified Method for Whole Exome Resequencing from Minimal Amounts of Starting DNA 
PLoS ONE  2012;7(4):10.1371/annotation/5b071c93-9fa8-4be6-9cbd-95aead146305.
PMCID: PMC3321049
18.  A Modified Method for Whole Exome Resequencing from Minimal Amounts of Starting DNA 
PLoS ONE  2012;7(3):e32617.
Next generation DNA sequencing (NGS) technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap) method generate datasets that are comparable to standard amount (SA) whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes.
PMCID: PMC3293839  PMID: 22403682
19.  Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions 
Journal of Molecular Biology  2012;416-318(2-12):300-318.
The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, 15N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (Rh = 25 Å) relative to the urea-denatured state (Rh ≥ 30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.
Graphical Abstract
► An unfolded variant of Im7 has been created in nondenaturing conditions. ► The variant is collapsed but lacks fixed secondary or tertiary structure. ► Transient helicity and collapse prime the protein towards folding.
PMCID: PMC3314952  PMID: 22226836
TS1, transition state 1; TS2, transition state 2; ColE7, colicin E7; SSP, secondary structure propensity; smFRET, single-molecule Förster resonance energy transfer; Im7, immunity protein 7; EDTA, ethylenediaminetetraacetic acid; HSQC, heteronuclear single quantum coherence; AUC, analytical ultracentrifugation; ITC, isothermal titration calorimetry; BMRB, Biological Magnetic Resonance Data Bank; NOE, nuclear Overhauser enhancement; AABUF, average area buried upon folding; PDB, Protein Data Bank; protein folding; NMR; unfolded ensemble; denatured state; immunity protein
20.  Mapping local structural perturbations in the native state of stefin B (cystatin B) under amyloid forming conditions 
Unlike a number of amyloid-forming proteins, stefins, and in particular stefin B (cystatin B) form amyloids under conditions where the native state predominates. In order to trigger oligomerization processes, the stability of the protein needs to be compromised, favoring structural re-arrangement however, accelerating fibril formation is not a simple function of protein stability. We report here on how optimal conditions for amyloid formation lead to the destabilization of dimeric and tetrameric states of the protein in favor of the monomer. Small, highly localized structural changes can be mapped out that allow us to visualize directly areas of the protein which eventually become responsible for triggering amyloid formation. These regions of the protein overlap with the Cu (II)-binding sites which we identify here for the first time. We hypothesize that in vivo modulators of amyloid formation may act similarly to painstakingly optimized solvent conditions developed in vitro. We discuss these data in the light of current structural models of stefin B amyloid fibrils based on H-exchange data, where the detachment of the helical part and the extension of loops were observed.
PMCID: PMC3469841  PMID: 23091450
Cu (II)-binding; precursors of amyloid; cystatin B; stefin B; proline isomerization
21.  Targeting Bone as a Therapy for Myeloma 
Cancer Microenvironment  2011;4(3):299-311.
Myeloma bone disease (BD) not only impairs quality of life, but is also associated with impaired survival. Studies of the biology underlying BD support the notion that the increased osteoclastogenesis and suppressed osteoblastogenesis, is both a consequence and a necessity for tumour growth and clonal expansion. Survival and expansion of the myeloma clone is dependent on its interactions with bone elements, thus targeting these interactions should have antimyeloma activities. Indeed both experimental and clinical findings indicate that bone-targeted therapies not only improve BD, but also create an inhospitable environment for myeloma cell growth and survival, favouring improved clinical outcome. This review summarizes recent progress in our understandings of the biology of myeloma BD, highlighting the role of osteoclasts and osteoblasts in this process and how they can be targeted therapeutically. Unravelling the mechanisms underlying myeloma-bone interactions will facilitate the development of novel therapeutic agents to treat BD, which as a consequence are likely to improve the clinical outcome of myeloma patients.
PMCID: PMC3234321  PMID: 21833747
Multiple myeloma; Bone disease; Osteoclast; Osteoblast; Therapy
22.  Effects of zoledronic acid versus clodronic acid on skeletal morbidity in patients with newly diagnosed multiple myeloma (MRC Myeloma IX): secondary outcomes from a randomised controlled trial 
The Lancet Oncology  2011;12(8):743-752.
Bisphosphonates are the standard of care for reducing the risk of skeletal-related events in patients with bone lesions from multiple myeloma. The MRC Myeloma IX study was designed to compare the effects of zoledronic acid versus clodronic acid in newly diagnosed patients with multiple myeloma. Here, we report the secondary outcomes relating to skeletal events.
Patients (≥18 years) with newly diagnosed multiple myeloma were enrolled from 120 centres in the UK and received intensive or non-intensive antimyeloma treatment. A computer-generated randomisation sequence was used to allocate patients in a 1:1 ratio, through an automated telephone service to intravenous zoledronic acid (4 mg every 21–28 days) or oral clodronic acid (1600 mg/day), and the drugs were continued at least until disease progression. No investigators, staff, or patients were masked to treatment allocation. The primary endpoints—overall survival, progression-free survival, and overall response rate—and adverse events have been reported previously. We assessed between-group differences with Cox proportional hazards models for time to first skeletal-related event and incidence of skeletal-related events. These were defined as fractures, spinal cord compression, radiation or surgery to bone, and new osteolytic lesions. Data were analysed until disease progression. Analyses were by intention to treat. This trial is registered, number ISRCTN68454111.
1960 patients were randomly assigned and analysed—981 in the zoledronic acid group and 979 in the clodronic acid group. This trial is fully enrolled, and follow-up continues. At a median follow-up of 3·7 years (IQR 2·9–4·7), patients in the zoledronic acid group had a lower incidence of skeletal-related events than did those in the clodronic acid group (265 [27%] vs 346 [35%], respectively; hazard ratio 0·74, 95% CI 0·62–0·87; p=0·0004). Zoledronic acid was also associated with a lower risk of any skeletal-related event in the subsets of patients with (233 [35%] of 668 vs 292 [43%] of 682 with clodronic acid; 0·77, 0·65–0·92; p=0·0038) and without bone lesions at baseline (29 [10%] of 302 vs 48 [17%] of 276 with clodronic acid; 0·53, 0·33–0·84; p=0·0068). Fewer patients in the zoledronic acid group had vertebral fractures than did those in the clodronic acid group (50 [5%] in the zoledronic acid group vs 88 [9%] in the clodronic acid group; p=0·0008), other fractures (45 [5%] vs 66 [7%]; p=0·04), and new osteolytic lesions (46 [5%] vs 95 [10%]; p<0·0001).
The results of this study support the early use of zoledronic acid rather than clodronic acid in patients with newly diagnosed multiple myeloma for the prevention of skeletal-related events, irrespective of bone disease status at baseline.
Medical Research Council (London, UK), Novartis, Schering Health Care, Chugai, Pharmion, Celgene, and Ortho Biotech.
PMCID: PMC3148431  PMID: 21771568
23.  Detection of Intraepithelial and Stromal Langerin and CCR5 Positive Cells in the Human Endometrium: Potential Targets for HIV Infection 
PLoS ONE  2011;6(6):e21344.
Both the upper (endocervix and uterus) and lower (ectocervix and vagina) female genital tract mucosa are considered to be target sites for sexual transmission of HIV. There are a few reports on the T cell and antigen-presenting cell distribution in human endometrial tissue however, there is little known about the expression of the HIV co-receptor CCR5 and HIV-binding C-type lectin receptors on endometrial cell subsets. We therefore assessed endometrial tissue sections from HIV seronegative women undergoing hysterectomy of a benign and non-inflammatory cause for phenotypic characterization of potential HIV target cells and receptors by immunohistochemistry. Langerin was expressed on intraepithelial CD1a+CD4+ and CD11c+CD4+ Langerhans cells. Furthermore, CCR5+CD4+CD3+ T cells, DC-SIGN+MR+CD11c+ myeloid dendritic cells and MR+CD68+ macrophages were found within or adjacent to the epithelium of the uterine lumen. In addition, occasional CD123+ BDCA-2+ plasmacytoid dendritic cells were detected deep in the endometrial stroma. Both T cells and several antigen-presenting cells were detected in lymphoid aggregate formations in close proximity to the epithelial lining. The finding of intraepithelial and stromal Langerin+ cells as well as CCR5+ CD4+ T cells is novel for human endometrium.
PMCID: PMC3126810  PMID: 21738639
24.  Thymic function and T cell parameters in a natural human experimental model of seasonal infectious diseases and nutritional burden 
The study exploits a natural human experimental model of subsistence farmers experiencing chronic and seasonally modified food shortages and infectious burden. Two seasons existed, one of increased deprivation and infections (Jul-Dec), another of abundance and low infections (Jan-Jun); referred to as the hungry/high infection and harvest/low infection seasons respectively. Prior analysis showed a 10-fold excess in infectious disease associated mortality in young adults born in the hungry/high infection versus harvest/low infection season, and reduced thymic output and T cell counts in infancy. Here we report findings on the role of early life stressors as contributors to the onset of T cell immunological defects in later life.
We hypothesised that season of birth effects on thymic function and T cell immunity would be detectable in young adults since Kaplan-Meier survival curves indicated this to be the time of greatest mortality divergence. T cell subset analyses by flow-cytometry, sjTRECs, TCRVβ repertoire and telomere length by PCR, were performed on samples from 60 males (18-23 y) selected to represent births in the hungry/high infection and harvest/low infection
Total lymphocyte counts were normal and did not differ by birth season. CD3+ and CD4+ but not CD8+ counts were lower for those born during the hungry/high infection season. CD8+ telomere length also tended to be shorter. Overall, CD8+ TCRVβ repertoire skewing was observed with 'public' expressions and deletions seen in TCRVβ12/22 and TCRVβ24, respectively but no apparent effect of birth season.
We conclude that, although thymic function was unchanged, the CD4+ and CD3+ counts, and CD8+ telomere length results suggested that aspects of adult T cell immunity were under the influence of early life stressors. The endemicity of CMV and HBV suggested that chronic infections may modulate immunity through T cell repertoire development. The overall implications being that, this population is at an elevated risk of premature immunosenescence possibly driven by a combination of nutritional and infectious burden.
PMCID: PMC3125341  PMID: 21676219
25.  Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response 
The EMBO Journal  2011;30(5):894-905.
Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response
In the endoplasmic reticulum, unfolded proteins stimulate Ire1 autophosphorylation and RNase activity. The crystal structure of the dephosphorylated kinase/RNase domain of human Ire1 bound to ADP provides insight into the autophosphorylation reaction.
Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N-terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C-terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1α bound to ADP, revealing the ‘phosphoryl-transfer' competent dimeric face-to-face complex, which precedes and is distinct from the back-to-back RNase ‘active' conformation described for yeast Ire1. We show that the Xbp1-specific ribonuclease activity depends on autophosphorylation, and that ATP-competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1α is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1α as a target for development of ATP-competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies.
PMCID: PMC3049214  PMID: 21317875
autophosphorylation; dimerisation; inhibition; signal transduction; UPR

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