Lower-rim ferrocenyl substituted calixarenes: new electrochemical sensors for anions

New ferrocene substituted calix[4 and 5]arenes have been prepared and the crystal structure of a lower-rim substituted bis ferrocene calix[4]arene (7) has been elucidated. The respective ferrocene/ferrocenium redox-couples of compounds 6 (a calix[4]arene tetra ferrocene amide) and 8 (a calix[5]arene pentaferrocene amide) are shown to be significantly cathodically perturbed in the presence of anions by up to 160 mV in the presence of dihydrogen phosphate

Defining the clinical genomic landscape for real-world precision oncology

Through the delivery of large international projects including ICGC and TCGA, knowledge of cancer genomics is reaching saturation point. Enabling this to improve patient outcomes now requires embedding comprehensive genomic profiling into routine oncology practice. Towards this goal, this study defined the biologically and clinically relevant genomic features of adult cancer through detailed curation and analysis of large genomic datasets, accumulated literature and biomarker-driven therapeutics in clinic and development. The characteristics and prevalence of these features were then interrogated in 2348 whole genome sequences, covering 21 solid tumour types, generated by the PCAWG project. This analysis highlights the predominant contribution of copy number alterations and identifies a critical role for disruptive structural variants in the inactivation of clinically important tumour suppressor genes, including PTEN and RB1, which are not currently captured by diagnostic assays. This study defines a set of essential genomic features for the characterisation of common adult cancers

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer

First Worldwide Proficiency Study on Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Complex Strains

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.Institute for Public Health and the Environment (RIVM), European Centre for Diseases Prevention and Control (ECDC

The Society for Immunotherapy of Cancer consensus statement on immunotherapy for the treatment of prostate carcinoma.

Prostate cancer is the most commonly diagnosed malignancy and second leading cause of cancer death among men in the United States. In recent years, several new agents, including cancer immunotherapies, have been approved or are currently being investigated in late-stage clinical trials for the management of advanced prostate cancer. Therefore, the Society for Immunotherapy of Cancer (SITC) convened a multidisciplinary panel, including physicians, nurses, and patient advocates, to develop consensus recommendations for the clinical application of immunotherapy for prostate cancer patients. To do so, a systematic literature search was performed to identify high-impact papers from 2006 until 2014 and was further supplemented with literature provided by the panel. Results from the consensus panel voting and discussion as well as the literature review were used to rate supporting evidence and generate recommendations for the use of immunotherapy in prostate cancer patients. Sipuleucel-T, an autologous dendritic cell vaccine, is the first and currently only immunotherapeutic agent approved for the clinical management of metastatic castrate resistant prostate cancer (mCRPC). The consensus panel utilized this model to discuss immunotherapy in the treatment of prostate cancer, issues related to patient selection, monitoring of patients during and post treatment, and sequence/combination with other anti-cancer treatments. Potential immunotherapies emerging from late-stage clinical trials are also discussed. As immunotherapy evolves as a therapeutic option for the treatment of prostate cancer, these recommendations will be updated accordingly

The frequency of detection of unexpected diabetes mellitus during haemoglobinopathy investigations

ABSTRACT Aims To establish the frequency of detection of previously undiagnosed diabetes mellitus as a result of detection of an increased glycated fraction of haemoglobin during high performance liquid chromatography (HPLC) for haemoglobinopathy diagnosis. Methods A prospective study was carried out over a 3-month period. During that period a total of 2094 patient samples were received for haemoglobinopathy investigation and were included in the study. Results Fifty samples were found to have an apparent increase in the glycated haemoglobin fraction and of these 38 were found to be from patients with known diabetes. Previously undiagnosed diabetes was discovered in 11 patients and it is likely that the twelfth patient also had diabetes. Conclusions The detection of evidence of undiagnosed diabetes during HPLC haemoglobinopathy investigations is not rare, there being four cases per month in this study. This incidental observation should be reported to clinical staff. A proportion of haemoglobin A undergoes posttranslational modification including glycosylation (non-enzymatic addition of glucose to the aminoterminal valine of the b chain) or glycation (less specifically, addition of carbohydrate to a protein). 1 Glycosylation occurs throughout the life-span of the red cell, at a rate determined by the ambient glucose concentration. Glycated haemoglobin is known as haemoglobin A 1 , of which 60e80% is glycosylated haemoglobin, haemoglobin A 1c . 1 In patients with diabetes mellitus, the proportion of haemoglobin that is haemoglobin A 1c has been found to be useful as an indication of the degree of hyperglycaemia during the preceding 3 months. In addition, since there are few other causes of an elevated haemoglobin A 1c , the finding of an elevated proportion can be useful in diagnosis. Glycated haemoglobin includes a labile fraction, which responds rapidly and transiently to raised blood glucose levels, and a stable fraction, to which the labile fraction is converted. 1 It is the stable fraction that is useful in judging long-term control of diabetes. Haemoglobin A 1c can be quantified by a variety of methods, of which high-performance liquid chromatography (HPLC) is increasingly used. When suspected haemoglobinopathies are investigated by cellulose acetate electrophoresis, the glycated fraction is not resolved and no specific abnormality is apparent in diabetic patients. However, when the technique used for such investigations is HPLC, the presence of a glycated fraction may be apparent. Our laboratory has previously drawn attention to the possibility that this may lead to the diagnosis of previously unsuspected diabetes mellitus. 2 Early diagnosis and good control of diabetes mellitus is important in reducing the end-organ damage that is characteristic of this disease, and it is therefore important for haematologists to alert clinical staff to the probability of this diagnosis when an increased proportion of glycated haemoglobin is observed. We therefore carried out a study to determine the frequency with which this is observed, and as a result of this study we developed a policy for notification of an increased glycated fraction to clinical staff. MATERIALS AND METHODS All tests were performed on peripheral blood samples anticoagulated with EDTA. HPLC for establishing relevant reference ranges and for haemoglobinopathy investigations was performed on a Bio-Rad Variant II instrument (Bio-Rad Laboratories, Hemel Hempstead, UK) using the b-Thalassaemia Short Program. Haemoglobin A 1c was quantified by HPLC using a Tosoh A1c 2.2 instrument. One hundred and two samples were obtained from fully informed young healthy volunteers of north European origin, in order to establish a reference range for haemoglobin A 2 . Data from the same samples were used to establish a reference range for peak 2 (P2), this being the peak with a retention time slightly longer than that of haemoglobin F, which we had previously noted to be increased in patients with an increased proportion of haemoglobin A 1c . The instruction manual of the Variant II states 'Diabetic specimens typically exhibit an elevated P2 peak'. In order to investigate the relationship between P2 on the Bio-Rad Variant II and haemoglobin A 1c on the Tosoh A1c 2.2, 93 samples from either healthy volunteers or patients with an elevated glycated fraction were studied in parallel on the two instruments. Results of all patients investigated by our haemoglobinopathy laboratory were surveyed over a 3-month period and when a sample was found to have a P2 fraction of 6% or greater, haemoglobin A 1c was measured and we investigated whether or not a diagnosis of diabetes mellitus had previously been established. If a review of request forms and laboratory records did not disclose this diagnosis, we contacted relevant clinical staff and requested review of clinical notes. If the patient had not been identified previously as suffering from diabetes mellitus, further tests were advised in order to confirm the diagnosis. Subsequently we followed-up the results of these confirmatory tests. Tests done were those usually carried out by the relevant clinical staff in order to confirm a diagnosis of diabetes

Application of the LymphGen classification tool to 928 clinically and genetically-characterised cases of diffuse large B cell lymphoma (DLBCL).

We recently published results of targeted sequencing applied to 928 unselected cases of DLBCL registered in the Haematological Malignancy Research Network (HMRN) registry (1). Clustering allowed us to resolve five genomic subtypes. These subtypes shared considerable overlap with those proposed in two independent genomic studies(2, 3), suggesting the potential to use genetics to stratify patients by both risk and biology. In the original studies, clustering techniques were applied to sample cohorts to reveal molecular substructure, but left open the challenge of how to classify an individual patient. This was addressed by the LymphGen classification tool (4). LymphGen assigns an individual case to one of six molecular subtypes. The tool accommodates data from exome or targeted sequencing, either with or without copy number variant (CNV) data. Separate gene expression data allows classification of a seventh, MYC-driven subtype defined by a double hit (DHL) or molecular high-grade (MHG) gene expression signature(5-7).HR was funded by a studentship from the Medical Research Council. DH was supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1). The Hodson laboratory receives core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute and core funding from the CRUK Cambridge Cancer Centre. HMRN is supported by BCUK 15037 and CRUK 18362