Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the Gs-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

The future of academic haematology.

Recent advances in the basic medical sciences, particularly cell biology and genomics, have great promise for the future development of all aspects of haematological practice. They will also impinge on the hitherto neglected fields of haematology, including haematology involving the care of the rapidly increasing number of elderly patients and the complex problems of haematological practice in the developing countries. To obtain the maximum benefit from these new developments it will be necessary to review the patterns of training of haematologists of the future at every level. In short, it will be important to try to design and develop various career pathways for training haematologists including those who wish to work full time in basic research, combine research with clinical practice, or commit all their time to clinical work and teaching.

The worm has turned: unexpected similarities between the transcription of enhancers and promoters in the worm and mammalian genomes.

Our understanding of biological processes in humans is often based on examination of analogous processes in other organisms. The nematode worm Caenorhabditis elegans has been a particularly valuable model, leading to Nobel prize winning discoveries in development and genetics. Until recently, however, the worm has not been widely used as a model to study transcription due to the lack of a comprehensive catalogue of its RNA transcripts. A recent study by Chen et al. uses next-generation sequencing to address this issue, mapping the transcription initiation sites in C. elegans and finding many unexpected similarities between the transcription of enhancers and promoters in the worm and mammalian genomes. As well as providing a valuable resource for researchers in the C. elegans community, these findings raise the possibility of using the worm as a model to investigate some key, current questions about transcriptional regulation that remain technically challenging in more complex organisms.

Identification of a pathway by which glucose regulates ß-catenin signalling via the cAMP/protein kinase A pathway in ß-cell models.

Pancreatic ß-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the ß-catenin signalling pathway plays an important role in regulating ß-cell function, but the mechanisms regulating ß-catenin signalling in these cells is not well understood. In the present study we show that ß-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and ß-TC6-F7 ß-cell models. We found a glucose-dependent increase in levels of ß-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of ß-catenin. This was associated with an increase in phosphorylation of ß-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on ß-catenin signalling. Finally, reducing ß-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate ß-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in ß-cells.

The evaluation of anaemia in an older primary care population: retrospective population-based study.

BACKGROUND: Anaemia is common in older people and the identification of potentially reversible haematinic deficiencies relies on appropriate investigation, often undertaken in primary care. AIM: To determine the laboratory prevalence of anaemia, the types of anaemia observed, and the biochemical and haematological investigations undertaken to characterise any associated haematinic abnormality in older primary care patients. DESIGN & SETTING: A retrospective primary care based study of patients aged >65 years undergoing a full blood count in Oxfordshire, UK between 1 January 2012 and 31 December 2013. METHOD: Consecutive patients aged >65 years with a full blood count were identified retrospectively from a laboratory database. Patient demographics, number of blood tests and additional laboratory investigations requested were recorded. World Health Organisation (WHO) criteria were used to define anaemia. RESULTS: In total 151 473 full blood counts from 53 890 participants were included: 29.6% of patients were anaemic. The majority had a normocytic anaemia (82.4%) and 46.0% of participants with anaemia had no additional investigations performed. The mean haemoglobin was lower in the anaemic group that underwent further investigation than those who did not (Hb 10.68 g/dl versus 11.24 g/dl, P<0.05): 33.2 % of patients with a microcytic anaemia (mean cell volume <80) did not have any markers of iron status measured. CONCLUSION: A large proportion of older adults in primary care with a recent blood test are anaemic, the majority with a normocytic anaemia, with evidence of inadequate investigation. Those with lower haemoglobin are more likely to be further investigated. Further work is needed to understand the approach to anaemia in older adults in primary care.

Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for ß-thalassemia.

ß-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and ß-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with ß-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with ß-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for ß-thalassemia.ß-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.

Genetic dissection of the α-globin super-enhancer in vivo.

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

Glicentin-related pancreatic polypeptide inhibits glucose-stimulated insulin secretion from the isolated pancreas of adult male rats.

Peptides derived from the glucagon gene Gcg, for example, glucagon and glucagon-like peptide 1 (GLP-1), act as physiological regulators of fuel metabolism and are thus of major interest in the pathogenesis of diseases, such as type-2 diabetes and obesity, and their therapeutic management. Glicentin-related pancreatic polypeptide (GRPP) is a further, 30 amino acid Gcg-derived peptide identified in human, mouse, rat, and pig. However, the potential glucoregulatory function of this peptide is largely unknown. Here, we synthesized rat GRPP (rGRPP) and a closely related peptide, rat GRPP-like peptide (rGRPP-LP), and investigated their actions in the liver and pancreas of adult male rats by employing isolated-perfused organ preparations. Rat GRPP and rGRPP-LP did not affect glucose output from the liver, but both elicited potent inhibition of glucose-stimulated insulin secretion (GSIS) from the rat pancreas. This action is unlikely to be mediated by glucagon or GLP-1 receptors, as rGRPP and rGRPP-LP did not stimulate cyclic adenosine monophosphate (cAMP) production from the glucagon or GLP-1 receptors, nor did they antagonize glucagon- or GLP-1-stimulated cAMP-production at either receptor. GRPP and GRPP-LP may be novel regulators of insulin secretion, acting through an as-yet undefined receptor.

Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat endothelium.

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.

Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment.

Gene expression during development and differentiation is regulated in a cell- and stage-specific manner by complex networks of intergenic and intragenic cis-regulatory elements whose numbers and representation in the genome far exceed those of structural genes. Using chromosome conformation capture, it is now possible to analyze in detail the interaction between enhancers, silencers, boundary elements and promoters at individual loci, but these techniques are not readily scalable. Here we present a high-throughput approach (Capture-C) to analyze cis interactions, interrogating hundreds of specific interactions at high resolution in a single experiment. We show how this approach will facilitate detailed, genome-wide analysis to elucidate the general principles by which cis-acting sequences control gene expression. In addition, we show how Capture-C will expedite identification of the target genes and functional effects of SNPs that are associated with complex diseases, which most frequently lie in intergenic cis-acting regulatory elements.

MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival.

BACKGROUND: MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM) is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0) of purified tumor (CD138+) cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS) and 9 controls. RESULTS: Unsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129) were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40%) suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59%) of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14) and t(11;14)) and del(13q). Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis) and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS) in MM, ten of which were significant by univariate (logrank) survival analysis. CONCLUSIONS: In summary, this work has identified aberrantly expressed microRNAs associated with the diagnosis, pathogenesis and prognosis of MM, data which will prove an invaluable resource for understanding the role of microRNAs in this devastating disease.

Paediatric tuberculosis in a Pacific Islands community in New Zealand.

OBJECTIVES: To describe the demographic, clinical and management aspects of an outbreak of tuberculosis (TB) within a paediatric Pacific Island community in Auckland, New Zealand, in 2002-2003. METHODS: The index and source case are described along with details of the extensive contact tracing that was undertaken in this community. RESULTS: A total of 24 children were diagnosed with TB over an 11-month period. All cases were found to be epidemiologically linked to the one source case, mother of the index case. This total included 22 children with pulmonary disease, 1 case of miliary disease (index case) and 1 of cervical adenitis. Only 58% had symptoms at diagnosis and only 5 presented to medical attention with symptoms, the remainder had symptoms disclosed after contact tracing occurred. The Mycobacterium tuberculosis isolate was fully sensitive and all children (excluding the child with miliary disease) received short course directly observed therapy. One child developed hepatotoxicity requiring modification of his drug regimen. CONCLUSIONS: Children are at high risk of developing active disease after exposure to TB. The study describes the minimal symptoms manifested in many of the children with significant radiological changes consistent with pulmonary TB. This highlights the need to consider Mantoux testing and chest X-rays for children presenting with persistent respiratory symptoms in high-risk populations. Issues of contact tracing and adherence were also a problem in this population.