Down syndrome is the most common genetic developmental disorder in humans and is caused by partial or complete triplication of human chromosome 21 (trisomy 21). It is a complex condition which results in multiple lifelong health problems, including varying degrees of intellectual disability and delays in speech, memory, and learning. As both length and quality of life are improving for individuals with Down syndrome, attention is now being directed to understanding and potentially treating the associated cognitive difficulties and their underlying biological substrates. These have included imaging and postmortem studies which have identified decreased regional brain volumes and histological anomalies that accompany early onset dementia. In addition, advances in genome‐wide analysis and Down syndrome mouse models are providing valuable insight into potential targets for intervention that could improve neurogenesis and long‐term cognition. As little is known about early brain development in human Down syndrome, we review recent advances in magnetic resonance imaging that allow non‐invasive visualization of brain macro‐ and microstructure, even in utero. It is hoped that together these advances may enable Down syndrome to become one of the first genetic disorders to be targeted by antenatal treatments designed to ‘normalize’ brain development.Magnetic resonance imaging can provide non‐invasive characterization of early brain development in Down syndrome.Down syndrome mouse models enable study of underlying pathology and potential intervention strategies.Potential therapies could modify brain structure and improve early cognitive levels.Down syndrome may be the first genetic disorder to have targeted therapies which alter antenatal brain development. Magnetic resonance imaging can provide non‐invasive characterization of early brain development in Down syndrome.Down syndrome mouse models enable study of underlying pathology and potential intervention strategies.Potential therapies could modify brain structure and improve early cognitive levels.Down syndrome may be the first genetic disorder to have targeted therapies which alter antenatal brain development.This article's abstract has been translated into Spanish and Portuguese.abstract to view the translations.Follow the links from the https://www.youtube.com/watch?v=x4x4iaP7ozk&feature=youtu.beVideo Podcast:  CHDCongenital heart defectsHsa21Human chromosome 21MmuMouse chromosomeDown syndrome is caused by partial or complete triplication of human chromosome 21  and is the most common genetic developmental disorder in humans. It is a complex condition which results in multiple lifelong health problems, including varying degrees of intellectual disability and delays in speech, memory, and learning. Worldwide, Down syndrome affects 1 in 1000 to 1100 live births annually. Whilst there have been significant improvements in non‐invasive prenatal screening,As Down syndrome is a multigene, multisystem disorder, accurately predicting neurocognitive abilities through the lifespan and understanding the high degree of variability across functional phenotypes remains a significant challenge. As a result, most clinical research in Down syndrome has been focused on understanding the pathogenesis of early onset dementia in adults, and clinical trials with pharmacological agents have been focused on improving cognition and delaying the development of dementia in adolescents and adults. Here, neuroimaging has become an increasingly useful modality to understand the progression of the underlying brain abnormalities and monitor the effects of potential therapeutic intervention. In contrast, published brain phenotypes during the fetal and neonatal period have been limited to only a handful of small postmortem case series. Therefore, whilst such studies have provided vital information about how early brain development is altered in Down syndrome, by nature, they cannot inform about the natural history of the abnormalities and crucially do not allow correlation of the identified brain phenotypes with subsequent outcome. In this review we describe how recent advances in developmental animal models of Down syndrome and non‐invasive imaging methods can fill this gap in knowledge by enabling the first in vivo studies in early human life.all levels of assessment: from gene expression, cellular responses, and subsequent brain development, to cognitive, motor, and behavioural phenotypes.Hsa21 contains 222 protein coding genes, and 325 non‐protein encoding genes.Variable but atypical behavioural and cognitive functioning emerges throughout the lifespan in Down syndrome. IQ ranges from mild to severe disability ,In comparison to typically developing controls, infants with Down syndrome may have only mild delays in learning and cognition during early infancy. Delayed or impaired cognitive and behavioural function then becomes more prominent from 2 years of age, with the rate of intellectual development slowing with increasing age.It has been widely reported that children and adults with Down syndrome have smaller whole brain volumes, and a smoother, simplified gyral appearance.Despite their small number of cases, postmortem studies have provided important information about the range and high variability of the neuropathological features evident in Down syndrome across the life‐span. These studies suggest that the known reductions in brain size (2D and 3D measures) and weight emerge during the fetal and newborn period.DYRK1A, APP, S100β, and OLIG2, all located on Hsa21.After birth (and described up to 14y) there is a profound decrease in neuronal number 20–50%) and altered morphology of dendritic spines across the cortical layers.% and altAdvances in genome‐wide analysis and the development of animal models have provided valuable insight into understanding gene dosage imbalances in disorders such as Down syndrome.a/A‐Ts[1716]65Dn/J) has historically been very important in the study of Down syndrome as it is trisomic for 90 protein coding genes on Mmu16 (approximately 55% of orthologous genes to Hsa21). However, Ts65Dn mice contain an extra copy of 60 genes (35 protein coding) located on Mmu17 (orthologous to Hsa6) that are not triplicated in people with Down syndrome and the resultant Ts65Dn phenotypes may be more severe than those seen in the human condition or possess spurious phenotypes not relevant to Down syndrome.The Ts65Dn mouse (B6EiC3Sn The Tc1 (B6129S‐Tc[HSA21]1TybEmcf/J) transchromosomic (trans‐species aneuploidy) mouse line contains a freely segregated copy of Hsa21. Although some chromosomal rearrangement and deletions have been identified in the construction process, it has allowed exploration of the relationship between specific Hsa21 genes (including those not found in the mouse) and phenotype.Studies in mouse models have primarily focused on understanding the pathology of the adult and ageing Down syndrome brain, despite knowledge that alterations in brain development are observed from fetal life. Comparison with human development can be challenging, as mice are postnatal brain developers with a gestational length of 19 to 21 days. The bulk of cortical neurogenesis occurs during the mouse embryonic period , but is ongoing into postnatal life in the hippocampus and cerebellum. The rate of cellular migration and maturation differ regionally, but around the time of (rodent) birth, postnatal day 1 to postnatal day 3, neural development is generally considered to be comparable to a preterm human infant of 23 to 32 weeks postmenstrual age. The brain growth spurt of rodents occurs at postnatal day 7 to postnatal day 10, which is comparable to a term human infant of 36 to 40 weeks postmenstrual age.Studies are being conducted in mouse models of Down syndrome to target dosage sensitive genes that are involved in defects and delays in neurogenesis and neurotransmission, oxidative stress, and neurodegeneration  has recently also been shown to be well tolerated in a Phase II clinical trial in young adults with Down syndrome without dementia.Current clinical trials in adolescents and adults with Down syndrome are aimed at improving cognition and delaying progression into Alzheimer disease. Two groups of common medications used to treat the symptoms of Alzheimer disease, acetylcholinesterase inhibitors  and N‐methyl‐D‐aspartate receptor antagonists (Memantine), are currently undergoing clinical trials in patients with Down syndrome.Growing evidence suggests that alterations in key cellular processes result in permanent modifications in structure from a very early stage in brain development. It is therefore possible that an early life therapeutic window exists, during which atypical brain development could be potentially modified before the abnormalities and neurocognitive impairment are fully established. In current clinical practice, commonly used early interventions include physiotherapy, occupational therapy, and speech and language therapy which may help to improve the acquisition of developmental milestones in infants and children with Down syndrome in the absence of any known effective pharmacological intervention. Here it is important to consider that the identification of novel candidate agents is difficult, given the absence of detailed understanding of the very early neurobiological trajectory in Down syndrome.There is a lack of understanding about when deviations in brain development arise in Down syndrome, how these relate to subsequent function, and whether they are further altered by additional congenital morbidities (e.g. cardiac defects). Such information is best monitored by in vivo studies that provide opportunities to follow development longitudinally.CHD (without Down syndrome) are generally associated with impaired clinical neurodevelopment and an underlying reduction in cortical grey matter volumes, gyrification index , and abnormal cortical microstructure in the neonatal period. These changes were further associated with reduced cerebral oxygen delivery.Two studies in both human and mouse models of Down syndrome have utilized transcriptomic analysis to characterize the specific gene networks and associated biological processes which are altered during prenatal and postnatal development.Whilst the aforementioned postmortem studies and animal models have provided significant insights into the neuropathology of Down syndrome, a true understanding of the natural history of the human condition and how the pathology relates to neurodevelopmental outcome is only possible through in vivo studies. Here, there is great potential for an enhanced understanding of in utero and neonatal brain development in Down syndrome through the application of recent advances in non‐invasive imaging. Although ultrasound provides valuable insights into gross fetal body and brain development, it cannot provide detailed information about region and tissue specific brain development and growth trajectories.Therefore magnetic resonance imaging (MRI) is an attractive alternative which is safe, does not use ionizing radiation, and can provide more extensive, detailed biometric data across gestation including information about both brain macro‐ and microstructure.Fetal MRI is challenging because of fetal motion, size, and position . However, in comparison to ultrasound, it offers excellent soft tissue contrast and benefits from a wide field of view which allows the whole fetus to be imaged up until term gestation. To combat the effects of fetal and maternal motion, significant advances have been made in acquisition and processing protocols  which can now provide high resolution and high signal to noise volumetric image data sets Fig. .87, 88, http://www.developingconnectome.org/) for non‐sedated sleeping infants, consisting of a neonatal head sized 32‐channel receive array coil and positioning system which significantly improves signal to noise ratio, an MRI safe trolley to minimize disturbance of the sleeping infant, additional ear protection, and a change in the start of magnetic resonance sequences to reduce the abrupt noise at the start of an acquisition sequence that may wake the infant.Whilst imaging a newborn or young infant also presents technical and practical difficulties, there are now magnetic resonance compatible incubators and population‐specific processing pipelines to overcome these. Examples include a neonatal brain imaging system developed for the developing human connectome project , data can also be collected and analysed when an individual is at rest . The latter can be used to identify spatial patterns of temporal correlation of intrinsic signal fluctuations . Altered patterns of functional connectivity are seen in neuropsychiatric conditions and therefore may provide a suitable biomarker for abnormal brain function and predicting later adverse neurodevelopment in Down syndrome.1H) (and less commonly other nuclei with an odd mass number [sodium 23Na or phosphorus 31P]) from a voxel placed on a specific region of interest. Proton magnetic resonance spectroscopy is most commonly used in human brain studies because of the abundance of hydrogen in human tissue. The resultant spectra demonstrate metabolite peaks at a specific frequency (parts per million). Specific brain metabolites that can be quantified include myo‐inositol , choline (cell membrane), creatine (energy metabolism), N‐acetyl aspartate , and lactate (anaerobic glycolysis).Magnetic resonance spectroscopy can non‐invasively quantify biochemical composition by sampling the resonant signal generated by hydrogen protons (Although current cognitive interventions target children and adults with Down syndrome, evidence suggests that deviations in brain development begin early in fetal life. However, to understand how to potentially intervene at this earlier time point, we need far greater knowledge about how the Down syndrome brain grows and develops, what causes the variability of neurodevelopmental outcomes, and the genotypic/phenotypic relationship that occurs in Down syndrome.Significant advances in fetal and neonatal MRI sequence acquisition, motion correction techniques, and analysis methods now allow detailed characterization of the spectrum of early imaging phenotypes.In addition, histological studies of human Down syndrome tissue at equivalent gestational ages can be used to determine the underlying neurobiological substrate for imaging phenotypes identified in the early developing brain in Down syndrome Fig. . This coThe combination of preclinical animal, human postmortem, and in vivo imaging methods can therefore provide comprehensive and vital new insights into aberrant brain development in Down syndrome. This also has the potential to provide non‐invasive imaging based surrogate markers to predict later neurodevelopmental outcome. In the future, this novel early human imaging data can also be used in clinical trials as biomarkers to monitor the effectiveness of new therapies intervening during antenatal or neonatal time‐points.


Chow et al., Chem. Sci., 2015, 6, 4148–4159.Correction for ‘Assessing the exchange coupling in binuclear lanthanide( A grant from the European Community's Seventh Framework Programme was inadvertently omitted in the Acknowledgements of this manuscript. The following information should be added to the Acknowledgements section:“The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement number 611488.”The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The nucleation mechanism of crystals of small organic molecules, postulated based on computer simulations, still lacks experimental evidence. In this study we designed an experimental approach to monitor the early stages of the crystallization of ibuprofen as a model system for small organic molecules. Ibuprofen undergoes liquid–liquid phase separation prior to nucleation. The binodal and spinodal limits of the corresponding liquid–liquid miscibility gap were analyzed and confirmed. An increase in viscosity sustains the kinetic stability of the dense liquid intermediate. Since the distances between ibuprofen molecules within the dense liquid phase are similar to those in the crystal forms, this dense liquid phase is identified as a precursor phase in the nucleation of ibuprofen, in which densification is followed by generation of structural order. This discovery may make it possible to enrich poorly soluble pharmaceuticals beyond classical solubility limitations in aqueous environments. The nucleation mechanism of crystals of small organic molecules was studied for the crystallization of ibuprofen as a model system. Ibuprofen undergoes liquid–liquid phase separation prior to nucleation and forms a dense liquid phase that is identified as a precursor phase in the nucleation of ibuprofen. These results may provide insight for the development of new pharmaceutical formulations. Crystallization, a natural phenomenon observed in everyday life, is crucial to many processes occurring in nature and in the chemical, pharmaceutical, and food industries. The majority of agrochemical and pharmaceutical products undergo many crystallization steps during their development and manufacture, where corresponding processes serve as versatile techniques in separation, purification, and product design.Nevertheless, the mechanistic understanding of phase separation and the formation of solid particles or liquid intermediates of the final crystalline systems is rather limited, especially for small organic molecules. Due to the analytical simplicity of classical nucleation theory (CNT), researchers have applied it extensively to solution crystallization. CNT considers that clusters of critical size form stochastically in supersaturated solutions due to the reversible addition of single molecules to unstable precritical clusters. Once they reach the critical size, their growth is thermodynamically favorable and as a result of this, crystal growth proceeds up to the final crystal.3 involves the formation of prenucleation clusters in solution, decrease in their dynamics and densification as the key step for phase separation, formation of a liquid phase, solidification, and finally crystallization.On the other hand, mechanisms of nonclassical nucleation were explored in simulations by ten Wolde and Frenkel.However, for small organic molecules the experimental evidence for metastable liquid phases within a two‐step nucleation or a prenucleation cluster pathway is rather poor. Supersaturated glycine solutions underwent laser‐induced nucleation at rates much faster than those of control solutions.S‐enantiomer.S‐enantiomer is employed.The poorly water‐soluble compound ibuprofen is a prominent model system and one of the most frequently prescribed essential analgesics in the world according to WHO.1H NMR spectroscopy, we located the binodal as well as the spinodal limits of the corresponding liquid–liquid miscibility gap at room temperature. Furthermore, we measured the interproton distances in the dense liquid phase by 2D 1H‐1H NOESY NMR experiments and compared them with interproton distances in the final crystal. This reveals that the liquid intermediates are dynamic species with intermolecular distances similar to those in crystals, where the concentration of the poorly soluble compound is significantly increased.Here, we aim to elucidate the underlying nucleation pathway of ibuprofen with emphasis on a liquid precursor phase in aqueous solution. We monitored the early stages of nucleation with a potentiometric titration assay taking advantage of the protolysis equilibrium of ibuprofen in combination with turbidimetry. Furthermore, we characterized the physicochemical properties of this precursor phase such as its molecular dynamics and viscosity. By quantifying the amount of ibuprofen bound within the dense liquid phase via This inherent property might provide a new means to improve the bioavailability of poorly water‐soluble compounds when liquid intermediates can be stabilized in pharmaceutical formulations.The supersaturation of protonated ibuprofen (IbuH) was increased by adding dilute hydrochloric acid solution to an ibuprofen sodium solution and decreasing the pH Scheme . Another2O and 1H NMR spectra were recorded subsequently. While signals of the ibuprofen molecule were present in samples taken before phase separation, additional peaks were present in those samples, in which liquid–liquid phase separation had already occurred. Interestingly, all of the recorded signals of ibuprofen exhibit secondary signals due to the separated phase, shifted by about 0.3 ppm  to about 0.5 ppm (for aromatic protons) /Vtotal) increases with a concurrent decrease of the amount of ibuprofen in the mother phase (n(IbuH)L1/Vtotal), whereby the total amount of ibuprofen remains constant; thus, the amount in both phases correlates linearly. Extrapolation of the corresponding linear fit yields the lowest concentration at which liquid–liquid phase separation can occur.For the determination of the locus of the liquid–liquid binodal limit, samples were drawn from titrations performed with the double‐dosing method in 5 vol. % Dy‐offset of the linear fit corresponds to the binodal limit of liquid–liquid miscibility gap. On the other hand, a saturation threshold can be identified at much higher supersaturations. This implies that the composition of the dense liquid phase does not change upon further increase in the concentration of IbuH, and thus the saturation threshold corresponds to the locus of the liquid–liquid spinodal limit.Hence, the S‐enantiomer of ibuprofen was employed since it exhibits a higher solubility than racemic ibuprofen.1H PFG‐STE diffusion NMR was employed as a non‐invasive, highly precise technique for the determination of diffusion coefficients.D for ibuprofen in the mother phase for several different samples drawn in the binodal regime  decreases with an increase in the amount of ibuprofen in the dense liquid phase. While the ratio of diffusion coefficients (D/D*) is 34 upon crossing the binodal limit, it increases to a ratio of 239, advancing further into the binodal regime (Table T1 and T2 relaxation times associated with the peaks (a) and (a*) in the one‐dimensional 1H NMR spectrum . According to Carper et al.τc , which may explain why the liquid phase is kinetically stabilized for a significant period of time before it crystallizes. Viscosity was proposed to play a key role in the formation of the ordered nucleus within a dense liquid intermediate by Vekilov et al.Consequently, rotational and translational motions of the molecules in the dense liquid phase do not sense the same viscosity since both parameters scale linearly with viscosity according to the Stokes–Einstein and Stokes–Debye equation, respectively. In other words, translational and rotational diffusion cannot be correlated with each other in this system. Similar observations were made by Pielak et al.1H‐1H NMR NOESY methodology (nuclear Overhauser effect spectroscopy) was employed. Solely the protons (a), (b), (c) are relevant here, because they cannot come closer due to the rigid aromatic ring structure and hence, they have a intermolecular distance higher than 5 Å in any conformation. Owing to the general limitations of NOESY methodology, the maximum separation that leads to cross peaks is about 5 Å.In order to gain quantitative information about inter‐ vs. intramolecular distances of ibuprofen molecules in the dense liquid phase, two‐dimensional homonuclear 1H NMR NOESY spectrum  and those of the isopropyl group  for the ibuprofen molecules present in the mother phase –(b*) and (a*)–(c*) in the dense liquid phase, although the intramolecular distance remains about 7 Å. Hence, this NOE signal necessarily arises from an intermolecular NOE with protons of other neighboring ibuprofen molecules closer than 5 Å. In order to obtain a more precise value for the intermolecular proton distance, the NOE signals can be used to determine interproton distances with high accuracy.g to Eq. :(1)dH-H=dH‐H is the intermolecular distance of two protons, IH‐H is the corresponding signal intensity in the 2D 1H‐1H NOESY spectrum, dref is the intramolecular distance in the reference molecule, and Iref is the corresponding signal intensity in the 1H NOESY spectrum. In the case of ibuprofen in the dense liquid phase, the fixed distance between protons (a) and (c) (2.456 Å) within the ibuprofen molecule is chosen as an intramolecular reference (dref) with the signal intensity of the (a*)–(c*) NOE signal (Iref). The results of this NOE interproton distance calculation are summarized in Table where S‐IbuH and the racemic RS‐IbuH –(c*) NOE signal there are also contributions of the (a*)–(c*) intermolecular NOE signals. But as the NOE signal intensity scales with m) and spinodal limits (0.71±0.01 mm) of the corresponding liquid–liquid miscibility gap were determined by means of 1H NMR spectroscopy due to the different chemical environment of the ibuprofen molecules in the two phases. The evaluation of molecular dynamics suggests that both translational and rotational diffusion of ibuprofen in the dense liquid phase are strongly hindered compared to the ibuprofen molecules in the mother phase. This shows that the ibuprofen molecules in the dense liquid phase, which is more concentrated than the mother phase, are in a state that is kinetically stabilized. Since rotational and translational diffusion processes are slowed down significantly, it affects the crystalline structure generation within the dense liquid phase and accounts for its metastability towards nucleation. The intermolecular distances in the dense liquid phase were found to be similar to those in the final crystal according to the measured interproton NOE signals. Consequently, the dense liquid phase can be regarded as a densified precursor phase for the nucleation of a crystalline phase. In the case of ibuprofen, these results show that densification is followed by structure generation and therefore implies a nonclassical nucleation pathway where the dense liquid phase is kinetically stabilized by the slow rotational and translational diffusion.In summary, nucleation of ibuprofen in aqueous solution proceeds via liquid–liquid phase separation as a precursor to crystals. The loci of the binodal  should be addressed to the authors.SupplementaryClick here for additional data file.


To determine the longitudinal construct validity of assessing hand OA progression on digital photographs over 7 years compared with progression determined from radiographs, clinical features and change in symptoms.Participants were community-dwelling older adults (≥50 years) in North Staffordshire, UK. Standardized digital hand photographs were taken at baseline and 7 years, and hand joints graded for OA severity using an established photographic atlas. Radiographic hand OA was assessed using the Kellgren and Lawrence grading system. Hand examination determined the presence of nodes, bony enlargement and deformity. Symptoms were reported in self-complete questionnaires. Radiographic and clinical progression and change in symptoms were compared to photographic progression. Differences were examined using analysis of covariance and Chi-Square tests.Of 253 individuals  the proportion with photographic progression at the joint and joint group-level was higher in individuals with radiographic or clinical progression compared to those without, although differences were not statistically significant. At the person-level, those with moderate photographic progression over 7 years had significantly higher summed radiographic and clinical scores (adjusted for baseline scores) compared to those with no or mild photographic progression. Similar findings were observed for change in symptoms, although differences were small and not statistically significant.Assessing hand OA on photographs shows modest longitudinal construct validity over 7 years compared with change in radiographic and clinical hand OA at the person-level. Using photographs to assess overall long-term change in a person with hand OA may be a reasonable alternative when hand examinations and radiographs are not feasible. Despite the high prevalence of hand osteoarthritis (OA) and the significant impact it has on pain experienced and interference with hand function little is still known about the course of the condition over time , 2.Photographs of the hands have been used in the assessment of OA in a number of studies –8 in an Photographic assessment of hand OA, which does not require repeated radiation exposure, reduces research clinic time and costs, and decreases inter-observer variation through having a single central reader, would be extremely useful if validated. It will allow longitudinal epidemiological studies to be undertaken in large samples, over wide and remote geographic areas, and also could be of benefit for clinical monitoring of the condition especially in the provision of remote health care using telemedicine.The aim of this study was to determine the longitudinal construct validity of assessing hand OA progression on digital photographs over a period of 7 years compared with hand OA progression determined from radiographs and from a hand examination of the clinical features over the same period. Additionally, hand OA progression on digital photographs will be examined to determine whether it is related to change in symptoms over 7 years.The Clinical Assessment Study of the Hand (CASHA) is a prospective observational cohort study. All individuals aged 50 years and over from two general practice registers in North Staffordshire, UK were invited to participate in a two-stage postal survey that collected data on demographics and characteristics of hand pain and hand problems in 2004–5. Respondents who indicated that they had experienced hand pain or hand problems in the last year were invited to attend a research clinic. Individuals who attended the baseline research clinics were contacted again at 7 years (2011–12) and invited to complete a postal questionnaire and attend a follow-up research clinic. Research clinics at both time points included: digital photographs of the hands, radiographs of the hands and a physical hand examination . Seven yO. The hand was pronated and placed in a fixed position on the gridded stand with forearm, wrist and fingers in a straight line and the hand resting in a natural position, i.e. with the fingers and thumb not held closely together or spanned. Figure Posterior digital photographs were taken of each hand separately using at baseline an Olympus Camedia C-4040 (Resolution 3.8 megapixels (MP)) and at follow-up either a Canon IXUS75 (resolution 7.1MP) or a Canon Powershot A480 (resolution 10.0MP). Hand photographs at both time points were collected in the same standardised position with the camera placed in a fixed position 38 cm (15 in.) above a gridded stand and were comparable to those used in the AGES-Reykjavik photographic scoring system . The parAn experienced reader (HJ) who was blinded to the clinical and radiographic data graded photographs from baseline and 7 years using the AGES-Reykjavik photographic scoring system. As per the scoring system, the presence and severity of hand OA were graded on unpaired photographs but with a known time sequence  . Five jon = 253) to determine inter-rater reliability. Intra-rater reliability was determined by (HJ) reading a sample of the hand photographs (n = 30) for a second time after at least four weeks.For the purpose of assessing reliability, a second experienced reader (GPH), also blinded to the clinical and radiographic data and the scores of the first reader, scored all the 7-year hand photographs  radiographs of the hands and wrists were taken at baseline and 7-year follow-up according to a standardised protocol . A singlIntra-rater reliability for the presence of hand OA in a joint was found to be excellent (mean Kappa (K) = 0.85 and percentage exact agreement (PEA) = 95%). Inter-rater reliability had previously been established with a second reader, an academic rheumatologist (RD) and found to be good (mean K = 0.79 and PEA = 95%) .Physical examination of the hands was undertaken at baseline and follow-up research clinics by trained physiotherapists and occupational therapists. The presence of nodes, hard tissue (bony) enlargement and deformity was determined in the second and third DIP, second and third PIP and first CMC joints in each hand and also any swelling present in the metacarpophalangeal (MCP) joints were recorded. Whilst we did not undertake formal reliability testing of the physical assessment in the study at baseline and 7 years, quality assurance and control were integral parts of the study as detailed in the study protocol . ClinicaParticipants were excluded from this analysis if they did not have digital hand photographs at both baseline and 7-year follow-up and if general practice or local rheumatology medical records or a musculoskeletal radiologist identified them as having inflammatory arthritis (rheumatoid or psoriatic arthritis) at baseline. They were also excluded if there was an indication of possible inflammatory arthritis or other serious pathology  on the digital hand photographs at baseline or 7-year follow-up, as determined by a consultant rheumatologist (HJ). Additionally, if a participant reached the maximum score at baseline for the photographic, radiographic scores or the maximum number of clinical features, then they were excluded from selected joint and person-level analyses as they were unable to progress.n = 30) using ICCs for the three joint groups.Reliability of using the photographic scoring system has previously been established , thus reNo reference standard is available for assessing progression on digital hand photographs so examining criterion validity was not possible. Therefore, longitudinal construct validity was investigated against a number of constructs. At the joint and joint group level the proportion of individuals who had shown photographic hand OA progression deom baseline to 7 years (increase in score ≥ 1) was calculated for individuals i) with (increase in score of ≥1) and without (change in score of < 1) radiographic progression between baseline and 7 years and ii) with (increase in score of ≥1) and without (change in score of < 1) progression of clinical features between baseline and 7 years. For a joint group, the highest change in in any joint between baseline and 7 years that was present within a joint group was used for photographic OA, radiographic OA and the number of clinical features. In addition, change scores were also calculated to represent the magnitude of change at each joint and in each joint group (7-year follow-up score – baseline score) for each construct . As data were not normally distributed, Spearman Rank correlation coefficients were used to assess how similar the constructs of radiographic OA and number of clinical features present were to the hand photographic scoring. Two a priori hypotheses were set regarding these correlations at the joint and joint group level:1) Change in photographic hand OA scores for each joint was expected to correlate more closely with the change in the number of clinical features than with the change in radiographic OA grade. 2) Correlations would be the same or slightly lower than was achieved at baseline for cross-sectional construct validity in each hand joint and joint group .At the person-level photographic hand OA scores were summed for the 10 assessed hand joints and change scores calculated . Individuals were divided into tertiles based on the distribution of data and categorised into those that had not progressed (change score ≤ 0), those that had undergone mild progression (change score = 1 to 2) and those that had undergone at least moderate progression (change score ≥ 3). Additionally, the number of joints that had undergone photographic hand OA progression over 7 years was examined using tertiles based on the distribution of data and categorised into no progression (0 joints), mild progression (1–2 joints) and at least moderate progression (≥3 joints). Analysis of variance was used to describe the mean ) summed radiographic OA score at 7-years, summed number of clinical features at 7-years, and 7-year AUSCAN pain (0–20), function (0–36), stiffness (0–4) and total (0–60) scores adjusted for baseline score for each tertile and to test for significance of differences between the tertiles and determine if change over time was similar between the different constructs. Percentages and Chi-Square Tests were used to explore differences in individuals perceived global assessment of change in their hand problem over 7-years  between the tertiles.n = 2 were excluded due to having the maximum baseline summed photographic hand OA score of 9 and n = 4 for having all ten hand joints affected with photographic hand OA at baseline.From the 432 eligible participants invited to take part at 7 years, a total of 376 participants completed the questionnaire (response rate = 87.0%) and 256 of whom attended the 7-year research clinics (response rate 59.3%)  were: slightly younger; less likely to have a manual occupation; thumb pain during activity in the last month; photographic hand OA at baseline; and moderate to severe radiographic OA (KL ≥ 3) in one or more hand joints at baseline; but were more likely to have attended further education  of participants experienced photographic progression in one or more hand joints over 7 years. Progression on the hand OA photographs was seen in each joint, but occurred more often in the DIP joints (14.4–22.6%) than the CMC (14.2–15.2%) and PIP joints (6.1–7.8%)  for all hand joints and most joint groups, but moderate for the PIP joint group (ICC = 0.54)  change in radiographic OA and ii) the change in the number of clinical features for joints and joint groups were mostly weak, although some were still statistically significant Table . Change Individuals undergoing at least moderate amounts of summed photographic hand OA progression (change score ≥ 3) over 7 years had significantly higher i) mean scores for radiographic hand OA and ii) the number of clinical features at 7 years after adjustment for baseline scores compared to those with no (change score ≤ 0) and mild (change score 1–2) photographic progression Table . Those uIndividuals who had ≥3 hand joints with photographic progression had significantly greater increases in radiographic OA and clinical features over 7 years compared to those with 1–2 joints or no joints that had progressed Table . IndividThis study is the first to investigate the longitudinal construct validity of assessing hand OA progression on digital photographs over a period of 7 years compared with progression determined on hand radiographs, clinical features on hand examination, and change in symptoms. We found that change in photographic assessment of hand OA at the person-level over 7 years does show longitudinal construct validity to changes in radiographic OA and clinical features. Individuals with the greatest amounts of photographic progression over 7 years had more pain and functional limitation than those with no or smaller amounts of progression, but differences were small. The results for longitudinal validity at the joint-level were less promising with weak correlations between changes in hand photographs and changes in radiographic OA or clinical features.Overall we found that 51% of individuals progressed on their hand photographs over 7 years. While rates of progression have not been reported previously for photographic progression or even clinical features, the rate we obtained is comparable to the progression reported in other studies of a similar length for radiographic change (53% over 6 years ), changeAt the person-level, those with moderate progression on the hand photographs had significantly higher scores for summed radiographic OA and clinical features compared with those classed as having none or mild progression. Moderate photographic progression in terms of summed change score and number of hand joints undergoing progression did also show stronger relationship to changes in symptoms, however, associations were non-significant. Change in the number of hand joints undergoing photographic hand OA progression seemed to be more sensitive than change in the summed photographic hand OA score. Mixed associations between symptoms and structural change have been reported in hand OA , 21 and Stronger relationships of photographic OA with radiographic OA and clinical features were seen at the person-level compared to the joint-level. While we found that overall about half of the individuals underwent any photographic progression in the hands, the numbers undergoing progression in the individual hand joints was much smaller (between 6.1–22.5). At the joint and joint group level, correlations between change in hand photographs and change in radiographic OA or clinical features were weak, but this was expected as the constructs were not similar. Another explanation of weaker correlation between photographic and clinical change is that only a single 2D photograph of the back of the hands was examined which may mean that some changes to the hands such as fixed flexion deformity could be missed. While this is similar to the single PA view taken radiographically it is possible that photographs taken from other views such as a lateral or even an oblique may be helpful and important for assessing clinical changes.While we were able to examine longitudinal construct validity, it was not possible to examine responsiveness as there is no accepted measure of clinically important change in hand OA, and no reference group that had not undergone any OA progression. The mean changes seen over 7 years in our general population sample were small, and it is possible that progression rates in a more severe clinical sample might have offered a better opportunity to examine longitudinal validity, although this could have limited generalisability of findings.We acknowledge a number of possible limitations of this study. Individuals that took part in the follow up at 7 years were less likely to have photographic hand OA at baseline than those without follow-up hand photographs, but we don’t envisage that this selective attrition is likely to have affected the relationships between the constructs within our study sample. Hand photographs were scored unpaired but with a known time sequence which has been suggested might lack sensitivity that could be obtained by reading in pairs and lead to an overestimation of change . HoweverThe assessment of photographic hand OA at the person-level over 7 years shows modest longitudinal construct validity in relation to radiographic OA and clinical features, but longitudinal validity at the joint-level was less favourable. Using hand photographs may be a reasonable method for examining overall change in a person with hand OA over the long term when radiographs or a hand examination is not feasible such as in large, populating-based studies or those undertaken over wide geographic areas.Additional file 1: Figure S1. Flow diagram of study participants


BPD). Systemic hypertension, arterial thickness and stiffness, and increased systemic afterload may all contribute to BPD pathophysiology by altering left ventricular (LV) function and increasing pulmonary venous congestion by lowering end‐diastolic compliance. This case series studied the usefulness of angiotensin‐converting enzyme (ACE) inhibition by measuring clinical and echocardiographic improvements in six consecutive infants with “severe” BPD unresponsive to conventional therapy. The range of gestation and birthweight were 23–29 weeks and 505–814 g, respectively. All required mechanical ventilation (including high‐frequency oscillation) and all but one were administered postnatal corticosteroids. Other treatments including sildenafil and diuretics made no clinical improvements. Captopril was started for systemic hypertension after cardiac and vascular ultrasounds which were repeated 5 weeks later. A significant reduction in oxygen  and ventilator requirements, and improved cardiovascular parameters were noted. This included a trend toward reduction in aorta intima media thickness  and an increased pulsatile diameter ). Improvements were observed for both systolic  and diastolic  function which was accompanied by increased pulmonary vein flow. Right ventricular output increased accompanied by a significant lowering of pulmonary vascular resistance. These findings suggest that improving respiratory and cardiac indices  warrants further exploration of ACE inhibition in BPD infants unresponsive to conventional therapy.Premature infants have a high incidence of bronchopulmonary dysplasia ( Bronchopulmonary dysplasia (BPD) is the most common and the most important respiratory consequence of preterm birth with incidence of approximately 60% in infants’ ≤25 weeks gestational age (GA) Chow . In infasystemic (left sided) pathology were limited to the management of systemic hypertension. The incidence of systemic hypertension has been variously reported between 7 and 43% and was associated with longer duration of hospital stay, home oxygen, and a higher mortality   reduction  are physiologically very suited to the proposed BPD pathophysiology. We evaluated a series of consecutive infants with severe BPD unresponsive to standard therapy and systemic hypertension, using focused functional ECHO. The objective was to generate hypothesis regarding the use of ACE inhibitors that would need to be tested in appropriately designed randomized controlled trials.EchoPAC™ . Standard ECHO indices were measured as described in detail previously . The study conformed to the standards set by the latest version of Declaration of Helsinki, except for registration in a database. Standard practice in our Unit is to start captopril in consultation with renal and cardiac physicians after informed discussion with the parents when systolic BP is consistently above 99th centile for PMA  to between 50 and 95th centile for PMA at repeat assessment.Table Table systemic arterial measures (afterload reduction) using ACE inhibitors was associated with significant improvement in cardiac function and clinical features (respiratory support). Assessment of LV function  and the systemic vasculature may be warranted in infants with severe BPD with persistent radiological features of pulmonary edema despite diuretic therapy or where pulmonary edema worsens and respiratory support requirements increase, in response to the management of PH with pulmonary vasodilators. Such physiology‐driven approach toward therapy should be explored in infants with severe BPD and hypertension, especially in those unresponsive to standard therapy.The study of arterial biomechanics and left heart function in infants with BPD is relatively recent compared to well‐studied monitoring and management of PH  have been implicated as possible mechanisms of systemic hypertension in this cohort  were found to be significantly thicker in BPD cases  and vasodilatation/antiproliferation  of the vascular wall. Figure β‐blockers, diuretics, and ACE inhibitors. While all drug classes were equally effective in reducing BP, only the ACE inhibitors improved endothelial function and endothelium‐dependent vasodilation  caused regression of the media to lumen ratio in rats  effects of ACE inhibition.Given the angiogenic function of angiotensin‐II, ACE seems to play a role in vascular remodeling, pulmonary hypertension, and in the pathologic mechanism of neonatal lung diseases  may improve outcomes of infants with severe BPD, unresponsive to standard therapy with diuretics and pulmonary vasodilators  are not routinely assessed using bedside ECHO. Assessments made much later in gestation (as in our cohort) probably mean prolonged duration of respiratory support and greater morbidity, longer duration of hospital stay and dedication of precious healthcare resources. As a future research direction for physiology‐driven care of this critically sick population, we propose that infants with severe BPD be assessed at close to 36 weeks corrected GA . Routine assessments of left heart function  and systemic arteries in addition to conventional right‐sided assessments will give greater insight into physiology‐driven care.None of the authors has any conflicts of interests.


Breast cancer (BC) is the most common cancer among women worldwide. With increasing survival rates, focus has expanded to long‐term adverse effects of adjuvant chemotherapy and/or aromatase inhibitors. Weight gain during chemotherapy has been well documented, but the underlying mechanisms remain unclear. A change in glucose and insulin metabolism is a possible consequence.We searched PubMed on the 4th of May 2018, and found eight articles that compared measurements of glucose and insulin before and after chemotherapy and/or aromatase inhibitors in woman with BC.A general trend of increased glucose and insulin is seen and likely to be caused by weight gain and/or changes in body composition as a consequence of adjuvant treatment of BC.Due to methodological limitations including short follow‐up times and small sample sizes, further studies are required to better describe metabolic consequences of adjuvant chemotherapy and/or aromatase inhibitors. Future studies could help identify patients in high‐risk of developing cardiometabolic disease after BC treatment. The most common regimens include sequential anthracycline‐cyclophosphamide and taxane (AC‐T); concurrent anthracycline‐cyclophosphamide and taxane (ACT); anthracycline‐cyclophosphamide without taxane (AC); cyclophosphamide, methotrexate, and fluorouracil (CMF); and docetaxel and cyclophosphamide (TC).It has been known since the 1970’s that women receiving adjuvant chemotherapy experience weight gain, commonly reported as 2‐5 kg but with great variance.Very few animal studies have examined the metabolic impact of chemotherapy; Story et alAltogether, studies suggest that women receiving adjuvant chemotherapy and/or aromatase inhibitors for BC experience metabolic alterations giving rise to a phenotype associated with increased risk of developing cardiometabolic disease as well as BC recurrence.The purpose of this systematic review is thus to assess the current available knowledge on impact of adjuvant chemotherapy and/or aromatase inhibitors in women with BC on metabolic alterations focusing on glucose and insulin metabolism.2This review has been written according to the PRISMA guidelines. All articles describing the effect of adjuvant chemotherapy and/or aromatase inhibitors in women with BC and how it affected glucose, insulin and/or insulin resistance by comparing measurements before and after treatment were identified and included Figure . No limi(Breast Neoplasms[mh] OR Breast Neoplasm*[ti] OR Breast Neoplasm*[ot] OR Breast Carcinoma[ti] OR Breast Carcinoma[ot] OR Breast cancer[ti] OR Breast cancer[ot])ANDAND(Body composition[mh] OR Body composition[ti] OR Body composition[ot] OR Body Weights and Measures[mh] OR Diabetes[ti] OR Diabetes[ot] OR Dyslipidemias[mh] OR Dyslipidemia*[ti] OR Dyslipidemia*[ot] OR Fat percentage[ti] OR Fat percentage[ot] OR Glucose Intolerance[mh] OR Glucose Intolerance[ti] OR Glucose Intolerance[ot] OR Hypercholesterolemia*[ti] OR Hypercholesterolemia*[ot] OR Hyperglycemia*[ti] OR Hyperglycemia*[ot] OR Hypertriglyceridaemia*[ti] OR Hypertriglyceridaemia*[ot] OR Insulin[ti] OR Insulin[ot] OR Insulin Resistance[ti] OR Insulin Resistance[ot] OR Insulin Sensitivity[ti] OR Insulin Sensitivity[ot] OR Metabolic Disease[mh] OR Metabolic syndrome[ti] OR Metabolic syndrome[ot] OR Metabolism[mh] OR Metabolism[ti] OR Blood glucose[mh] OR Body Mass Index[mh] OR Metabolic syndrome[mh] OR Metabolic syndrome[ti] OR Metabolic syndrome[ot] OR Triglycerides[mh] OR Waist‐Hip Ratio[mh])We searched PubMed (Medline) on 4th of May 2018 with the following search terms:3As shown in Figure Adjuvant chemotherapy regimen varied between studies; four of the studies used a combination of an alkylating agent (cyclophosphamide or carboplatin) along with anthracyclines (doxorubicin or epirubicin) and taxanes (docetaxel or paclitaxel). Chala et al use a regimen of alkylating agent with anthracyclines and fluorouracil and Hickish et al vary between both of the abovementioned combinations. Two studies do not specify treatment. In three of the studies it is indicated that some patients received endocrine treatment, Dieli‐Conwright et alThe eight articles were published between 2006 and 2016, with a prospective study design in six and a retrospective study design in the two remaining studies. The general trend of the studies was to use measurements from before the first cycle of chemotherapy and compare to measurements during or shortly after the last cycle. The follow‐up periods range from 3 to 40 months, but with only two studies having an average follow‐up time of more than 7 months. Four of the studies reported a significant increase in weight/BMI whereas three studies did not find a significant change in body weight  was measured in all studies. HbA1c is a measure of glycation of hemoglobin, and as red blood cells have a lifespan of up to 120 days, it represents a measure of the average blood glucose concentration during the past 8‐12 weeks. Most studies found no significant change from before to after adjuvant chemotherapy and/or aromatase inhibitors. However, two studies found a significant increase in glucose and HbA1c respectivelyInsulin was measured in six studies and was significantly increased after adjuvant chemotherapy in four of these. Contrary to this Chala et al found a significant decrease in insulin at 120 minutes during an oral glucose tolerance (OGTT) test compared to before adjuvant therapy. Homeostatic model assessment of insulin resistance (HOMA‐IR) was calculated in five of the studies, and was significantly increased in four of the studies.44.1Although only a limited number of studies have looked at the impact of adjuvant chemotherapy and/or aromatase inhibitors on glucose and insulin metabolism in women with BC, it appears that a negative effect is the most common observation. The eight studies in focus do have severe limitations. The short follow‐up times make it difficult to predict long‐term consequences of metabolic changes. Likewise, are several of the studies small in sample size and some are retrospective studies.2 which correspond with the fact that increased weight, is a risk factor of BC. However, none of the studies included a control group, thus no comparable background information including weight and BMI exist on aged‐matched women without BC.The pre‐ and post‐treatment weight and/or BMI were reported in seven of the eight studies. Four of the studies reported a significant increase in weight and/or BMI in response to treatment.Weight gain is common among aging women, especially during the menopausal transition. According to several studies, the average weight gain in women during midlife is approximately 0.4‐0.7 kg/y independent of initial body weight and ethnicity, however with a large individual variation.The generalizability of the studies to current BC patients is limited by the differing treatment, with only four studies using regimens that reflect current practice with the combination of an alkylating agent combined with an anthracycline and a taxane. Furthermore, many studies lack detailed descriptions concerning treatment, especially with regard to endocrine treatment. The adverse effects on metabolism are poorly understood and rarely the focus in clinical or animal studies of chemotherapy effect in cancer. Finally, has weight gain not been clearly linked to any specific regimen or single chemotherapeutic agent.4.2The long‐term effects of metabolic changes induced by chemotherapy are difficult to assess, but in a study of more than 3000 women Saquib et alAmong the studies on the glucose metabolism, only two have follow‐up periods of more than a year, with Guinan et al4.3The underlying cause of the metabolic alterations including changes in glucose and insulin metabolism is not clear and are most likely only part of the metabolic changes occurring as a consequence of adjuvant chemotherapy and/or aromatase inhibitors of BC patients. However, the previously mentioned weight gain and/or changes in body composition are likely to be factors contributing to the metabolic changes. Weight change during chemotherapy may be caused by treatment‐related side‐effects such as fatigue, physical inactivity, sarcopenia and altered appetite. The increase in circulating lipidsIt is likely that certain subgroups may be at increased risk of unfavorable changes in body composition following chemotherapy. Previous studies have suggested that premenopausal women may gain more weight during treatment of BC than postmenopausal womenAdiposity may increase the risk of metabolic disease, and in recent years adipokines and primarily leptin has been investigated. Leptin is a hormone produced in the fat tissue and is positively correlated with body fat mass.Interleukins has been shown by Paz et al4.4With obesity being an independent risk factor for development of BC in postmenopausal women,4.5Exercise training and dietary interventions have been suggested to alleviate or minimize treatment‐related side effects to BC. Recent systematic reviews and meta‐analysis have shown significant effects of physical exercise on anthropometric measures and quality of life.5Women undergoing medical treatment for BC, appear to become metabolically deranged to some extent. This is mainly evident by a small increase in weight and/or change in body composition, but the underlying mechanism has not yet been thoroughly described. Earlier explanations like overeating,None declared.


Streptococcus pyogenes CRISPR/Cas system has found widespread applications as a gene‐editing and regulatory tool for the simultaneous delivery of the Cas9 protein and guide RNAs into the cell, thus making the recognition of specific DNA sequences possible. The recent study that shows that Cas9 can also bind to and cleave RNA in an RNA‐programmable manner is suggestive of potential utility of this system as a universal nucleic‐acid recognition tool. To increase the signal intensity of the CRISPR/Cas system, a signal amplification technique has to be exploited appropriately; this requirement is also a challenge for the detection of DNA or RNA. Furthermore, the CRISPR/Cas system may be used to detect point mutations or single‐nucleotide variants because of the specificity of the recognition between the target sequence and the CRISPR/Cas system. These lines of evidence make this technique capable of detecting pathogens during infection via analysis of their DNA or RNA. Thus, here we summarize applications of the CRISPR/Cas system to the recognition and detection of DNA and RNA molecules as well as the signal amplification. We also describe its potential ability to detect mutations and single‐nucleotide variants. Finally, we sum up its applications to testing for pathogens and potential barriers for its implementation.The  The researchers found it possible to combine these two RNAs into a chimeric single guide RNA (known as sgRNA) that can effectively direct Cas9 to specific DNA targets. The rules used by Cas9 to search for a DNA target are not complicated, the specificity is determined only by a 20‐nucleotide (nt) sequence in the sgRNA that hybridizes with the target DNA in the presence of a DNA protospacer‐adjacent motif (PAM) in the complementary region close to the target site.Clustered regions of interspersed palindromic repeats with Cas9 (CRISPR/Cas9) is an antiviral defense system first found in 2MUC4 gene and concluded that 26 to 36 sgRNAs are sufficient to detect a non‐repetitive genomic locus by means of CRISPR. They also labelled different regions of the same gene or different genes with multiple sgRNAs and reported that the spots of imaging via two genes increased whereas those of the same gene did not, suggesting that CRISPR can label multiple genomic elements at the same time. On the basis of this observation, Wulan DengS. pyogenes Cas9 whose PAM is required only for the “NGG” sequence, many other bacterial Cas9 proteins recognize more complicated PAM sequences, which makes the target sequence more demanding. Consequently, to circumvent these issues, Yi Fu et alDNA fluorescence in situ hybridization (FISH) has been widely used to visualize sequence‐specific genes for research and diagnostic purposes. Nonetheless, it requires heat treatments and formamide to denature double‐stranded DNA (dsDNA) to enable probe hybridization; these treatments may affect integrity of the biological structure and organization of the genome. Accordingly, Baohui ChenIn view of the development of DNA detection technologies, from repetitive to non‐repetitive sequences, the challenge was to enhance the signal released from the locus of the target sequence labelled with re‐engineered sgRNA/dCas9–viral RNA‐binding protein–fluorescent protein complex. Comparison of the detection of repetitive and non‐repetitive sequences revealed that the former yields an obvious signal amplification effect because its abundant copies can combine with much more sgRNA molecules, which should produce a stronger signal. Nonetheless, the latter was not able to produce such a strong signal this way. Therefore, the researchers devised two other methods to amplify the signal: one involves transduction of large amounts of sgRNAs targeting the same gene in different regions, and the other involves attaching more fluorescent proteins to one single sgRNA. The limitations of the first method are the following: (a) with the increase in the number of sgRNAs, the off‐target effects will become more serious and ineluctable; and (b) it is technically difficult to deliver so many sgRNAs into a cell. It seemed that the second method is suitable for detection of a single non‐repetitive sequence but still required four sgRNAs, and lattice light sheet microscopy was needed to obtain a reliable signal. From the single loci to simultaneous multilocus detection, various improvements have been made to add various fluorescent colours though the method was still restricted to six colours. Accordingly, there is still much room for improvement.GAPDH transcripts from a cell lysate under physiological conditions. At first, they obtained only two GAPDH‐specific RNA fragments. But after complete elimination of RNase H–mediated RNA cleavage, they successfully isolated intact GAPDH mRNA and observed that in the absence of a PAMmer, GAPDH mRNA can still be isolated though with lower efficacy. These data indicate that the Cas9/gRNA complex binds to GAPDH mRNA through direct RNA–RNA hybridization. These results mean that this approach can help to purify endogenous untagged RNA transcripts.One study indicates that the HNH nuclease domain of Cas9 is homologous to other HNH domains that cleave RNA substrates.S. pyogenes Cas9 mutant (dCas9) to deploy a large RNA cargo to targeted DNA loci by incorporating the cargo into the sgRNA. In other words, the sgRNA became a longer sgRNA that contains a sequence of interest such as artificial aptamers, pools of random sequences, and natural long non‐coding RNAs. The sgRNA may bind to genomic loci so that finally we could find out where the RNA sequence of interest is localized. The limitation of this method is that the length of inserted RNA should be at least 4.8 kb. This approach is different from the procedure for inserting the RNA sequence into sgRNA to display RNA domains. The Doudna lab quickly demonstrated that nuclease‐inactive CRISPR/Cas9 can bind RNA in a nucleic‐acid–programmed manner and allows for endogenous RNA tracking in live cellsThe study on CRISPR/Cas9 targeting of RNA began to gain popularity after Doudna's study. David M. Shechner3It is well known that CRISPR/Cas9 has high efficacy of site‐specific gene targeting, but its potential off‐target effects have raised major concerns regarding its application in many respects. What's more, in Luhan Yang et alKRAS, PIK3CA and IDH1 in human cancer cell lines by means of the activity‐decreased RGENs. Moreover, they can also detect point mutations in the BRAF and NRAS genes using RGENs that recognize the “NAG” PAM sequence.Techniques have been developed to detect mutations, for example, Surveyor nuclease assays and T7 endonuclease 1 (T7E1) assays. Both are enzyme mismatch cleavage assays for detection of a single‐base mismatch or small indels and target and cleave mismatched heteroduplex dsDNA. Then, the digested DNA fragments can be distinguished by agarose gel electrophoresis to determine whether there is a mutation. Nonetheless, the limitation of the two assays is that they cannot differentiate homozygous biallelic mutant clones from wild‐type clones and heterozygous monoallelic mutant clones from heterozygous biallelic mutant clones. Jong Min Kim et alKRT12 that causes Meesmann's epithelial corneal dystrophy and can lead to the occurrence of a novel PAM. This meant that they could design a sgRNA complementary to the sequence adjacent to this mutation‐derived PAM to target and cleave the mutated gene. They took advantage of this approach in treating Meesmann's epithelial corneal dystrophy. Another teamKRAS gene to solve the problem of acquired drug resistance to a MEK signalling inhibitor. Both confirmed that the mutation‐ or SNP‐derived PAM could be put to good use in the treatment of some dominantly inherited diseases because the sgRNA/Cas9 complex can specifically disrupt the mutant allele, whereas expression of the wild‐type allele would remain unaffected.It is not easy to control the recognition and cleavage of the RNA‐guided Cas9 nuclease because its specificity is dependent on the length of the sgRNAKRAS, PIK3CA and IDH1 in human cancer cell lines have been identified. As for the novel PAM, the cleavage activity of Cas9 is often employed to disrupt the mutated gene while the wild‐type allele is unaffected. This method is more likely to cure some heterozygous mutations causing an autosomal dominant (AD) single‐allele disease because of the subsequent homology‐directed repair after cleavage by Cas9.Let us review the two assays that are aimed at recognizing an SNV that happened in a genome. An SNV generally has two kinds of consequences: one is the appearance of a new target gene that is different from the former target gene by a single base (relevant to the high specificity of the modified sgRNA/Cas9 complex). The other is the emergence of a novel PAM of Cas9 if there is an ‘‘NGG’’ sequence resulting from the SNV. As for the new target gene, researchers find that by introduction of a single‐base mismatch into the guide RNA, the cleavage specificity of Cas9 can be improved probably because of the decreased enzymatic activity of RGEN, which is quite interesting and needs to be clarified. This assay could be used to distinguish between a mutant type and wild‐type, and thus, three recurrent oncogenic point mutations in genes 4Emerging infectious diseases, such as Zika virus (ZIKV) infections in Latin America and Southeast Asia, have rapidly drawn attention to the need to develop simplified diagnostic tests for clinical practice in some underdeveloped geographic areas. Some authors have published papers about applying the CRISPR/Cas system to the molecular diagnosis to detect the presence of virus such as Zika virus. Keith PardeeStaphylococcus aureus (MRSA) using a CRISPR‐associated protein 9/single‐guide RNA (dCas9/sgRNA) complex as a targeting material to capture genomic DNA of MRSA by specific recognition of the mecA gene (which is implicated in the resistance to methicillin in MRSAS. aureus (MSSA) strains does not contain the mecA gene, the well‐designed dCas9/sgRNA complex should not affect MSSA. Next, the MSSA and MRSA could be separated by Ni‐NTA magnetic nanobeads attached to dCas9 utilizing a magnet. Finally, SYBR Green I (SG I) is added to stain dsDNA and the fluorescence intensity can reflect the concentration of MRSA. It was also confirmed that test samples can be cell lysates without isolation of genomic DNA, this method does not require PCR and therefore is practical and rapid. On the other hand, some researchers have already found a new mecA homologue (mecALGA251) sharing only 70% nucleotide homology with mecA,mecA, and there is a spot of MSSA strains that contains the mecA genemecA gene not specific to MRSA.In contrast, Deng Wulan et alAfter the DoudnaThe principles of the three methods are quite different and all three involve a combination of the CRISPR/Cas or modified CRISPR/Cas system with some other technique to achieve a detection goal. The main features of the three methods are summarized in Table 5Despite the great achievements in CRISPR/Cas technology, there are still many hurdles and limitations in its application of molecular detection. Firstly, the proper recognition of a target site by the sgRNA requires a PAM sequence6Since the discovery of the CRISPR/Cas system, it has been widely used for genomic editing to treat some mutation‐induced diseases. Nevertheless, there is still too much controversy regarding the applications of CRISPR/Cas9 in medical treatments due to the risk of “off‐target” effects. From Yanfang Fu'sThe authors declare no conflict of interests.


LC).Despite the incidence and recurrence rates of breast cancer, there are currently no biomarkers to predict which cases will develop into lobular carcinoma , hypoechoic echogenicity (88%), poorly circumscribed margin (95%), posterior acoustic enhancement (93%) and absent calcifications (81%). Using Fisher's extract test, it was found that stromal fibrosis, single file type pattern, atypical lobular hyperplasia and LC Grade II were significantly correlated with irregular shape and hypoechoic echogenicity.Morphologic ultrasound characteristics most frequently reported in the dataset of positively diagnosed LC of the breast.A prognostic predictor tool can be designed from this study's findings which can then be used in practice to raise awareness of the unique morphometric markers related to  Lobular carcinoma (LC) is the second most common carcinoma of the breast (5–20% incidence) and has a high risk of recurrence in the contralateral breast (50–70%).Despite the incidence and recurrence rates of breast cancer, there are currently no biomarkers to predict which cases will develop into LC.Current research hallmarks mammography as the gold standard for breast cancer detection with a sensitivity rate of 85%. However, sensitivity is reduced to 68% with dense type breast tissue.From a pathological and histological point of view, strict morphological criteria for LC and its various subtypes are uncommon.The question arises whether there are certain morphometric properties of LC which are significantly correlated with positively diagnosed LC. Also, could the development of a prognostic predictor tool using such morphometric properties improve diagnosis of LC?LC has a diffuse growth pattern, which relates to its low detection rate with mammography.Ultrasound plays an integral role in the improved sensitivity and diagnosis of LC.Ultrasound findings of LC vary. Terms such as weak internal echogenicity, a heterogeneous (complex), hypoechoic mass, an irregular distribution or an angular, ill‐defined mass are used.It has been established that high‐frequency ultrasound is the preferred imaging modality for diagnosis of LC since mammography has limited sensitivity and specificity.A cross‐sectional descriptive study was conducted at two private radiology practices which both serve as dedicated women's wellness centres. Two radiologists are responsible for performing and interpreting the high‐frequency ultrasound examinations of the breast on the Siemens Acuson X300 5–13 MHz  and the Toshiba Aplio 300 5–14 MHz linear array probes . Data were collected retrospectively from patient files and prospectively from re‐observation of ultrasound images. Ethical clearance for use of retrospective ultrasound images was sought from Medical Research Ethics Committee of University of Pretoria.Both radiologists have extensive knowledge regarding breast ultrasound of over 25 years.The researcher handled the data securely and confidentially. Patient confidentiality was upheld through anonymity of personal information. No identifying information was used or reported on. Patient names were replaced with unique study numbers on the high‐frequency ultrasound images presented to the interpreters.The Research Ethics Committee of the Faculty of Health Sciences waived the necessity of individual patient consent. However, consent was obtained from the radiology practices for access to the data which was essential for completion of the study.Ultrasound images were retrieved from a local PACS system and were analysed on a five megapixel Barco workstation by two interpreters. All images were interpreted according to the guidelines of the BI‐RADS® lexicon. Interpreters completed the relevant section of the data collection sheet according to the findings on the high‐frequency ultrasound images. Interpreters were blinded to the histology results, ultrasound BIRADS classification and previous ultrasound reports.Histopathological findings from retrospective results were all descriptions from specimens with terminology: atypical lobular hyperplasia, comedo necrosis, cystic dilated terminal lobular units, stromal fibrosis, epithelial hyperplasia, signet ring appearance, classic/single file/ single columns of linear cells or ribbons.Once the interpreters had completed the relevant section of the data collection sheet, the researcher completed the remaining sections. Findings from high‐frequency ultrasound images were correlated with histology findings and ultrasound BIRADS classification to determine which morphometric characteristics were most accurate predictors of LC.Consecutive sampling of all cases of histologically positive malignancies in the breast, diagnosed through ultrasound‐guided core biopsies from December 2016 retrospectively to January 2013 was undertaken. One hundred of the 1052 cases of patients with confirmed breast malignancy were confirmed LC cases. The ultrasound images of the confirmed LC cases were retrieved from the image database. The histology reports for each case were then extracted from the radiology database.Two radiologists evaluated the ultrasound images in terms of morphological characteristics which corresponded to the lesions on the high‐frequency ultrasound images for each case. Benign and malignant morphologic characteristics provided in the data collection sheet were derived from previous studies.Histologic descriptions were grouped into five subsets; epithelial hyperplasia and stromal fibrosis see Fig. , signet Dependent variables included ultrasound morphometric characteristics of LC. Independent variables include the histopathological results and age group of each patient. Confounding variables include the possibility of inter‐rater variability with the image analysis performed on the ultrasound images.A power calculation was done to meet the primary aim, considering the 5–20% incidence of LCA sample size of 100 was deemed appropriate to obtain a 95% confidence interval with a 5% margin of error, based on previous epidemiological studies conducted in the field of study.P‐value less than or equal to 0.05 was considered significant by means of Fisher's exact tests for all correlations that were analysed. Data analysis included frequencies and proportions to describe categorical variables.Data analysis was performed in consultation with a biostatistician and a For the ultrasound morphology descriptors, the chi‐square test was used to test for associations between the categorical variables.P‐value less or equal to 0.05 was considered as statistically significant results.For the comparison between morphology and histology descriptors, ultrasound characteristics were noted for each data set unit and correlated with the histopathology results. Fisher's exact test with a One‐way ANOVA was used to test for differences across age for the histological factors, namely Bonferroni correction and Bartlett's test for equal variances to assess whether there was homogeneity in variances.The assumption was made that the image interpretation of the selected interpreters were accurate. All analysis was done using STATA 14 data analysis software.The age range of the sample varied from 29 to 92 years with a mean age of 58.56 ±1.33) years. The frequency of the morphologic descriptions reported by the two interpreters is included in Table .33 yearsThe most frequently reported ultrasound morphology of LC cases included: an irregular (ill defined) ultrasound shape (86%) see Fig. , hypoechThe histologic grade and description extracted from the histopathologic reports appear in Table LC Grade II (33%) as well as atypical lobular hyperplasia/ LC in situ (39%) were groups with the highest frequency for histological grading as seen in Table Morphologic characteristics that were the most common findings, were a hypoechoic mass as well as an irregular shape. This finding is supported by previous morphologic descriptions of LC.P ≤ 0.05) to atypical lobular hyperplasia  and the histological descriptions of single file/ribbons/single file patterns as well as epithelial hyperplasia or stromal fibrosis.An irregular ultrasound shape was significantly correlated  was found. For histologic descriptions, there was a significant correlation between a hypoechoic ultrasound echogenicity, single file/ribbons/single file pattern as well as epithelial hyperplasia/stromal fibrosis.In terms of histologic grading, a significant correlation between hypoechoic echogenicity and atypical lobular hyperplasia as well as LC Grade II  and hypoechoic ultrasound echogenicity (88%). These findings correlate with other studies, which described LC lesions as a hypoechoic,% and hypHistology descriptions unique to LC were single file type pattern which coincides with previous literature stating scattered columns of infiltrated malignant cells surrounding normal breast tissue in a diffuse pattern. However, this may not be seen consistently in all patientsWeaknesses of the study included the use of a small sample size, as well as the interpreters not blinded to the study data; reducing its statistical significance.The study is limited by its retrospective approach and acknowledges previous research regarding LC, in lieu of novel findings.Larger sample sizes should be utilised in future research to improve the results related to inter interpreter agreement. With regards to the methodology; retrospective studies prove a possible selection bias with sample populations; future studies should attempt a prospective data collection approach.The use of both diagnostic and screening programmes in breast clinics should be implemented as standard protocol to improve patient predictor outcome. It should also be noted in practice, the importance of ultrasound as adjunct to mammography based on the current study's findings and should be practiced as part of a standard diagnostic screening programme tool.In conclusion unique morphologic characteristics are related to histological findings of LC during ultrasound based diagnosis.There is no financial support or relationships that may pose conflict of interest.


To measure the variation in prescribing of second‐line non‐insulin diabetes drugs.We evaluated time trends for the period 1998 to 2016, using England's publicly available prescribing datasets, and stratified these by the order in which they were prescribed to patients using the Clinical Practice Research Datalink. We calculated the proportion of each class of diabetes drug as a percentage of the total per year. We evaluated geographical variation in prescribing using general practice‐level data for the latest 12 months (to August 2017), with aggregation to Clinical Commissioning Groups. We calculated percentiles and ranges, and plotted maps.Prescribing of therapy after metformin is changing rapidly. Dipeptidyl peptidase‐4 (DPP‐4) inhibitor use has increased markedly, with DPP‐4 inhibitors now the most common second‐line drug (43% prescriptions in 2016). The use of sodium‐glucose co‐transporter‐2 (SGLT‐2) inhibitors also increased rapidly . There was wide geographical variation in choice of therapies and average spend per patient. In contrast, metformin was consistently used as a first‐line treatment in accordance with guidelines.In England there is extensive geographical variation in the prescribing of diabetes drugs after metformin, and increasing use of higher‐cost DPP‐4 inhibitors and SGLT‐2 inhibitors compared with low‐cost sulphonylureas. Our findings strongly support the case for comparative effectiveness trials of current diabetes drugs. There is wide variation in cost between these treatment options, with metformin and sulphonylureas costing an average of ~£4 to £6 per item prescribed in 2015/2016, and SGLT‐2 and DPP‐4 inhibitors ~£40 per item.The aim of the present study was to determine variation in prescribing patterns and prescribing costs of antidiabetic treatments both geographically, across practices in England, and over time, by using three different datasets, summarized in Table 22.1We used 3 sources of data: the Clinical Practice Research Datalink (CPRD), a UK‐representative database of anonymized primary care electronic health records;2.2We extracted clinical and prescription records for 207 338 people with type 2 diabetes from the CPRD  who were prescribed a first‐ to fourth‐line oral diabetes drug, classified in the BNF 6.1.2 as “other antidiabetic drugs,” over the period 1998 to 2016 and had not previously been prescribed insulin. A detailed description of CRPD data ascertainment has been previously reported.2.3The annual PCA datasets contain one row for each treatment and dose, for all prescriptions issued in NHS primary care and dispensed in community settings in England, describing the number of prescriptions dispensed and the total cost. PCA data were processed as previously described.2.4The monthly prescribing datasets published by NHS Digital contain one row for each treatment and dose, in each prescribing organization in NHS primary care in England, describing the number of prescriptions issued and the total cost. We limited this to organizations with setting code “4”– general practices, according to the NHS Digital dataset of practice characteristics,2.52 = 0.9965). We calculated items prescribed/cost per person with diabetes for England by dividing prescribing figures per 1000 population by the prevalence rate per 1000. CCG and practice‐level prevalence figures were obtained from the Quality Outcomes Framework (QOF)Estimates of percentage national prevalence of type 2 diabetes for 2000 to 2013 were obtained from an analysis of practices in The Health Improvement Network (THIN) database,2.6In the CPRD we categorized drug prescriptions using BNF codes and Medcode keyword searches of “product name” and “drug substance name.” The CPRD includes full prescription records but no data on drug dispensation. New drug prescriptions (and their corresponding start dates) were defined as the first‐ever prescription of a drug in each class for each patient, even if only prescribed once. Patients were considered to have stopped a drug if there was a gap in prescribing of that drug of at least 6 months.In the PCA and practice‐level datasets we extracted the prescribing data for paragraph 6.1.2, “other antidiabetic drugs.” Drugs were each assigned to the appropriate class  based on their chemical name Table . In CPRD2.7stata v14.0, and trends charts were produced using Excel. Deciles of practice‐level prescribing trends across all practices were calculated for each available month and plotted as time trend charts using Python. After limiting to and aggregating the latest available 12 months, summary tables of CCG and practice prescribing were produced in Python.We calculated prescribing rates per class of drug by dividing the number of items prescribed by the total number of antidiabetic items (BNF 6.1.2) prescribed. Trend charts from PCA data were produced in Excel by summing items or cost per patient with type 2 diabetes over each class per year. In the CPRD, we calculated the proportion of new prescriptions of each drug for each calendar year and line of therapy as the total number of new prescriptions of the drug/total number of new prescriptions. CPRD data extraction and analysis was conducted in 2.8http://openprescribing.net/analyse, by selecting all the chemicals within each class as numerator and BNF Paragraph 6.1.2 as denominator. The results were converted to percentages (from per 1000). The data source for http://openprescribing.net is the monthly practice‐level prescribing dataset described above. It includes all practices with status “4” , but does not exclude closed and dormant practices. The map of spend per patient across CCGs was produced using Tableau Open software.Maps of antidiabetic items prescribed by all CCGs in England for a single month snapshot were created using 2.9http://openprescribing.net are provided in the Supporting Information. The Tableau workbook mapping the costs per patient is available online at https://public.tableau.com/views/Diabetesmap/Dashboard1?:embed=y&:display_count=yes. Practice‐level data were analysed and charts produced using Python scipy.stats, matplotlib.pyplot and seaborn modules. Complete codes are provided in the Supporting Information and PCA data extract in FigShare.PCA and practice‐level data were extracted using SQL in Google BigQuery. The links to each map for CCG prescribing on 33.1From the CPRD we included data on 392 764 new first‐ to fourth‐line antidiabetic drug prescriptions for 207 338 unique patients in the period 1998 to 2016. Of these, 48% (191 550) were for a first‐line drug, 31% (124 673) for a second‐line, 14% (57 679) for a third‐line and 5% (18 862) for a fourth‐line drug. Metformin was the most common newly initiated drug prescribed , followed by sulphonylureas (27%), TZDs (10%), DPP‐4 inhibitors (10%), GLP‐1 analogues (2%) and SGLT‐2 inhibitors (2%). All PCA data were extracted successfully. From the practice‐level data for the latest 12 months, 7261 practices belonging to all 207 current CCGs were included, after excluding 342 with a status of closed (68) or dormant (274). In total this covered 48 million registered patients (aged ≥15 years), of which ~3.1 million were registered as having diabetes. For decile calculations for the period 2010 to 2017, in total 8155 practices were included.3.2The CPRD data show that metformin has been favoured as first‐line treatment since 2001  was accompanied by only a 5% increase in items dispensed (4.6‐4.8). This rise corresponds with increased usage of DPP‐4 and SGLT‐2 inhibitors. An increase in the price of TZDs, caused by a shortage of generic pioglitazoneDespite remaining the most widely used second‐line therapy, sulphonylureas contribute less to total cost per patient than most of the other classes because of their generic availability Figure B,C. Rece3.4We investigated how the level of prescribing of each antidiabetic drug class varied across England, summarizing the proportions prescribed by each CCG over a 12‐month period Table . There wThe spend on antidiabetic drugs per patient with diabetes over the latest 12 months ranged from £60 to £200 across CCGs Figure . Lower c3.5As expected, variation was greater across practices than when aggregated to CCGs Table ; the pro4We have assessed variation in NHS primary care prescribing of diabetes treatments both geographically, across CCGs and practices in England, and over time. There is wide regional variation in choice of second‐line therapy, reflecting the absence of clear evidence or guidelines to inform treatment choice. The more specific guidelines concerning metformin are well adhered to overall, with relatively little variation in metformin use across regions and over time. Recent prescribing increasingly favours the newer more expensive treatments, leading to a rapid increase in cost of prescribing over recent years.A key strength of the present analysis is that it uses three datasets, with overlapping strengths and weaknesses. The CPRD contains data on individual patients, which permits selection of only those with type 2 diabetes and investigation of the order in which medicines were prescribed for individual patients. However, the CPRD covers only a small subset of all prescribing, and does not permit exploration of individual institutions' prescribing at the level of identifiable CCGs and practices, while the PCA and practice‐level datasets cover the complete data for all primary care prescribing in England, not a sample, down to the level of all practices and CCGs, and PCA data cover all national‐level prescribing back to 1998.While prescriptions issued by a hospital clinic or private practice, or dispensed in hospital, are not included in our data, almost all prescriptions for glucose‐lowering agents in the United Kingdom are issued through a general practice. Even agents started on the recommendation of a hospital or community endocrinologist or other specialist will almost always be prescribed through general practice and captured in our data, with the exception being inpatient and emergency prescribing. Our data are therefore an accurate representation of UK prescribing practice. Using number of items prescribed in PCA and practice‐level data does not distinguish between different lengths of courses being prescribed , therefore drugs prescribed in shorter courses may amount to a greater total number of items. Converting into average daily quantities or defined daily doses would be an improvement, but a comprehensive dataset to render this calculation for all medications does not exist, and antidiabetes drugs may be given in different dosages. Using quantity instead would help to overcome this problem, but does not allow fair comparison between drugs given in different dosing regimens, or between items prescribed in different units of measurement such as liquids (mL) and tablets. In the CPRD, individual prescribing data were available, including dose and prescription frequency, and the finding of consistent results across CPRD and PCA data is a strength of the present study. Although the data do not indicate adherence to therapy, the focus of our study was on prescribing choices.Correcting for diabetes prevalence allowed us to investigate variation in prescribing independently from the increasing number of people living with the condition. National type 2 diabetes prevalence data for 1998 to 2016 were extrapolated from estimates from THIN practices.Similarly to the United Kingdom, type 2 diabetes guidelines from the American College of Physicians, the European Association for the Study of Diabetes and the American Diabetes Association all leave the choice of therapy after metformin largely to the practitionerWe found that metformin has been favoured as first‐line treatment since 2001, reflecting guidance and previous reports.The relationship between type 2 diabetes prescribing levels and HbA1c control across practices participating in the NDA (>50%) has recently been studied.Previous work has shown that responses to unclear guidelines can be variable. When a common antipsychotic drug had its licence severely restricted but no specific advice was given on which alternative drug should be prescribed, in Scotland chlorpromazine was the most common replacement, whereas in England it was a combination of chlorpromazine and two newer drugs,We found unexplained variation in choice of non‐metformin treatment, in the context of absence of clear advice in guidelines and current evidence. Aside from clinicians' personal choices, there may be a variety of external influences, including local policy, price changes, marketing, financial arrangements with drug companies, media reports, access to educational material, and drug safety alerts. The present findings raise various prospects and opportunities in diabetes research. Firstly, they suggest that a randomized trial of choice of second‐line medication would be clinically useful to resolve outstanding uncertainty on the best treatment for an extremely common clinical presentation. There is no such study ongoing in the United Kingdom, which seems a remarkable oversight, given that diabetes is the single biggest cost area for prescribing in NHS England. The one such study ongoing in the United States, GRADE, does not include SGLT‐2 inhibitor therapy.http://openprescribing.net project is an openly accessible data service which highlights prescribing variation in primary care, and allows practices and commissioners to monitor their own prescribing behaviour for key prescribing measures and any chemical of interest, using statistical process control techniques to send alerts automatically to practices when they deviate from national changes in behaviour (including on diabetes prescribing). We have previously argued that greater investment in disseminating evidence, auditing its implementation, and using variation in practice to target clinicians for educational interventions may all prove to be cost‐effective mechanisms to ensure that health services use treatments effectively and cost‐effectively.Thirdly, in the absence of guidance on which second‐line treatment is best, and with guidance only suggesting that the lowest cost options within each class are preferred, we found extensive variation in prescribing costs between CCGs. The total spend was £414 m over 12 months, but with a potential saving of £113 m if all CCGs had prescribed at the same per‐patient cost as the most efficient decile of CCGs; however, a full cost‐effectiveness analysis would require consideration of differences in side effects and cardiovascular outcomes across the different drug classes as well as consideration of non‐medicinal treatments. Our In conclusion, with a lack of good evidence to guide choice of second‐line treatment for diabetes, we found evidence of extensive variation in choice of drug, and prescription volumes for new treatments rising as they appear on the market, in the absence of good comparative effectiveness data.Appendix 1. Additional Tables and FiguresTable S1. Comparison of key features of each dataset used in our study, and the list of figures and tables for which each dataset is used.Table S2. Assignment of BNF drug names to diabetes drug classes.Table S3. Proportion of each drug class prescribed across England's practices, September 2016 to August 2017.Table S4. Links to updated maps as shown in Figure Figure S1. Prescribing trends for individual chemicals within each class of anti‐diabetes drug in England, 1998 to 2016.Figure S2. Map of total spend on anti‐diabetic drugs per patient and prescribing level of newer anti‐diabetic drugs, by CCG.Appendix 2. SQL and Python Codes for data extraction and analysis.Click here for additional data file.