Sympathetically cooled highly charged ions in a radio-frequency trap with superconducting magnetic shielding.

We sympathetically cool highly charged ions (HCI) in Coulomb crystals of Doppler-cooled Be+ ions confined in a cryogenic linear Paul trap that is integrated into a fully enclosing radio-frequency resonator manufactured from superconducting niobium. By preparing a single Be+ cooling ion and a single HCI, quantum logic spectroscopy toward frequency metrology and qubit operations with a great variety of species are enabled. While cooling down the assembly through its transition temperature into the superconducting state, an applied quantization magnetic field becomes persistent, and the trap becomes shielded from subsequent external electromagnetic fluctuations. Using a magnetically sensitive hyperfine transition of Be+ as a qubit, we measure the fractional decay rate of the stored magnetic field to be at the 10-10 s-1 level. Ramsey interferometry and spin-echo measurements yield coherence times of >400 ms, demonstrating excellent passive magnetic shielding at frequencies down to DC.

Variation in open access vildagliptin use in Waikato patients with type 2 diabetes.

AIM: To determine what the variation was in the initial use of vildagliptin in patients with type 2 diabetes following approval of open access funding in October 2018, including by ethnicity, gender, age, funding model and patient HbA1c levels. METHODS: Data were collected from 31 general practices for all adult patients with type 2 diabetes. National Health Index-matched medication data were obtained from the national Pharmaceutical Collection. Patients were included for analysis if they had received at least one diabetes medication in the 12 months prior to funding approval for vildagliptin. The proportion of patients who initiated vildagliptin therapy following open access funding approval was then evaluated, as was the time taken until the first dispensing (days since funding approval). RESULTS: A total of 724 of 3,971 (18.2%) of patients initiated vildagliptin therapy; mean time to first dispensing was 192.1±112.4 days. In logistic regression, Asian patients were more likely and Maori less likely to receive vildagliptin than Europeans. Younger patients and those with an HbA1c of >64mmol/mol were also more likely to initiate therapy. Vildagliptin use by general practice ranged from 0.0-82.4%. CONCLUSIONS: Despite open access funding, there was inequity in the initial use of vildagliptin. Substantial variation by general practice indicates that practitioner education may be needed to ensure appropriate and early adoption of new diabetes medications.

Outcomes from colonoscopy following referral from New Zealand general practice: a retrospective analysis.

BACKGROUND: New Zealand has high rates of colorectal cancer (CRC) but poor outcomes. Most patients with CRC are diagnosed following referral from general practice, where a general practitioner (GP) assesses symptoms according to national guidelines. All referred patients are then re-prioritised by the hospital system. The first objective of this study was to identify what proportion of patients referred by general practice to surgical/gastroenterology at Waikato District Health Board (DHB) had a colonoscopy. The second objective was to determine what proportion of these referrals have an underlying CRC and the factors associated with the likelihood of this diagnosis. METHODS: This study is a retrospective analysis of e-referral data for patients aged 30-70+ who were referred from 75 general practices to general surgery, gastroenterology or direct to colonoscopy at Waikato DHB, 01 January 2015-31 December 2017. Primary and secondary outcome measures included the proportion and characteristics of patients who were having colonoscopy, and of those, who were diagnosed with CRC. Data were analysed using chi square and logistic regression. RESULTS: 6718/20648 (32.5%) patients had a colonoscopy and 372 (5.5%) of these were diagnosed with CRC. The probability of having CRC following a colonoscopy increased with age (p value < 0.001). Females (p value < 0.001), non-Maori (p value < 0.001), and patients with a high suspicion of cancer (HSCan) label originating from their GP were more likely to have a colonoscopy, while the odds ratio of Maori having a colonoscopy was 0.66 (95% CI 0.60-0.73). The odds ratio of a CRC diagnosis following colonoscopy was 1.67 (95% CI 1.35-2.07) for men compared to women, and 2.34 (95% CI 1.70-3.22) for those with a GP HSCan label. Of the 585 patients referred with a GP HSCan, 423 (72.3%) were reprioritised by the hospital and 55 patients had their diagnosis unnecessarily delayed. CONCLUSIONS: If a GP refers a patient with an HSCan, and the patient receives a colonoscopy, then the likelihood of having CRC is almost 15.0%. This would suggest that these patients should be routinely prioritised without further triage by the hospital. Further research is needed to understand why Maori are less likely to receive a colonoscopy following referral from general practice.

Glycaemic control across the lifespan in a cohort of New Zealand patients with type 1 diabetes mellitus.

BACKGROUND: It is well known that tight glycaemic control reduces all-cause mortality and the development of microvascular complications in patients with type 1 diabetes mellitus (T1D), but that effective glycaemic control is difficult to achieve in different age groups. Currently, the state of glycaemic control across the lifespan in patients with T1D in New Zealand is not known. AIM: To determine the differences in glycaemic control with age, gender, rurality and ethnicity in patients with T1D in the Waikato region of New Zealand. METHODS: Retrospective review of clinical records of all patients with T1D on the Waikato Regional Diabetes Database in December 2017 (n = 1303). Glycaemic control was determined by the most recent HbA1c in the past 2 years. RESULTS: Median (25%, 75%) HbA1c was 67 (59, 81) mmol/mol (8.3%) and highest in those aged 15-29 years. Values exceeded clinical recommendations in 85.3% of all patients. Median HbA1c was lower in patients on insulin pump therapy than on multiple daily injections (63 (7.9%) versus 69 mmol/mol (8.5%); P < 0.001), though insulin pumps were significantly less likely to be used by Maori (P = 0.003) and men (P < 0.0001). Worsening glycaemic control was associated with increasing social deprivation (P < 0.001) but was not influenced by rural/urban living. CONCLUSIONS: Poor glycaemic control in Waikato patients with T1D is likely due to inequities in health care, including reduced access to insulin pump therapy, particularly in Maori and socially deprived populations.

Metformin adherence in patients with type 2 diabetes and its association with glycated haemoglobin levels.

INTRODUCTION Metformin is the initial medication of choice for most patients with type 2 diabetes. Non-adherence results in poorer glycaemic control and increased risk of complications. AIM The aim of this study was to characterise metformin adherence and association with glycated haemoglobin (HbA1c) levels in a cohort of patients with type 2 diabetes. METHODS Prescription and dispensing data were used for this study. Primary care clinical and demographic data were collected from 10 general practices (October 2016-March 2018) and linked to pharmaceutical dispensing information. Metformin adherence was initially measured by calculating the proportion of patients who had optimal medication cover for at least 80% of days (defined as a medication possession ratio (MPR) of ≥0.8), calculated using dispensing data. Prescription adherence was assessed by comparing prescription and dispensing data. The association between non-adherence (MPR <0.8) and HbA1c levels was also assessed. RESULTS Of the 1595 patients with ≥2 metformin prescriptions, the mean MPR was 0.87. Fewer Maori had an MPR ≥0.8 than New Zealand European (63.8% vs. 81.2%). Similarly, Maori received fewer metformin prescriptions (P=0.02), although prescription adherence did not differ by ethnicity. Prescription adherence was lower in younger patients (P=0.002). Mean HbA1c levels were reduced by 4.8 and 5.0mmol/mol, respectively, in all and Maori patients with an MPR ≥0.8. Total prescription adherence reduced HbA1c by 3.2mmol/mol (all P<0.01). DISCUSSION Ethnic disparity exists for metformin prescribing, leading to an overall reduction in metformin coverage for Maori patients. This needs to be explored further, including understanding whether this is a patient preference or health system issue.

Structural Basis of Protein Kinase R Autophosphorylation.

The RNA-activated protein kinase, PKR, is a key mediator of the innate immunity response to viral infection. Viral double-stranded RNAs induce PKR dimerization and autophosphorylation. The PKR kinase domain forms a back-to-back dimer. However, intermolecular ( trans) autophosphorylation is not feasible in this arrangement. We have obtained PKR kinase structures that resolves this dilemma. The kinase protomers interact via the known back-to-back interface as well as a front-to-front interface that is formed by exchange of activation segments. Mutational analysis of the front-to-front interface support a functional role in PKR activation. Molecular dynamics simulations reveal that the activation segment is highly dynamic in the front-to-front dimer and can adopt conformations conducive to phosphoryl transfer. We propose a mechanism where back-to-back dimerization induces a conformational change that activates PKR to phosphorylate a "substrate" kinase docked in a front-to-front geometry. This mechanism may be relevant to related kinases that phosphorylate the eukaryotic initiation factor eIF2α.

Interaction of PKR with single-stranded RNA.

Although the antiviral kinase PKR was originally characterized as a double-stranded RNA activated enzyme it can be stimulated by RNAs containing limited secondary structure. Single-stranded regions in such RNAs contribute to binding and activation but the mechanism is not understood. Here, we demonstrate that single-stranded RNAs bind to PKR with micromolar dissociation constants and can induce activation. Addition of a 5'-triphosphate slightly enhances binding affinity. Single-stranded RNAs also activate PKR constructs lacking the double-stranded RNA binding domain and bind to a basic region adjacent to the N-terminus of the kinase. However, the isolated kinase is not activated by and does not bind single-stranded RNA. Photocrosslinking measurements demonstrate that that the basic region interacts with RNA in the context of full length PKR. We propose that bivalent interactions with the double stranded RNA binding domain and the basic region underlie the ability of RNAs containing limited structure to activate PKR by enhancing binding affinity and thereby increasing the population of productive complexes containing two PKRs bound to a single RNA.

Activation of PKR by short stem-loop RNAs containing single-stranded arms.

Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem-loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5'-triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5'- and 3'-single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation.

RNA-activated protein kinase (PKR) is a key component of the interferon-induced antiviral pathway in higher eukaryotes. Upon recognition of viral dsRNA, PKR is activated via dimerization and autophosphorylation. PKR contains two N-terminal dsRNA binding domains (dsRBD) and a C-terminal kinase domain. The dsRBDs and the kinase are separated by a long, unstructured ∼80-amino acid linker in the human enzyme. The length of the N-terminal portion of the linker varies among PKR sequences, and it is completely absent in one ortholog. Here, we characterize the effects of deleting the variable region from the human enzyme to produce PKRΔV. The linker deletion results in quantitative but not qualitative changes in catalytic activity, RNA binding, and conformation. PKRΔV is somewhat more active and exhibits more cooperative RNA binding. As we previously observed for the full-length enzyme, PKRΔV is flexible in solution and adopts a range of compact and extended conformations. The conformational ensemble is biased toward compact states that might be related to weak interactions between the dsRBD and kinase domains. PKR retains RNA-induced autophosphorylation upon complete removal of the linker, indicating that the C-terminal, basic region is also not required for activity.

Benefits of automated crystallization plate tracking, imaging, and analysis.

We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems.