Small-scale approach for precrystallization screening in GPCR X-ray crystallography.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets. Knowledge of their 3D structures is critical to understanding mechanisms of drug action. Low cellular expression, purification yield, and in vitro instability are substantial hurdles to the successful determination of GPCR structure. Intense effort is required to optimize a receptor's protein sequence and purification procedure, increasing the complexity of the precrystallization process. Here, we present a procedure for a small-scale precrystallization screen that involves detecting GPCR expression levels in Spodoptera frugiperda (Sf9) culture by flow cytometry and evaluating GPCR stability by size-exclusion chromatography and UV absorbance measurements. The example procedure uses the smallest volumes of Sf9 cell culture that will yield sufficient quantities of purified protein for intrinsic UV absorbance analysis and is amenable to medium throughput with the same constructs and conditions that would be scaled up for crystallization trials. The protocol takes 8 d to complete and requires expertise in cell culture, protein purification, and chromatography.

Meridional flow in the Sun's convection zone is a single cell in each hemisphere.

The Sun's magnetic field is generated by subsurface motions of the convecting plasma. The latitude at which the magnetic field emerges through the solar surface (as sunspots) drifts toward the equator over the course of the 11-year solar cycle. We use helioseismology to infer the meridional flow (in the latitudinal and radial directions) over two solar cycles covering 1996-2019. Two data sources are used, which agree during their overlap period of 2001-2011. The time-averaged meridional flow is shown to be a single cell in each hemisphere, carrying plasma toward the equator at the base of the convection zone with a speed of ~4 meters per second at 45° latitude. Our results support the flux-transport dynamo model, which explains the drift of sunspot-emergence latitudes through the meridional flow.

De Novo Genome Sequence Assemblies of Gossypium raimondii and Gossypium turneri.

Cotton is an agriculturally important crop. Because of its importance, a genome sequence of a diploid cotton species (Gossypium raimondii, D-genome) was first assembled using Sanger sequencing data in 2012. Improvements to DNA sequencing technology have improved accuracy and correctness of assembled genome sequences. Here we report a new de novo genome assembly of G. raimondii and its close relative G. turneri The two genomes were assembled to a chromosome level using PacBio long-read technology, HiC, and Bionano optical mapping. This report corrects some minor assembly errors found in the Sanger assembly of G. raimondii We also compare the genome sequences of these two species for gene composition, repetitive element composition, and collinearity. Most of the identified structural rearrangements between these two species are due to intra-chromosomal inversions. More inversions were found in the G. turneri genome sequence than the G. raimondii genome sequence. These findings and updates to the D-genome sequence will improve accuracy and translation of genomics to cotton breeding and genetics.

Optometric referrals to retina specialists: evaluation and triage via teleophthalmology.

A retrospective noncomparative consecutive case series was conducted to evaluate the clinical outcomes of a novel teleophthalmology program linking optometrists to retina specialists in Alberta, Canada. One hundred seventy-one patients, referred by optometrists via teleophthalmology to a group retina practice between June 2004 and May 2006 underwent stereoscopic, mydriatic digital photography. Images were transmitted to a secure Web server and analyzed by a retina specialist. Diagnosis and recommendations were sent back to the optometrist and, if necessary, patients were referred for additional testing and clinical evaluation. A chart review of all clinical encounters was performed and the data was tabulated. Demographic features, diagnosis, testing, treatment, distance and time traveled by patient, durations between telemedicine referral, teleophthalmology consultation, in-person consultation, testing, and treatment were recorded. One hundred seventy patients were assessed via teleophthalmology for a total of 190 consultations. Eighty-nine patients (52.0%) required conventional in-person consultation with a referral completion success of 92.1% (82 patients). Fifty of these patients underwent additional diagnostic testing including fluorescein angiography (41), optical coherence tomography (14), laboratory testing (5), visual fields (2), carotid Doppler ultrasound (2), and ocular ultrasound (2). Twenty-five patients required surgical or medical treatment including focal argon laser (10), photodynamic therapy (8), panretinal photocoagulation (2), vitrectomy (2), scleral buckle (1), and other procedures (8). Average wait time between telemedicine referral and teleophthalmology review of images by the retina specialist was 1.9 days (maximum = 20 days). For those patients requiring office evaluation, the average wait time between teleophthalmology referral and in-person evaluation was 25.1 days. Twenty-one of the 25 patients (84.0%) requiring treatment underwent examination, testing, and treatment in a single day. When compared to conventional consultation methods, teleophthalmology reduced average travel distance and time by 219.1 km and 2.7 hours, respectively. Teleophthalmology reduced office visits to the retina specialist by 48% while improving the efficiency of clinical examination, testing, and treatment. Patients benefited through reduced travel time and distance.

A corrole nanobiologic elicits tissue-activated MRI contrast enhancement and tumor-targeted toxicity.

Water-soluble corroles with inherent fluorescence can form stable self-assemblies with tumor-targeted cell penetration proteins, and have been explored as agents for optical imaging and photosensitization of tumors in pre-clinical studies. However, the limited tissue-depth of excitation wavelengths limits their clinical applicability. To examine their utility in more clinically-relevant imaging and therapeutic modalities, here we have explored the use of corroles as contrast enhancing agents for magnetic resonance imaging (MRI), and evaluated their potential for tumor-selective delivery when encapsulated by a tumor-targeted polypeptide. We have found that a manganese-metallated corrole exhibits significant T1 relaxation shortening and MRI contrast enhancement that is blocked by particle formation in solution but yields considerable MRI contrast after tissue uptake. Cell entry but not low pH enables this. Additionally, the corrole elicited tumor-toxicity through the loss of mitochondrial membrane potential and cytoskeletal breakdown when delivered by the targeted polypeptide. The protein-corrole particle (which we call HerMn) exhibited improved therapeutic efficacy compared to current targeted therapies used in the clinic. Taken together with its tumor-preferential biodistribution, our findings indicate that HerMn can facilitate tumor-targeted toxicity after systemic delivery and tumor-selective MR imaging activatable by internalization.

Using health trainers to promote self-management of chronic pain: can it work?

BACKGROUND: In 2011, the Sheffield Primary Care Trust piloted a Health Trainer (HT) programme targeted specifically to people with chronic pain. The programme aimed to determine whether patients presenting to primary care with chronic pain would benefit from self-management support, thereby reducing the burden on primary care and secondary care services. METHODS: We conducted a formative mixed-methods evaluation of the pilot programme, focusing on four aspects of implementation: general practitioner (GP) referral to the programme, HT's ability to use cognitive behavioural (CB) approaches, short-term outcomes for clients and adequacy of resources. Qualitative data were collected via interviews with GPs, HTs and the chronic pain team; supervision sessions with HTs; and client case studies. Quantitative data were collected on satisfaction with training, HT's self-reported confidence to implement CB and clients' self-rated well-being before and after participation. RESULTS: A total of 143 clients with pain for 1 year or more were referred, exceeding the projected 90 referrals by over 50%. A total of 70% of the clients came from the most deprived areas of Sheffield, 40% were listed as permanently sick/disabled and only 20% were working. Qualitative analysis indicated that the CB training was delivered as intended. Clients reported that 75% of their goals were either achieved or partly achieved, and at follow-up 43% of them reported maintaining strategies for self-management. Financial resources were supplemented by indirect resources, including GP 'champions' with a special interest in pain, and a multidisciplinary chronic pain team. The prior history of working with community organizations was critical in ensuring credibility in client communities and addressing client needs. CONCLUSION: A HT programme promoting self-management of chronic pain can be successfully implemented when supported by community organisations. Preliminary data indicate that the programme can be instrumental in helping clients to actively participate in identifying their own problems, set achievable goals for self-management and successfully manage the challenges of everyday life. SUMMARY POINTS: Community-based Health Trainer programmes can be successfully established to promote self-management of chronic pain among clients in the deprived areas using multidisciplinary pain management teams. Utilising a community organization infrastructure that has experience of delivering successful programmes was instrumental in ensuring credibility of the initiative and access for clients. Health trainers can integrate cognitive behavioral training with their existing skills to work with clients who have chronic pain.

Relationships of intraosseous and systemic pressure waveforms in a Swine model.

BACKGROUND: Despite some focus on the use of intraosseous (IO) catheters to obtain laboratory samples, very little is known about the potential for obtaining other forms of clinical data. Largely unstudied is the relationship between IO pressures (IOPs) and systemic hemodynamic pressures such as mean arterial pressure (MAP) and central venous pressures (CVP). OBJECTIVES: The objective was to explore the relationship between hemodynamic parameters (blood pressures) measured through an IO catheter and intravascular catheters placed in the arterial and central venous circulation. METHODS: Eight pigs (Sus scrofa) weighing 30 to 45 kg were sedated with a short-acting agent, intubated with a cuffed endotracheal tube, and anesthetized with 2% to 3% isoflurane. Intravascular catheters were placed into the femoral or carotid artery and the femoral or jugular vein for MAP and CVP measurements. IO catheters, 15 mm for the sternum and 25 mm for the long bones, were placed percutaneously into the proximal tibia, proximal femur, proximal humerus, right proximal ulna, and/or sternum. Pressures were recorded during normotension, hypotension, and hypertension. RESULTS: Averaged across all eight animals, the means (ranges) for baseline systemic pressures were as follows: MAP = 66.5 (55.6 to 76.7) mm Hg, tibia IOP = 17.4 (9.3 to 34.5) mm Hg, femur IOP =18.4 (3.3 to 33.1) mm Hg, humerus IOP = 15.7 (2.8 to 28.9) mm Hg, ulna IOP = 16.0 (7.9 to 25.6) mm Hg, sternum IOP = 5.7 (-0.5 to 47.9) mm Hg, and CVP = 2.7 mm Hg (-3.3 to 7.9) mm Hg. The best median correlation occurred between femur IOP and mean MAP (r = 0.65). The four highest correlations between IOP and MAP were associated with mean femur IOP. Only one IO site had a correlation coefficient over 0.50 for CVP. The long bones tended to correlate better with the MAP and the sternum tended to correlate better with the CVP. Nonlinearity was observed in the actively rising pressure phases, which can be explained by a hysteresis model. CONCLUSIONS: The relationship between IOP and MAP or CVP is variable by site, with the MAP and CVP tending to be estimated by the femur and sternum, respectively. The relationship to actively rising pressures is nonlinear and a hysteresis model is proposed to explain the phase change. Further experimentation is needed to refine the IOP relationship to the MAP and CVP and assess the potential of these measurements to provide clinically relevant information.

Disparities in healthcare utilisation rates for Aboriginal and non-Aboriginal Albertan residents, 1997-2006: a population database study.

BACKGROUND: It is widely recognised that significant discrepancies exist between the health of indigenous and non-indigenous populations. Whilst the reasons are incompletely defined, one potential cause is that indigenous communities do not access healthcare to the same extent. We investigated healthcare utilisation rates in the Canadian Aboriginal population to elucidate the contribution of this fundamental social determinant for health to such disparities. METHODS: Healthcare utilisation data over a nine-year period were analysed for a cohort of nearly two million individuals to determine the rates at which Aboriginal and non-Aboriginal populations utilised two specialties (Cardiology and Ophthalmology) in Alberta, Canada. Unadjusted and adjusted healthcare utilisation rates obtained by mixed linear and Poisson regressions, respectively, were compared amongst three population groups - federally registered Aboriginals, individuals receiving welfare, and other Albertans. RESULTS: Healthcare utilisation rates for Aboriginals were substantially lower than those of non-Aboriginals and welfare recipients at each time point and subspecialty studied [e.g. During 2005/06, unadjusted Cardiology utilisation rates were 0.28% (Aboriginal, n = 97,080), 0.93% (non-Aboriginal, n = 1,720,041) and 1.37% (Welfare, n = 52,514), p = <0.001]. The age distribution of the Aboriginal population was markedly different [2.7%≥65 years of age, non-Aboriginal 10.7%], and comparable utilisation rates were obtained after adjustment for fiscal year and estimated life expectancy [Cardiology: Incidence Rate Ratio 0.66, Ophthalmology: IRR 0.85]. DISCUSSION: The analysis revealed that Aboriginal people utilised subspecialty healthcare at a consistently lower rate than either comparatively economically disadvantaged groups or the general population. Notably, the differences were relatively invariant between the major provincial centres and over a nine year period. Addressing the causes of these discrepancies is essential for reducing marked health disparities, and so improving the health of Aboriginal people.