Optimisation of recombinant production of active human cardiac SERCA2a ATPase

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca2+ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins

Targeted microbubbles: a novel application for the treatment of kidney stones

Kidney stone disease is endemic. Extracorporeal shockwave lithotripsy was the first major technological breakthrough where focused shockwaves were used to fragment stones in the kidney or ureter. The shockwaves induced the formation of cavitation bubbles, whose collapse released energy at the stone, and the energy fragmented the kidney stones into pieces small enough to be passed spontaneously. Can the concept of microbubbles be used without the bulky machine? The logical progression was to manufacture these powerful microbubbles ex vivo and inject these bubbles directly into the collecting system. An external source can be used to induce cavitation once the microbubbles are at their target; the key is targeting these microbubbles to specifically bind to kidney stones. Two important observations have been established: (i) bisphosphonates attach to hydroxyapatite crystals with high affinity; and (ii) there is substantial hydroxyapatite in most kidney stones. The microbubbles can be equipped with bisphosphonate tags to specifically target kidney stones. These bubbles will preferentially bind to the stone and not surrounding tissue, reducing collateral damage. Ultrasound or another suitable form of energy is then applied causing the microbubbles to induce cavitation and fragment the stones. This can be used as an adjunct to ureteroscopy or percutaneous lithotripsy to aid in fragmentation. Randall's plaques, which also contain hydroxyapatite crystals, can also be targeted to pre-emptively destroy these stone precursors. Additionally, targeted microbubbles can aid in kidney stone diagnostics by virtue of being used as an adjunct to traditional imaging methods, especially useful in high-risk patient populations. This novel application of targeted microbubble technology not only represents the next frontier in minimally invasive stone surgery, but a platform technology for other areas of medicine

Delta chirality ruthenium ‘light-switch’ complexes can bind in the minor groove of DNA with five different binding modes

[Ru(phen)2(dppz)]2+ has been studied since the 1990s due to its “light-switch” properties. It can be used as a luminescent DNA probe, with emission switched on through DNA binding. The luminescence observed is dependent on the solvent accessibility of the pyrazine nitrogen atoms, and therefore is sensitive to changes in both binding site of the cation and chromophore orientation. The compound is also chiral, and there are distinct differences between the enantiomers in terms of the emission behaviour when bound to a variety of DNA sequences. Whilst a number of binary DNA-complex X-ray crystal structures is available, most include the Λ enantiomer, and there is very little structural information about binding of the Δ enantiomer. Here we present the first X-ray crystal structure of a Δ enantiomer bound to well-matched DNA, in the absence of the other, Λ, enantiomer. We show how the binding site observed here can be related to a more general pattern of motifs in the crystallographic literature and propose that the Δ enantiomer can bind with five different binding modes, offering a new hypothesis for the interpretation of solution data

Field Test of CO2 Injection in a Vertical Middle Bakken Well to Evaluate the Potential for Enhanced Oil Recovery and CO2 Storage

In 2017, an injection test was conducted in a vertical well completed in the Middle Member of the Bakken Formation. The objectives of the field test were to quantitatively determine the injectivity of an unstimulated Bakken reservoir and the ability of injected CO2 to mobilize oil.https://commons.und.edu/eerc-publications/1011/thumbnail.jp

Patient clinical documentation in telehealth environment: Are we collecting appropriate and sufficient information for best practice?

BACKGROUND: During the COVID-19 pandemic, the use of telehealth for patient visits grew rapidly and served an important role as a valuable and necessary resource. Although clinical documentation is critical for telehealth patient visits, there is limited information about how healthcare facilities manage telehealth patient visit documentation, technology used for telehealth visits, and challenges encountered with telehealth patient visit documentation. This study aimed to assess the use of telehealth during the pandemic, the quality of clinical documentation in telehealth practice and to identify challenges and issues encountered with telehealth patient visits in order to develop a strategy for best practices for telehealth documentation and data management. METHODS: Data were collected for this cross-sectional study in January-February 2021 via a self-designed survey of administrators/managers from physicians\u27 offices and mental health facilities. Survey questions included four categories: health organization demographic information; telehealth visits; clinical documentation for telehealth visit; and challenges and barriers related to telehealth documentation technology use. RESULTS: Of 76 respondents, more than half (62%) of the healthcare facilities started using telehealth for patient visits within one year of the onset of the COVID-19 pandemic, with 94% of respondents indicating an increased use of telehealth for patient visits since the pandemic. The most common types of telehealth patient care provided during the pandemic included pediatrics, primary care, cardiology, and women\u27s health. The most consistent data documentation of telehealth visits included: date of service, patient identification number, communication methods, patient informed consent, diagnosis and impression, evaluation results, and recommendations. The telehealth visit data was most commonly used for patient care and clinical practice, billing and reimbursement, quality improvement and patient satisfaction, and administrative planning. The top barriers to telehealth use by the healthcare professionals included patient challenges with telehealth services, such as inequities in quality of technology, lack of patient understanding, and lack of patient satisfaction; this was followed by frustration with constant updates of telehealth guidelines and procedures, understanding required telehealth documentation for reimbursement purposes, payer denial for telehealth visits, and legal and risk issues. CONCLUSIONS: Findings from this study can assist government entities, policymakers, and healthcare organizations in developing and advocating best practices in telehealth usage and clinical documentation improvement strategies

Adaptação de longo prazo ao treinamento cíclico induzido eletricamente em indivíduos com severa lesão na medula espinhal

Indivíduos com lesão da medula espinhal (LME) mais freqüentemente adquirem essa condição na juventude e são relegados a uma vida de maior ou menor inatividade física. Em adição às implicações primárias da LME, indivíduos com LME severa são estigmatizados e relegados a uma condição de vida física inativa. É desconhecido se essas condições relatadas são potencialmente reversíveis e o objetivo do presente estudo foi, portanto, examinar os efeitos do exercício em indivíduos com LME. Então, 10 indivíduos (6 com tetraplegia e 4 com paraplegia; idade de 27 a 45 anos; tempo de lesão de 3 a 23 anos) foram treinados por 1 ano em cicloergometria com estimulação elétrica controlada por feedback. Eles treinaram 3 vezes por semana (média 2,3 vezes), 30 minutos em cada sessão. Os músculos glúteos, isquiotibiais e quadríceps foram estimulados por eletrodos colocados na superfície da pele sobre seus pontos motores. Durante o primeiro treino, uma variação substancial na performance foi observada entre os pacientes. A maioria dos indivíduos foi capaz de realizar o exercício por 30 minutos na primeira sessão, mas dois indivíduos foram capazes de realizar o exercício por apenas poucos minutos. Depois do treino de 1 ano, todos os indivíduos foram capazes de realizar 30 minutos contínuos de treino e o trabalho produzido teve aumento de 4 ± 1 (média de “erro-padrão” EP) para 17 ± 2 kJ por sessão de treino (P < 0,05). A taxa de captação máxima de O2 durante o exercício com estimulação elétrica aumentou de 1,20 ± 0,08 l/min, mensurada depois de poucas semanas de exercício, para 1,43 ± 0,09 l/min após 1 ano de treinamento (P < 0,05). Imagens de corte com ressonância magnética foram feitas na coxa para avaliar a massa muscular, que teve um aumento de 12% (média, P < 0,05) em 1 ano de treinamento. Em biópsias feitas antes do exercício, vários estados de atrofia foram observados nas fibras musculares dos indivíduos, um fenômeno que foi parcialmente normalizado em todos os pacientes depois do treinamento. É sabido que a distribuição do tipo de fibra no músculo esquelético é alterada para fibras do tipo II B (contração rápida, rapidamente fatigável, glicolíticas) dentro dos primeiros 2 anos após a lesão medular. Nessa avaliação, os músculos continham 63% de miosina de cadeia pesada (MHC) isoforme II B, 33% de MHC isoforme II A (contração rápida e resistentes à fadiga) e menos de 5% de MHC isoforme I (fibras de contração lenta) antes do treinamento. Uma transformação para obterem-se fibras com proteínas contráteis mais resistentes à fadiga foi encontrada após 1 ano de treinamento. A porcentagem de MHC isoforme II A aumentou para 61% do total de proteínas contráteis e houve uma diminuição de 32% nas fibras rapidamente fatigáveis do tipo MHC isoforme II, enquanto as MHC isoformes I somente compunham 7% da quantidade total de MHC. Essa alteração foi acompanhada de um aumento de 100% na atividade enzimática da citrato sintetase, como um indicador da capacidade oxidativa mitocondrial. Conclui-se que as alterações na performance, nesse exercício e nas características do músculo esquelético, associadas à inatividade que ocorre em indivíduos com LME, são reversíveis, mesmo até 20 anos após a lesão. Sucede que o treino com exercícios induzidos por estimulação elétrica dos músculos paralisados é uma efetiva ferramenta de reabilitação que deveria ser oferecida aos indivíduos com LME no futuro.Spinal cord injured (SCI) individuals most often contract their injury at a young age and are deemed to a life of more or less physical inactivity. In addition to the primary implications of the SCI, severe SCI individuals are stigmatized by conditions related to their physically inactive lifestyle. It is unknown if these inactivity related conditions are potentially reversible and the aim of the present study was, therefore, to examine the effect of exercise on SCI individuals. Ten such individuals (six with tetraplegia and four with paraplegia; age 27-45 years; time since injury 3-23 years) were exercise trained for 1 year using an electrically induced computerized feedback controlled cycle ergometer. They trained for up to three times week (mean 2.3 times), 30 min on each occasion. The gluteal, hamstring and quadriceps muscles were stimulated via lectrodes placed on the skin over their motor points. During lie first training bouts, a substantial variation in performance was seen between the subjects. A majority of them were capable of performing 30 min of exercise in the first bout, however, two individuals were only able to perform a few minutes of exercise. After training for 1 year all of the subjects were able to perform 30 min of continuous training and the work output had increased from 4±1 (mean±SE) to 17±2 kilo Joules per training bout (P<0.05). The maximal oxygen uptake during electrically induced exercise increased from 1.20±0.08 litres per minute measured after a few weeks habituation to the exercise to 1.43±0.09 litres per minute after training for 1 year (P<0.05). Magnetic resonance cross sectional images of the thigh were performed to estimate muscle mass and an increase of 12% (mean, P<0.05) was seen in response to 1 year of training. In biopsies taken before exercise various degrees of atrophy were observed in the individual muscle fibres, a phenomenon that was partially normalized in all subjects after training. The fibre type distribution in skeletal muscles is known to shift towards type IIB fibres (fast twitch, fast fatiguable, glycolytic fibres) within the first 2 years after the spinal cord injury. The muscle in the present investigation contained 63% of myosin heavy chain (MHQ isoform IIB, 33% MHC isoform IIA (fast twitch, fatigue resistant) and less than 5% MHC isoform I (slow twitch) before training. A shift towards more fatigue resistant contractile proteins was found after 1 year of training. The percentage of MHC isoform IIA increased to 61% of all contractile protein and a corresponding decrease to 32% was seen in the fast fatiguable MHC isoform IIB, where as MHC 1 only comprised 7% of the total amount of MHC. This shift was accompanied by a doubling of the enzymatic activity of citrate synthase, as an indicator of mitochondrial oxidative capacity. It is concluded that inactivity-associated changes in exercise perfomance capacity and skeletal muscle occurring in SCI individuals after injury are reversible, even up to over 20 years after the injury. It follows that electrically induced exercise training of the paralysed limbs is an effective rehabilitation tool that should be offered to SCI individuals in the future

Avian seed dispersal may be insufficient for plants to track future temperature change on tropical mountains

AIM: Climate change causes shifts in species ranges globally. Terrestrial plant species often lag behind temperature shifts, and it is unclear to what extent animal-dispersed plants can track climate change. Here, we estimate the ability of bird-dispersed plant species to track future temperature change on a tropical mountain. LOCATION: Tropical elevational gradient (500–3500 m.a.s.l.) in the Manú biosphere reserve, Peru. TIME PERIOD: From 1960–1990 to 2061–2080. TAXA: Fleshy-fruited plants and avian frugivores. METHODS: Using simulations based on the functional traits of avian frugivores and fruiting plants, we quantified the number of long-distance dispersal (LDD) events that woody plant species would require to track projected temperature shifts on a tropical mountain by the year 2070 under different greenhouse gas emission scenarios [representative concentration pathway (RCP) 2.6, 4.5 and 8.5]. We applied this approach to 343 bird-dispersed woody plant species. RESULTS: Our simulations revealed that bird-dispersed plants differed in their climate-tracking ability, with large-fruited and canopy plants exhibiting a higher climate-tracking ability. Our simulations also suggested that even under scenarios of strong and intermediate mitigation of greenhouse gas emissions (RCP 2.6 and 4.5), sufficient upslope dispersal would require several LDD events by 2070, which is unlikely for the majority of woody plant species. Furthermore, the ability of plant species to track future changes in temperature increased in simulations with a low degree of trait matching between plants and birds, suggesting that plants in generalized seed-dispersal systems might be more resilient to climate change. MAIN CONCLUSION: Our study illustrates how the functional traits of plants and animals can inform predictive models of species dispersal and range shifts under climate change and suggests that the biodiversity of tropical mountain ecosystems is highly vulnerable to future warming. The increasing availability of functional trait data for plants and animals globally will allow parameterization of similar models for many other seed-dispersal systems

Antiproton Production in p+Ap+A Collisions at AGS Energies

Inclusive and semi-inclusive measurements are presented for antiproton ($\bar{p}$) production in proton-nucleus collisions at the AGS. The inclusive yields per event increase strongly with increasing beam energy and decrease slightly with increasing target mass. The $\bar{p}$ yield in 17.5 GeV/c p+Au collisions decreases with grey track multiplicity, $N_g$, for $N_g>0$, consistent with annihilation within the target nucleus. The relationship between $N_g$ and the number of scatterings of the proton in the nucleus is used to estimate the $\bar{p}$ annihilation cross section in the nuclear medium. The resulting cross section is at least a factor of five smaller than the free $\bar{p}-p$ annihilation cross section when assuming a small or negligible formation time. Only with a long formation time can the data be described with the free $\bar{p}-p$ annihilation cross section.Comment: 8 pages, 6 figure