Description of serum symmetric dimethylarginine concentration and of urinary SDS-AGE pattern in dogs with ACTH dependent hyperadrenocorticism

Serum symmetric dimethylarginine (SDMA) and patterns of urinary protein separated by sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE) have not been investigated as biomarkers in dogs with ACTH-dependent hyperadrenocorticism (ADHAC). This exploratory prospective study aimed to evaluate SDMA, serum creatinine (sCR), and SDS-AGE in dogs with ADHAC with and without proteinuria (ADHAC-P and ADHAC-nP, respectively). Thirty-five pet dogs classified as ADHAC-P (n=16), ADHAC-nP (n=6) and healthy (n=13) were included. Renal biomarkers were evaluated in all dogs at diagnosis. Baseline concentration of SDMA was not significantly different between the three groups (P = 0.15) whereas sCr was significantly lower in dogs in ADHAC dogs compared to healthy dogs (88.0 µmol/L [70.4-132.6; 79.2-114.4]) whether they had proteinuria or not (P = 0.014 and 0.002, respectively). However, baseline concentrations of sCr and SDMA were not significantly different between dogs with ADHAC-P dogs (SDMA, 8 µg/dL [5-12; 7-9]; sCr, 57.2 µmol/L [35.2-212.2; 52.8-92.4]) and ADHAC-nP dogs (SDMA, 8.5 µg/dL [7-13; 8-10]; sCr, 70.4 µmol/L [61.6-79.2; 61.6-70.4]) (P = 0.35 and P = 0.41, respectively). Proteinuria in dogs with ADHAC-P was mainly of glomerular origin (SDS-AGE pattern: glomerular in 10/16 dogs; mixed glomerular/tubular in four dogs). In our study, SDMA was neither significantly different in dogs with ADHAC whether they were proteinuric or not, nor between ADHAC and healthy dogs. Urinary electrophoresis provides additional information to the UPC and further investigations are needed to determine whether it may help identify dogs with ADHAC-P requiring specific antiproteinuric treatment.

Improved adherence to hip fracture standards reduces mortality after hip fractures.

BACKGROUND: Hip fractures are increasing in incidence due to increasing life expectancy. Mortality continues to improve but it is important to explore which factors are responsible for driving improvements. METHODS: A cohort of hip fracture patients predating SARS-CoV-2 was examined to determine the predictors of adherence to the six Irish Hip Fracture Standards (IHFS) and the impact of adherence on short (30 day) and long term (1 year) mortality. Our primary aim was assess the impact of a single HFS and cumulative number of HFS on mortality after hip fracture. Our secondary aim was to determine the impact of the HFS which are intrinsically linked to specialist Geriatric care. RESULTS: Across 962 patients, over 5 years, the factors which were associated with adherence to HFS were female gender, increasing ASA grade and being nursed on an orthopaedic ward. Patients with increasing ASA were more likely to have met HFS 4-6 (Geriatrician review HFS4, bone health HFS5 & specialist falls assessment HFS6), less likely to have surgery within 48 h are more likely to develop a pressure ulcer. If the patient was not nursed on an orthopaedic ward all HFS were less likely to be met. At 30 days HFS 4-6 were associated with a statistically significant odds ratio (OR) of being alive, while at one year HFS 1 (admitted to an orthopaedic ward within 4 h), 5 and 6 were associated with a statistically significant OR of being alive. As increasing numbers of hip fracture standards were met patients were more likely to be alive at 30 days and one year. CONCLUSION: This study has identified that improved adherence to hip fracture standards are associated with improved mortality at 30 days and one year.

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode.

Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps and size-based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells. For V590, chromatography with mixed mode cation exchange resins returned final product yields of ∼50% and logarithmic reduction values (LRVs) of 1.7->3.4 and 2.5-3.0 for host cell DNA (hcDNA) and host cell proteins (HCPs), respectively. For Measles, chromatography with mixed mode anion exchange resins returned final product yields of ∼50% and LRVs of 1.6 and 2.2 for hcDNA and HCPs, respectively. For both V590 and Measles processing, the employed resins cleared a key HCP, fibronectin, which could foul the UF/DF unit operation, and thusly enabling it to further reduce HCPs and to formulate the final LVV products. This integrated purification process utilizes the complementary action of the two unit operations and its applicability across LVVs supports its consideration for their processing.

Cortical stimulation leads to shortened myelin sheaths and increased axonal branching in spared axons after cervical spinal cord injury.

Neural activity and learning lead to myelin sheath plasticity in the intact central nervous system (CNS), but this plasticity has not been well-studied after CNS injury. In the context of spinal cord injury (SCI), demyelination occurs at the lesion site and natural remyelination of surviving axons can take months. To determine if neural activity modulates myelin and axon plasticity in the injured, adult CNS, we electrically stimulated the contralesional motor cortex at 10 Hz to drive neural activity in the corticospinal tract of rats with sub-chronic spinal contusion injuries. We quantified myelin and axonal characteristics by tracing corticospinal axons rostral to and at the lesion epicenter and identifying nodes of Ranvier by immunohistochemistry. Three weeks of daily stimulation induced very short myelin sheaths, axon branching, and thinner axons outside of the lesion zone, where remodeling has not previously been reported. Surprisingly, remodeling was particularly robust rostral to the injury which suggests that electrical stimulation can promote white matter plasticity even in areas not directly demyelinated by the contusion. Stimulation did not alter myelin or axons at the lesion site, which suggests that neuronal activity does not contribute to myelin remodeling near the injury in the sub-chronic period. These data are the first to demonstrate wide-scale remodeling of nodal and myelin structures of a mature, long-tract motor pathway in response to electrical stimulation. This finding suggests that neuromodulation promotes white matter plasticity in intact regions of pathways after injury and raises intriguing questions regarding the interplay between axonal and myelin plasticity.

Environmental life cycle assessment of production of the high intensity sweetener steviol glycosides from Stevia rebaudiana leaf grown in Europe: The SWEET project.

Purpose: There is an increasing interest in the use of non-nutritive sweeteners to replace added sugar in food and beverage products for reasons of improving consumer health. Much work has been done to understand safety of sweeteners, but very little on sustainability. To address that gap, this study presents the results of a life cycle assessment (LCA) of production of rebaudioside A 60%, 95% pure (RA60) steviol glycoside mix from Stevia rebaudiana leaf grown in Europe. Methods: An attributional cradle-to-factory-gate life cycle assessment was conducted on growing of stevia leaves and extraction of steviol glycosides in Europe. Primary data were used from a case study supply chain. Results are reported in impact categories from the ReCiPe 2016 (H) method, with focus given to global warming potential, freshwater eutrophication, water consumption, and land use. Impacts are expressed both in terms of production mass and sweetness equivalence, a common metric for understanding high intensity sweetener potency. Sweetness equivalence of RA60 is typically 200 to 300 times that of sugar. Comparison of environmental impact is made to sugar (sucrose) produced from both cane and beets. The research is part of the EU project SWEET (sweeteners and sweetness enhancers: impact on health, obesity, safety, and sustainability). Results and discussion: Global warming potential for production of RA60 was found to be 20.25 kgCO2-eq/kgRA60 on a mass basis and 0.081 kgCO2-eq/kgSE on a sweetness equivalence basis. Field production of stevia leaves was found to be the main source of impact for most impact categories, and for all four focus categories. Extraction of the RA60 was the main source of impact for the others. Leaf processing and seedling propagation were minor contributors to life cycle impact. Removal of international transport from the supply chain reduced global warming potential by 18.8%. Compared with sugar on a sweetness equivalence basis, RA60 has approximately 5.7% to 10.2% the impact for global warming potential, 5.6% to 7.2% the impact for land use, and is lower across most other impact categories. Conclusion: This is the first LCA of steviol glycoside mix RA60 produced from leaf in Europe. The results indicate that RA60 can be used to reduce environmental impact of providing a sweet taste by replacing sugar across all impact categories. However, it is important to note that specific formulations in which RA60 is used will have a bearing on the final environmental impact of any food or beverage products. For solid foods, this requires further research. Supplementary Information: The online version contains supplementary material available at 10.1007/s11367-022-02127-9.

Patient-reported health outcomes of SARS-CoV-2-tested patients presenting to emergency departments: a propensity score-matched prospective cohort study.

OBJECTIVE: This study aimed to compare the long-term physical and mental health outcomes of matched severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive and SARS-CoV-2-negative patients controlling for seasonal effects. STUDY DESIGN: This was a retrospective cohort study. METHODS: This study enrolled patients presenting to emergency departments participating in the Canadian COVID-19 Emergency Department Rapid Response Network. We enrolled consecutive eligible consenting patients who presented between March 1, 2020, and July 14, 2021, and were tested for SARS-CoV-2. Research assistants randomly selected four site and date-matched SARS-CoV-2-negative controls for every SARS-CoV-2-positive patient and interviewed them at least 30 days after discharge. We used propensity scores to match patients by baseline characteristics and used linear regression to compare Veterans RAND 12-item physical health component score (PCS) and mental health component scores (MCS), with higher scores indicating better self-reported health. RESULTS: We included 1170 SARS-CoV-2-positive patients and 3716 test-negative controls. The adjusted mean difference for PCS was 0.50 (95% confidence interval [CI]: -0.36, 1.36) and -1.01 (95% CI: -1.91, -0.11) for MCS. Severe disease was strongly associated with worse PCS (ß = -7.4; 95% CI: -9.8, -5.1), whereas prior mental health illness was strongly associated with worse MCS (ß = -5.4; 95% CI: -6.3, -4.5). CONCLUSION: Physical health, assessed by PCS, was similar between matched SARS-CoV-2-positive and SARS-CoV-2-negative patients, whereas mental health, assessed by MCS, was worse during a time when the public experienced barriers to care. These results may inform the development and prioritization of support programs for patients.

Feasibility study of computational occupational dosimetry: evaluating a proof-of-concept in an endovascular and interventional cardiology setting.

Individual monitoring of radiation workers is essential to ensure compliance with legal dose limits and to ensure that doses are As Low As Reasonably Achievable. However, large uncertainties still exist in personal dosimetry and there are issues with compliance and incorrect wearing of dosimeters. The objective of the PODIUM (Personal Online Dosimetry Using Computational Methods) project was to improve personal dosimetry by an innovative approach: the development of an online dosimetry application based on computer simulations without the use of physical dosimeters. Occupational doses were calculated based on the use of camera tracking devices, flexible individualised phantoms and data from the radiation source. When combined with fast Monte Carlo simulation codes, the aim was to perform personal dosimetry in real-time. A key component of the PODIUM project was to assess and validate the methodology in interventional radiology workplaces where improvements in dosimetry are needed. This paper describes the feasibility of implementing the PODIUM approach in a clinical setting. Validation was carried out using dosimeters worn by Vascular Surgeons and Interventional Cardiologists during patient procedures at a hospital in Ireland. Our preliminary results from this feasibility study show acceptable differences of the order of 40% between calculated and measured staff doses, in terms of the personal dose equivalent quantity Hp(10), however there is a greater deviation for more complex cases and improvements are needed. The challenges of using the system in busy interventional rooms have informed the future needs and applicability of PODIUM. The availability of an online personal dosimetry application has the potential to overcome problems that arise from the use of current dosimeters. In addition, it should increase awareness of radiation protection among staff. Some limitations remain and a second phase of development would be required to bring the PODIUM method into operation in a hospital setting. However, an early prototype system has been tested in a clinical setting and the results from this two-year proof-of-concept PODIUM project are very promising for future development.

Regeneration of Capto Core 700 resin through high throughput and laboratory scale studies and impact on production of a SARS-CoV-2 vaccine candidate.

During the development of a SARS-CoV-2 vaccine candidate, at the height of the COVID-19 pandemic, raw materials shortages, including chromatography resins, necessitated the determination of a cleaning in place (CIP) strategy for a multimodal core-shell resin both rapidly and efficiently. Here, the deployment of high throughput (HT) techniques to screen CIP conditions for cleaning Capto Core 700 resin exposed to clarified cell culture harvest (CCCH) of a SARS-CoV-2 vaccine candidate produced in Vero adherent cell culture are described. The best performing conditions, comprised of 30% n-propanol and ≥0.75 N NaOH, were deployed in cycling experiments, completed with miniature chromatography columns, to demonstrate their effectiveness. The success of the CIP strategy was ultimately verified at the laboratory scale. Here, its impact was assessed across the entire purification process which also included an ultrafiltration/diafiltration step. It is shown that the implementation of the CIP strategy enabled the re-use of the Capto Core 700 resin for up to 10 cycles without any negative impact on the purified product. Hence, the strategic combination of HT and laboratory-scale experiments can lead rapidly to robust CIP procedures, even for a challenging to clean resin, and thus help to overcome supply shortages.

Application of cation exchange chromatography in bind and elute and flowthrough mode for the purification of enteroviruses.

Members of the enterovirus genus are promising oncolytic agents. Their morphogenesis involves the generation of both genome-packed infectious capsids and empty capsids. The latter are typically considered as an impurity in need of removal from the final product. The separation of empty and full capsids can take place with centrifugation methods, which are of low throughput and poorly scalable, or scalable chromatographic processes, which typically require peak cutting and a significant trade-off between purity and yield. Here we demonstrate the application of packed bed cation exchange (CEX) column chromatography for the separation of empty capsids from infectious virions for a prototype strain of Coxsackievirus A21. This separation was developed using high throughput chromatography techniques and scaled up as a bind and elute polishing step. The separation was robust over a wide range of operating conditions and returned highly resolved empty and full capsids. The CEX step could be operated in bind and elute or flowthrough mode with similar selectivity and returned yields greater than 70% for full mature virus particles. Similar performance was also achieved using a selection of other bead based CEX chromatography media, demonstrating general applicability of this type of chromatography for Coxsackievirus A21 purification. These results highlight the wide applicability and excellent performance of CEX chromatography for the purification of enteroviruses, such as Coxsackievirus A21.

The very small angle neutron scattering instrument at the National Institute of Standards and Technology.

A description and the performance of the very small angle neutron scattering diffractometer at the National Institute of Standards and Technology are presented. The measurement range of the instrument extends over three decades of momentum transfer q from 2 × 10-4 to 0.7 Å-1. The entire scattering angle range from 8 × 10-5 to π/6 rad (30°) can be measured simultaneously using three separate detector carriages on rails holding nine 2D detector arrays. Versatile choices of collimation options and neutron wavelength selection allow the q resolution and beam intensity to be optimized for the needs of the experiment. High q resolution is achieved using multiple converging-beam collimation with circular pinholes combined with refractive lenses and prisms. Relaxed vertical resolution with much higher beam intensity can be achieved with narrow slit collimation and a broad wavelength range chosen by truncating the moderator source distribution below 4 Šwith a Be crystalline filter and above 8 Šwith a supermirror deflector. Polarized beam measurements with full polarization analysis are also provided by a high-performance supermirror polarizer and spin flipper, capable of producing flipping ratios of over 100, along with a high-efficiency 3He polarization analyzer.

In vitro reconstitution of substrate S-acylation by the zDHHC family of protein acyltransferases.

Protein S-acylation, more commonly known as protein palmitoylation, is a biological process defined by the covalent attachment of long chain fatty acids onto cysteine residues of a protein, effectively altering the local hydrophobicity and influencing its stability, localization and overall function. Observed ubiquitously in all eukaryotes, this post translational modification is mediated by the 23-member family of zDHHC protein acyltransferases in mammals. There are thousands of proteins that are S-acylated and multiple zDHHC enzymes can potentially act on a single substrate. Since its discovery, numerous methods have been developed for the identification of zDHHC substrates and the individual members of the family that catalyse their acylation. Despite these recent advances in assay development, there is a persistent gap in knowledge relating to zDHHC substrate specificity and recognition, that can only be thoroughly addressed through in vitro reconstitution. Herein, we will review the various methods currently available for reconstitution of protein S-acylation for the purposes of identifying enzyme-substrate pairs with a particular emphasis on the advantages and disadvantages of each approach.

Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

Idiopathic subglottic stenosis in Saskatchewan Hutterite population.

BACKGROUND: By studying the odds of developing idiopathic subglottic stenosis in the isolated and genetically unique Hutterite population, this study sought to strengthen the hypothesis that an underlying genetic predisposition may exist for its development. METHODS: A retrospective chart review examined the medical records of all adult patients treated for idiopathic subglottic stenosis in Saskatchewan between 2008 and 2018. Cases were segregated into Hutterite and non-Hutterite. RESULTS: Four out of 36 cases of idiopathic subglottic stenosis occurred among Hutterites. The odds of a Hutterite developing idiopathic subglottic stenosis are 21.89 times higher than for non-Hutterites. Positive family history was only observed in the Hutterite population. CONCLUSION: The study strengthens the hypothesis that genetics may play a role in the aetiology of idiopathic subglottic stenosis by demonstrating that the genetically and socially unique Hutterites are more likely to develop this rare disease. This study is the first to demonstrate that a specific subpopulation is at a higher risk for developing idiopathic subglottic stenosis.

A Multicentre Retrospective Study of Fulvestrant Use and Efficacy in Advanced/Metastatic Breast Cancer.

AIMS: Fulvestrant is a selective oestrogen receptor (ER) degrader used in postmenopausal women with hormone receptor-positive advanced breast cancer. The study aim was to analyse demographics and outcomes of UK patients treated with fulvestrant monotherapy at nine representative centres. MATERIALS AND METHODS: Medical records of 459 patients with locally advanced or metastatic ER-positive, HER2-negative breast cancer treated with fulvestrant between August 2011 and November 2018 at nine UK centres were reviewed. Data were collated on demographics, progression-free survival, overall survival and disease response at first radiological assessment following fulvestrant initiation. Patients still alive by December 2018 were censored. RESULTS: Data from 429 of the 459 patients identified were eligible for inclusion in the analysis. The median age was 69 (range 21-95) and 64% (n = 275) had Eastern Cooperative Oncology Group performance status 0-1. Bone was the most commonly involved metastatic site (72%, n = 306). However, 295 (69%) patients had visceral involvement. Patients had received a median 2 (range 0-5) prior lines of endocrine therapy and median 0 (range 0-6) prior chemotherapies. Fulvestrant was first-line therapy in 43 patients (10%). The median duration of treatment was 5 months (range 1-88). The median progression-free survival was 5.5 months. In 51% of 350 patients radiologically assessed, there was evidence of disease response to fulvestrant. Fifteen per cent of these had a complete/partial response. Fulvestrant was discontinued predominantly due to disease progression, with 3% discontinued solely due to adverse events. The median overall survival for the whole cohort was 22.5 months (range 0-88). CONCLUSIONS: This is one of the largest studied cohorts of breast cancer patients treated with fulvestrant. This heavily endocrine-pretreated population reflects real-life use in the UK. Within this context, our retrospective data show that patients can experience maintained disease response when treated with fulvestrant, supporting the importance of equitable availability for all UK patients.

Identifying the best predictive diagnostic criteria for psoriasis in children (< 18 years): a UK multicentre case-control diagnostic accuracy study (DIPSOC study).

BACKGROUND: In children, psoriasis can be challenging to diagnose. Difficulties arise from differences in the clinical presentation compared with adults. OBJECTIVES: To test the diagnostic accuracy of previously agreed consensus criteria and to develop a shortlist of the best predictive diagnostic criteria for childhood psoriasis. METHODS: A case-control diagnostic accuracy study in 12 UK dermatology departments (2017-2019) assessed 18 clinical criteria using blinded trained investigators. Children (< 18 years) with dermatologist-diagnosed psoriasis (cases, N = 170) or a different scaly inflammatory rash (controls, N = 160) were recruited. The best predictive criteria were identified using backward logistic regression, and internal validation was conducted using bootstrapping. RESULTS: The sensitivity of the consensus-agreed criteria and consensus scoring algorithm was 84·6%, the specificity was 65·1% and the area under the curve (AUC) was 0·75. The seven diagnostic criteria that performed best were: (i) scale and erythema in the scalp involving the hairline, (ii) scaly erythema inside the external auditory meatus, (iii) persistent well-demarcated erythematous rash anywhere on the body, (iv) persistent erythema in the umbilicus, (v) scaly erythematous plaques on the extensor surfaces of the elbows and/or knees, (vi) well-demarcated erythematous rash in the napkin area involving the crural fold and (vii) family history of psoriasis. The sensitivity of the best predictive model was 76·8%, with specificity 72·7% and AUC 0·84. The c-statistic optimism-adjusted shrinkage factor was 0·012. CONCLUSIONS: This study provides examination- and history-based data on the clinical features of psoriasis in children and proposes seven diagnostic criteria with good discriminatory ability in secondary-care patients. External validation is now needed.

Expanding the use of dolutegravir-based antiretroviral therapy in multidrug-resistant TB.

The wider availability of dolutegravir (DTG) containing HIV therapy for patients living with multidrug-resistant TB (MDR-TB) presents several advantages. DTG-based antiretroviral therapy (ART) has superior potency, reduces pill burden, and may reduce overall treatment-related toxicity, giving it the potential to improve outcomes in both diseases. While the uptake of DTG-based ART in programs where drug-resistant TB is treated remains unknown, there is early evidence from three programs that uptake is increasing. The use of DTG-based ART should be scaled-up, beginning with antiretroviral-naïve or virologically suppressed patients initiating MDR-TB treatment.