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A detailed methodology for a three-dimensional, self-structuring bone model that supports the differentiation of osteoblasts towards osteocytes and the production of a complex collagen-rich mineralised matrix.
BACKGROUND: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). METHODS: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. RESULTS: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. CONCLUSIONS: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.
Vital Signs: Health Disparities in Hemodialysis-Associated Staphylococcus aureus Bloodstream Infections - United States, 2017-2020.
INTRODUCTION: Racial and ethnic minorities are disproportionately affected by end-stage kidney disease (ESKD). ESKD patients on dialysis are at increased risk for Staphylococcus aureus bloodstream infections, but racial, ethnic, and socioeconomic disparities associated with this outcome are not well described. METHODS: Surveillance data from the 2020 National Healthcare Safety Network (NHSN) and the 2017-2020 Emerging Infections Program (EIP) were used to describe bloodstream infections among patients on hemodialysis (hemodialysis patients) and were linked to population-based data sources (CDC/Agency for Toxic Substances and Disease Registry [ATSDR] Social Vulnerability Index [SVI], United States Renal Data System [USRDS], and U.S. Census Bureau) to examine associations with race, ethnicity, and social determinants of health. RESULTS: In 2020, 4,840 dialysis facilities reported 14,822 bloodstream infections to NHSN; 34.2% were attributable to S. aureus . Among seven EIP sites, the S. aureus bloodstream infection rate during 2017-2020 was 100 times higher among hemodialysis patients (4,248 of 100,000 person-years) than among adults not on hemodialysis (42 of 100,000 person-years). Unadjusted S. aureus bloodstream infection rates were highest among non-Hispanic Black or African American (Black) and Hispanic or Latino (Hispanic) hemodialysis patients. Vascular access via central venous catheter was strongly associated with S. aureus bloodstream infections (NHSN: adjusted rate ratio [aRR] = 6.2; 95% CI = 5.7-6.7 versus fistula; EIP: aRR = 4.3; 95% CI = 3.9-4.8 versus fistula or graft). Adjusting for EIP site of residence, sex, and vascular access type, S. aureus bloodstream infection risk in EIP was highest in Hispanic patients (aRR = 1.4; 95% CI = 1.2-1.7 versus non-Hispanic White [White] patients), and patients aged 18-49 years (aRR = 1.7; 95% CI = 1.5-1.9 versus patients aged ≥65 years). Areas with higher poverty levels, crowding, and lower education levels accounted for disproportionately higher proportions of hemodialysis-associated S. aureus bloodstream infections. CONCLUSIONS AND IMPLICATIONS FOR PUBLIC HEALTH PRACTICE: Disparities exist in hemodialysis-associated S. aureus infections. Health care providers and public health professionals should prioritize prevention and optimized treatment of ESKD, identify and address barriers to lower-risk vascular access placement, and implement established best practices to prevent bloodstream infections.
Vital Signs: Health Disparities in Hemodialysis-Associated Staphylococcus aureus Bloodstream Infections - United States, 2017-2020.
INTRODUCTION: Racial and ethnic minorities are disproportionately affected by end-stage kidney disease (ESKD). ESKD patients on dialysis are at increased risk for Staphylococcus aureus bloodstream infections, but racial, ethnic, and socioeconomic disparities associated with this outcome are not well described. METHODS: Surveillance data from the 2020 National Healthcare Safety Network (NHSN) and the 2017-2020 Emerging Infections Program (EIP) were used to describe bloodstream infections among patients on hemodialysis (hemodialysis patients) and were linked to population-based data sources (CDC/Agency for Toxic Substances and Disease Registry [ATSDR] Social Vulnerability Index [SVI], United States Renal Data System [USRDS], and U.S. Census Bureau) to examine associations with race, ethnicity, and social determinants of health. RESULTS: In 2020, 4,840 dialysis facilities reported 14,822 bloodstream infections to NHSN; 34.2% were attributable to S. aureus. Among seven EIP sites, the S. aureus bloodstream infection rate during 2017-2020 was 100 times higher among hemodialysis patients (4,248 of 100,000 person-years) than among adults not on hemodialysis (42 of 100,000 person-years). Unadjusted S. aureus bloodstream infection rates were highest among non-Hispanic Black or African American (Black) and Hispanic or Latino (Hispanic) hemodialysis patients. Vascular access via central venous catheter was strongly associated with S. aureus bloodstream infections (NHSN: adjusted rate ratio [aRR] = 6.2; 95% CI = 5.7-6.7 versus fistula; EIP: aRR = 4.3; 95% CI = 3.9-4.8 versus fistula or graft). Adjusting for EIP site of residence, sex, and vascular access type, S. aureus bloodstream infection risk in EIP was highest in Hispanic patients (aRR = 1.4; 95% CI = 1.2-1.7 versus non-Hispanic White [White] patients), and patients aged 18-49 years (aRR = 1.7; 95% CI = 1.5-1.9 versus patients aged ≥65 years). Areas with higher poverty levels, crowding, and lower education levels accounted for disproportionately higher proportions of hemodialysis-associated S. aureus bloodstream infections. CONCLUSIONS AND IMPLICATIONS FOR PUBLIC HEALTH PRACTICE: Disparities exist in hemodialysis-associated S. aureus infections. Health care providers and public health professionals should prioritize prevention and optimized treatment of ESKD, identify and address barriers to lower-risk vascular access placement, and implement established best practices to prevent bloodstream infections.
Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties.
BACKGROUND: Cells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body. METHODS: A blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested. RESULTS: Chondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties. CONCLUSIONS: A DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.
National Healthcare Safety Network 2018 Baseline Neonatal Standardized Antimicrobial Administration Ratios.
BACKGROUND: The microbiologic etiologies, clinical manifestations, and antimicrobial treatment of neonatal infections differ substantially from infections in adult and pediatric patient populations. In 2019, the Centers for Disease Control and Prevention developed neonatal-specific (Standardized Antimicrobial Administration Ratios SAARs), a set of risk-adjusted antimicrobial use metrics that hospitals participating in the National Healthcare Safety Network's (NHSN's) antimicrobial use surveillance can use in their antibiotic stewardship programs (ASPs). METHODS: The Centers for Disease Control and Prevention, in collaboration with the Vermont Oxford Network, identified eligible patient care locations, defined SAAR agent categories, and implemented neonatal-specific NHSN Annual Hospital Survey questions to gather hospital-level data necessary for risk adjustment. SAAR predictive models were developed using 2018 data reported to NHSN from eligible neonatal units. RESULTS: The 2018 baseline neonatal SAAR models were developed for 7 SAAR antimicrobial agent categories using data reported from 324 neonatal units in 304 unique hospitals. Final models were used to calculate predicted antimicrobial days, the SAAR denominator, for level II neonatal special care nurseries and level II/III, III, and IV NICUs. CONCLUSIONS: NHSN's initial set of neonatal SAARs provides a way for hospital ASPs to assess whether antimicrobial agents in their facility are used at significantly higher or lower rates compared with a national baseline or whether an individual SAAR value is above or below a specific percentile on a given SAAR distribution, which can prompt investigations into prescribing practices and inform ASP interventions.
On/off switchable physical stimuli regulate the future direction of adherent cellular fate.
The utilization of cell-manipulating techniques reveals information about biological behaviors suited to address a wide range of questions in the field of life sciences. Here, we introduced an on/off switchable physical stimuli technique that offers precise stimuli for reversible cell patterning to allow regulation of the future direction of adherent cellular behavior by leveraging enzymatically degradable alginate hydrogels with defined chemistry and topography. As a proof of concept, targeted muscle cells adherent to TCP exhibited a reshaped structure when the hydrogel-based physical stimuli were applied. This simple tool offers easy manipulation of adherent cells to reshape their morphology and to influence future direction depending on the characteristics of the hydrogel without limitations of time and space. The findings from this study are broadly applicable to investigations into the relationships between cells and physiological extracellular matrix environments as well as has potential to open new horizons for regenerative medicine with manipulated cells.
Antimicrobial Use in US Hospitals: Comparison of Results From Emerging Infections Program Prevalence Surveys, 2015 and 2011.
BACKGROUND: In the 2011 US hospital prevalence survey of healthcare-associated infections and antimicrobial use 50% of patients received antimicrobial medications on the survey date or day before. More hospitals have since established antimicrobial stewardship programs. We repeated the survey in 2015 to determine antimicrobial use prevalence and describe changes since 2011. METHODS: The Centers for Disease Control and Prevention's Emerging Infections Program sites in 10 states each recruited ≤25 general and women's and children's hospitals. Hospitals selected a survey date from May-September 2015. Medical records for a random patient sample on the survey date were reviewed to collect data on antimicrobial medications administered on the survey date or day before. Percentages of patients on antimicrobial medications were compared; multivariable log-binomial regression modeling was used to evaluate factors associated with antimicrobial use. RESULTS: Of 12 299 patients in 199 hospitals, 6084 (49.5%; 95% CI, 48.6-50.4%) received antimicrobials. Among 148 hospitals in both surveys, overall antimicrobial use prevalence was similar in 2011 and 2015, although the percentage of neonatal critical care patients on antimicrobials was lower in 2015 (22.8% vs 32.0% [2011]; P = .006). Fluoroquinolone use was lower in 2015 (10.1% of patients vs 11.9% [2011]; P < .001). Third- or fourth-generation cephalosporin use was higher (12.2% vs 10.7% [2011]; P = .002), as was carbapenem use (3.7% vs 2.7% [2011]; P < .001). CONCLUSIONS: Overall hospital antimicrobial use prevalence was not different in 2011 and 2015; however, differences observed in selected patient or antimicrobial groups may provide evidence of stewardship impact.
Assessment of the Appropriateness of Antimicrobial Use in US Hospitals.
IMPORTANCE: Hospital antimicrobial consumption data are widely available; however, large-scale assessments of the quality of antimicrobial use in US hospitals are limited. OBJECTIVE: To evaluate the appropriateness of antimicrobial use for hospitalized patients treated for community-acquired pneumonia (CAP) or urinary tract infection (UTI) present at admission or for patients who had received fluoroquinolone or intravenous vancomycin treatment. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional study included data from a prevalence survey of hospitalized patients in 10 Emerging Infections Program sites. Random samples of inpatients on hospital survey dates from May 1 to September 30, 2015, were identified. Medical record data were collected for eligible patients with 1 or more of 4 treatment events (CAP, UTI, fluoroquinolone treatment, or vancomycin treatment), which were selected on the basis of common infection types reported and antimicrobials given to patients in the prevalence survey. Data were analyzed from August 1, 2017, to May 31, 2020. EXPOSURE: Antimicrobial treatment for CAP or UTI or with fluoroquinolones or vancomycin. MAIN OUTCOMES AND MEASURES: The percentage of antimicrobial use that was supported by medical record data (including infection signs and symptoms, microbiology test results, and antimicrobial treatment duration) or for which some aspect of use was unsupported. Unsupported antimicrobial use was defined as (1) use of antimicrobials to which the pathogen was not susceptible, use in the absence of documented infection signs or symptoms, or use without supporting microbiologic data; (2) use of antimicrobials that deviated from recommended guidelines; or (3) use that exceeded the recommended duration. RESULTS: Of 12 299 patients, 1566 patients (12.7%) in 192 hospitals were included; the median age was 67 years (interquartile range, 53-79 years), and 864 (55.2%) were female. A total of 219 patients (14.0%) were included in the CAP analysis, 452 (28.9%) in the UTI analysis, 550 (35.1%) in the fluoroquinolone analysis, and 403 (25.7%) in the vancomycin analysis; 58 patients (3.7%) were included in both fluoroquinolone and vancomycin analyses. Overall, treatment was unsupported for 876 of 1566 patients (55.9%; 95% CI, 53.5%-58.4%): 110 of 403 (27.3%) who received vancomycin, 256 of 550 (46.6%) who received fluoroquinolones, 347 of 452 (76.8%) with a diagnosis of UTI, and 174 of 219 (79.5%) with a diagnosis of CAP. Among patients with unsupported treatment, common reasons included excessive duration (103 of 174 patients with CAP [59.2%]) and lack of documented infection signs or symptoms (174 of 347 patients with UTI [50.1%]). CONCLUSIONS AND RELEVANCE: The findings suggest that standardized assessments of hospital antimicrobial prescribing quality can be used to estimate the appropriateness of antimicrobial use in large groups of hospitals. These assessments, performed over time, may inform evaluations of the effects of antimicrobial stewardship initiatives nationally.
Distinct genetic pathways define pre-malignant versus compensatory clonal hematopoiesis in Shwachman-Diamond syndrome.
To understand the mechanisms that mediate germline genetic leukemia predisposition, we studied the inherited ribosomopathy Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with high risk of myeloid malignancies at an early age. To define the mechanistic basis of clonal hematopoiesis in SDS, we investigate somatic mutations acquired by patients with SDS followed longitudinally. Here we report that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. We show that germline SBDS deficiency establishes a fitness constraint that drives selection of somatic clones via two distinct mechanisms with different clinical consequences. EIF6 inactivation mediates a compensatory pathway with limited leukemic potential by ameliorating the underlying SDS ribosome defect and enhancing clone fitness. TP53 mutations define a maladaptive pathway with enhanced leukemic potential by inactivating tumor suppressor checkpoints without correcting the ribosome defect. Subsequent development of leukemia was associated with acquisition of biallelic TP53 alterations. These results mechanistically link leukemia predisposition to germline genetic constraints on cellular fitness, and provide a rational framework for clinical surveillance strategies.
Adiponectin signalling in bone homeostasis, with age and in disease.
Adiponectin is the most abundant circulating adipokine and is primarily involved in glucose metabolism and insulin resistance. Within the bone, osteoblasts and osteoclasts express the adiponectin receptors, however, there are conflicting reports on the effects of adiponectin on bone formation and turnover. Many studies have shown a pro-osteogenic role for adiponectin in in vivo murine models and in vitro: with increased osteoblast differentiation and activity, alongside lower levels of osteoclastogenesis. However, human studies often demonstrate an inverse relationship between adiponectin concentration and bone activity. Moreover, the presence of multiple isoforms of adiponectin and multiple receptor subtypes has the potential to lead to more complex signalling and functional consequences. As such, we still do not fully understand the importance of the adiponectin signalling pathway in regulating bone homeostasis and repair in health, with age and in disease. In this review, we explore our current understanding of adiponectin bioactivity in the bone; the significance of its different isoforms; and how adiponectin biology is altered in disease. Ultimately, furthering our understanding of adiponectin regulation of bone biology is key to developing pharmacological and non-pharmacological (lifestyle) interventions that target adiponectin signalling to boost bone growth and repair in healthy ageing, following injury or in disease.
Opportunities and Challenges in Functional Genomics Research in Osteoporosis: Report From a Workshop Held by the Causes Working Group of the Osteoporosis and Bone Research Academy of the Royal Osteoporosis Society on October 5th 2020.
The discovery that sclerostin is the defective protein underlying the rare heritable bone mass disorder, sclerosteosis, ultimately led to development of anti-sclerostin antibodies as a new treatment for osteoporosis. In the era of large scale GWAS, many additional genetic signals associated with bone mass and related traits have since been reported. However, how best to interrogate these signals in order to identify the underlying gene responsible for these genetic associations, a prerequisite for identifying drug targets for further treatments, remains a challenge. The resources available for supporting functional genomics research continues to expand, exemplified by "multi-omics" database resources, with improved availability of datasets derived from bone tissues. These databases provide information about potential molecular mediators such as mRNA expression, protein expression, and DNA methylation levels, which can be interrogated to map genetic signals to specific genes based on identification of causal pathways between the genetic signal and the phenotype being studied. Functional evaluation of potential causative genes has been facilitated by characterization of the "osteocyte signature", by broad phenotyping of knockout mice with deletions of over 7,000 genes, in which more detailed skeletal phenotyping is currently being undertaken, and by development of zebrafish as a highly efficient additional in vivo model for functional studies of the skeleton. Looking to the future, this expanding repertoire of tools offers the hope of accurately defining the major genetic signals which contribute to osteoporosis. This may in turn lead to the identification of additional therapeutic targets, and ultimately new treatments for osteoporosis.
SIRT1 directly activates autophagy in human chondrocytes.
Osteoarthritis (OA) is the most common form of arthritis worldwide with no effective treatment. Ageing is the primary risk factor for OA. We sought to investigate if there is a distinct and functional convergence of ageing-related mechanisms SIRT1 and autophagy in chondrocytes. Our results show that, levels of SIRT1 are decreased in human normal aged and OA cartilage compared with young cartilage. Moreover, silencing SIRT1 in chondrocytes lead to decreased expression of chondrogenic markers but did not alter the expression of catabolic proteases. In contrast, activation of SIRT1 increased autophagy in chondrocytes by the deacetylation of lysine residues on crucial autophagy proteins (Beclin1, ATG5, ATG7, LC3). This activation was shown to be mTOR/ULK1 independent. Our results indicate that maintenance of autophagy in chondrocytes by SIRT1 is essential for preserving cartilage integrity throughout life and therefore is a target for drug intervention to protect against OA.
SIRT1 directly activates autophagy in human chondrocytes.
Osteoarthritis (OA) is the most common form of arthritis worldwide with no effective treatment. Ageing is the primary risk factor for OA. We sought to investigate if there is a distinct and functional convergence of ageing-related mechanisms SIRT1 and autophagy in chondrocytes. Our results show that, levels of SIRT1 are decreased in human normal aged and OA cartilage compared with young cartilage. Moreover, silencing SIRT1 in chondrocytes lead to decreased expression of chondrogenic markers but did not alter the expression of catabolic proteases. In contrast, activation of SIRT1 increased autophagy in chondrocytes by the deacetylation of lysine residues on crucial autophagy proteins (Beclin1, ATG5, ATG7, LC3). This activation was shown to be mTOR/ULK1 independent. Our results indicate that maintenance of autophagy in chondrocytes by SIRT1 is essential for preserving cartilage integrity throughout life and therefore is a target for drug intervention to protect against OA.
Diagnosis and management of de novo non-specific spinal infections: European Association of Neurosurgical Societies (EANS) Spine Section Delphi consensus recommendations.
INTRODUCTION: The management of de novo non-specific spinal infections (spondylodiscitis - SD) remains inconsistent due to varying clinical practices and a lack of high-level evidence, particularly regarding the indications for surgery. RESEARCH QUESTION: This study aims to develop consensus recommendations for the diagnosis and management of SD, addressing diagnostic modalities, surgical indications, and treatment strategies. MATERIAL AND METHODS: A Delphi process was conducted with 26 experts from the European Association of Neurosurgical Societies (EANS). Sixtytwo statements were developed on diagnostic workup, management decisions, surgical techniques, non-surgical treatment, and follow-up and submitted to the panel of experts. RESULTS: Consensus was reached on 38 of 62 statements. MRI was confirmed as the gold standard for diagnosis. Regarding surgical indications, the panel agreed that any new neurological deficit, even subtle, warrants surgical consideration. Motor deficits with a motor score (MRC) below 4 and bladder or bowel dysfunction were unanimously considered clear indications for surgery. For spinal deformity and instability, thresholds such as kyphosis >20°, scoliosis >10°, and vertebral body collapse >50% were established to guide surgical decision-making. Minimally invasive surgery (MIS) was endorsed whenever feasible, and a 12 week antibiotic treatment regimen was favored in cases of complicated infections. DISCUSSION AND CONCLUSION: This EANS consensus provides updated recommendations for SD management, incorporating recent evidence on improved outcomes with surgical therapy. While these guidelines offer a more structured approach to clinical decision-making, further research is required to optimize surgical timing and validate the long-term impact of these treatment strategies.
AO Spine Clinical Practice Recommendations for Diagnosis and Management of Degenerative Cervical Myelopathy: Evidence Based Decision Making - A Review of Cutting Edge Recent Literature Related to Degenerative Cervical Myelopathy.
Study DesignLiterature review of key topics related to degenerative cervical myelopathy (DCM) with critical appraisal and clinical recommendations.ObjectiveThis article summarizes several key current topics related to the management of DCM.MethodsRecent literature related to the management of DCM was reviewed. Four articles were selected and critically appraised. Recommendations were graded as Strong or Conditional.ResultsArticle 1: The Relationship Between pre-operative MRI Signal Intensity and outcomes. Conditional recommendation to use diffusion-weighted imaging MR signal changes in the cervical cord to evaluate prognosis following surgical intervention for DCM. Article 2: Efficacy and Safety of Surgery for Mild DCM. Conditional recommendation that surgery is a valid option for mild DCM with favourable clinical outcomes. Article 3: Effect of Ventral vs Dorsal Spinal Surgery on Patient-Reported Physical Functioning in Patients With Cervical Spondylotic Myelopathy: A Randomized Clinical Trial. Strong recommendation that there is equipoise in the outcomes of anterior vs posterior surgical approaches in cases where either technique could be used. Article 4: Machine learning-based cluster analysis of DCM phenotypes. Conditional recommendation that clinicians consider pain, medical frailty, and the impact on health-related quality of life when counselling patients.ConclusionsDCM requires a multidimensional assessment including neurological dysfunction, pain, impact on health-related quality of life, medical frailty and MR imaging changes in the cord. Surgical treatment is effective and is a valid option for mild DCM. In patients where either anterior or posterior surgical approaches can be used, both techniques afford similar clinical benefit albeit with different complication profiles.
An ALPK3 truncation variant causing autosomal dominant hypertrophic cardiomyopathy is partially rescued by mavacamten.
The ALPK3 gene encodes alpha-protein kinase 3, a cardiac pseudo-kinase of unknown function. Heterozygous truncating variants (ALPK3tv) can cause dominant adult-onset hypertrophic cardiomyopathy (HCM). Here we confirm an excess of ALPK3tv in sarcomere-gene negative HCM patients. Moreover, we generated a novel knock-in mouse model carrying an ALPK3tv (K201X). Homozygous animals displayed hypertrophy and systolic dysfunction. Heterozygous animals demonstrated no obvious baseline; however, they had an aggravated hypertrophic response upon chronic adrenergic challenge. Isolated, unloaded cardiomyocytes from heterozygous and homozygous mice showed reduced basal sarcomere length with prolonged relaxation, whilst calcium transients showed increased diastolic calcium levels. Protein kinase A-mediated phosphorylation, including that of cardiac troponin I, was significantly decreased. In agreement with the cellular HCM phenotype, reduced ratios of myosin heads in the super-relaxed state were measured. Contractile and calcium handling defects were partly corrected by treatment with mavacamten, a novel myosin inhibitor. For the first time with a non-sarcomere HCM variant, we have demonstrated hallmark changes in cardiac contractility and calcium handling. Mavacamten is able to partially rescue the cellular phenotype, hence could be beneficial to HCM patients with ALPK3tv. Moreover, our data points at a potential role of ALPK3 as a modulator of protein kinase A signalling.
Characterisation of infantile cardiomyopathy in Alström syndrome using ALMS1 knockout induced pluripotent stem cell derived cardiomyocyte model.
Alström syndrome (AS) is an inherited rare ciliopathy characterised by multi-organ dysfunction and premature cardiovascular disease. This may manifest as an infantile-onset dilated cardiomyopathy with significant associated mortality. An adult-onset restrictive cardiomyopathy may also feature later in life. Loss of function pathogenic variants in ALMS1 have been identified in AS patients, leading to a lack of ALMS1 protein. The biological role of ALMS1 is unknown, particularly in a cardiovascular context. To understand the role of ALMS1 in infantile cardiomyopathy, the reduction of ALMS1 protein seen in AS patients was modelled using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), in which ALMS1 was knocked out. MuscleMotion analysis and calcium optical mapping experiments suggest that ALMS1 knockout (KO) cells have increased contractility, with altered calcium extrusion and impaired calcium handling dynamics compared to wildtype (WT) counterparts. Seahorse metabolic assays showed ALMS1 knockout iPSC-CMs had increased glycolytic and mitochondrial respiration rates, with ALMS1 knockout cells portraying increased energetic demand and respiratory capacity than WT counterparts. Using senescence associated β-galactosidase (SA-β gal) staining assay, we identified increased senescence of ALMS1 knockout iPSC-CMs. Overall, this study provides insights into the molecular mechanisms in AS, particularly the role of ALMS1 in infantile cardiomyopathy in AS, using iPSC-CMs as a 'disease in a dish' model to provide insights into multiple aspects of this complex disease.
Cardiac function and energetics in mice with combined genetic augmentation of creatine and creatine kinase activity.
Improving energy provision in the failing heart by augmenting the creatine kinase (CK) system is a desirable therapeutic target. However, over-expression of the creatine transporter (CrT-OE) has shown that very high creatine levels result in cardiac hypertrophy and dysfunction. We hypothesise this is due to insufficient endogenous CK activity to maintain thermodynamically favourable metabolite ratios. If correct, then double transgenic mice (dTg) overexpressing both CrT and the muscle isoform of CK (CKM-OE) would rescue the adverse phenotype. In Study 1, overexpressing lines were crossed and cardiac function assessed by invasive haemodynamics and echocardiography. This demonstrated that CKM-OE was safe, but too few hearts had creatine in the toxic range. In Study 2, a novel CrT-OE line was generated with higher, homogeneous, creatine levels and phenotyped as before. Myocardial creatine was 4-fold higher in CrT-OE and dTg hearts compared to wildtype and was associated with hypertrophy and contractile dysfunction. The inability of dTg hearts to rescue this phenotype was attributed to downregulation of CK activity, as occurs in the failing heart. Nevertheless, combining both studies in a linear regression analysis suggests a modest positive effect of CKM over a range of creatine concentrations. In conclusion, we confirm that moderate elevation of creatine is well tolerated, but very high levels are detrimental. Correlation analysis lends support to the theory that this may be a consequence of limited CK activity. Future studies should focus on preventing CKM downregulation to unlock the potential synergy of augmenting both creatine and CK in the heart.
Clinical practice guidelines for the care of patients with a chronic subdural haematoma: multidisciplinary recommendations from presentation to recovery.
INTRODUCTION: A chronic subdural haematoma (cSDH) is an encapsulated collection of fluid and blood degradation products in the subdural space. It is increasingly common, affecting older people and those living with frailty. Currently, no guidance exists to define optimal care from onset of symptoms through to recovery. This paper presents the first consensus-built recommendations for best practice in the care of cSDH, co-designed to support each stage of the patient pathway. METHODS: Guideline development was led by a multidisciplinary Steering Committee with representation from diverse clinical groups, professional associations, patients, and carers. Literature searching to identify relevant evidence was guided by core clinical questions formulated through facilitated discussion with specially convened working groups. A modified Delphi exercise was undertaken to build consensus on draft statements for inclusion in the guideline using survey methodology and an in-person meeting. The proposed guideline was subsequently endorsed by the Society for British Neurological Surgeons, Neuroanaesthesia and Critical Care Society, Association of Anaesthetists, British Association of Neuroscience Nurses, British Geriatric Society, and Centre for Perioperative Care. RESULTS: We identified that high quality evidence was generally lacking in the literature, although randomised controlled trial (RCT) data were available to inform specific recommendations on aspects of surgical technique and use of corticosteroids. The final guideline represents the outcome of synthesising available evidence, consensus-built expert opinion and patient involvement. The guideline comprises 67 recommendations across eight major themes, covering: presentation and diagnosis, neurosurgical triage and shared decision-making, non-operative management, perioperative management (including anticoagulation), timing of surgery, intraoperative and postoperative care, rehabilitation and recovery. CONCLUSIONS: We present the first multidisciplinary guideline for the care of patients with cSDH. The recommendations reflect a paradigm shift in the care of cSDH, recognising and formalising the need for multidisciplinary and collaborative clinical management, communication and decision-making delivered effectively across secondary and tertiary care.