ABSTRACT
Nucleosomes are the basic compaction unit of chromatin and nucleosome structure and their higher-order assemblies regulate genome accessibility. Many post-translational modifications alter nucleosome dynamics, nucleosome-nucleosome interactions, and ultimately chromatin structure and gene expression. Here, we investigate the role of two post-translational modifications associated with actively transcribed regions, H3K36me3 and H4K5/8/12/16ac, in the contexts of tri-nucleosome arrays that provide a tractable model system for quantitative single-molecule analysis, while enabling us to probe nucleosome-nucleosome interactions. Direct visualization by AFM imaging reveals that H3K36me3 and H4K5/8/12/16ac nucleosomes adopt significantly more open and loose conformations than unmodified nucleosomes. Similarly, magnetic tweezers force spectroscopy shows a reduction in DNA outer turn wrapping and nucleosome-nucleosome interactions for the modified nucleosomes. The results suggest that for H3K36me3 the increased breathing and outer DNA turn unwrapping seen in mononucleosomes propagates to more open conformations in nucleosome arrays. In contrast, the even more open structures of H4K5/8/12/16ac nucleosome arrays do not appear to derive from the dynamics of the constituent mononucleosomes, but are driven by reduced nucleosome-nucleosome interactions, suggesting that stacking interactions can overrule DNA breathing of individual nucleosomes. We anticipate that our methodology will be broadly applicable to reveal the influence of other post-translational modifications and to observe the activity of nucleosome remodelers.
Subject(s)
Histones , Nucleosomes , Protein Processing, Post-Translational , Nucleosomes/metabolism , Nucleosomes/chemistry , Histones/metabolism , Histones/chemistry , Epigenesis, Genetic , Histone Code , DNA/metabolism , DNA/chemistry , Nucleic Acid Conformation , Methylation , Microscopy, Atomic Force/methodsABSTRACT
This longitudinal prospective controlled multicenter study aimed to monitor immunity generated by three exposures caused by breakthrough infections (BTI) after COVID-19-vaccination considering pre-existing cell-mediated immunity to common-corona-viruses (CoV) which may impact cellular reactivity against SARS-CoV-2. Anti-SARS-CoV-2-spike-IgG antibodies (anti-S-IgG) and cellular reactivity against Spike-(S)- and nucleocapsid-(N)-proteins were determined in fully-vaccinated (F) individuals who either experienced BTI (F+BTI) or had booster vaccination (F+Booster) compared to partially vaccinated (P+BTI) and unvaccinated (U) from 1 to 24 weeks post PCR-confirmed infection. High avidity anti-S-IgG were found in F+BTI compared to U, the latter exhibiting increased long-lasting pro-inflammatory cytokines to S-stimulation. CoV was associated with higher cellular reactivity in U, whereas no association was seen in F. The study illustrates the induction of significant S-specific cellular responses in F+BTI building-up basic immunity by three exposures. Only U seem to benefit from pre-existing CoV immunity but demonstrated inflammatory immune responses compared to F+BTI who immunologically benefit from enhanced humoral and cellular immunity after BTI. This study demonstrates that individuals with hybrid immunity from COVID-19-vaccination and BTI acquire a stable humoral and cellular immune response that is maintained for at least 6 months. Our findings corroborate recommendations by health authorities to build on basic immunity by three S-protein exposures.
Subject(s)
Antibodies, Viral , COVID-19 Vaccines , COVID-19 , Immunity, Cellular , Spike Glycoprotein, Coronavirus , Adult , Aged , Female , Humans , Male , Middle Aged , 2019-nCoV Vaccine mRNA-1273/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , BNT162 Vaccine/immunology , BNT162 Vaccine/administration & dosage , Breakthrough Infections/immunology , Breakthrough Infections/prevention & control , Coronavirus Nucleocapsid Proteins/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , COVID-19 Vaccines/administration & dosage , Cytokines/immunology , Immunization, Secondary , Immunoglobulin G/blood , Longitudinal Studies , Phosphoproteins/immunology , Prospective Studies , Spike Glycoprotein, Coronavirus/immunology , VaccinationABSTRACT
How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.
ABSTRACT
How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. We investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner qualitatively agree with our data. We speculate that 'monkey-bar' mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.
ABSTRACT
Hypophosphatasia (HPP) is a rare disease affecting bone mineralization. Adults with HPP have an increased occurrence of low-energy fractures, which cannot be explained by reduced bone mass assessed by dual energy X-ray absorptiometry. The bone phenotype in adults with HPP requires further studies investigating bone strength and bone structural parameters. INTRODUCTION: Hypophosphatasia (HPP) is a rare inherited disorder of bone and mineral metabolism, characterized by broad-ranging clinical manifestations and severity. However, studies investigating the clinical spectrum in adults with HPP compared to a control group are scarce. The aim of this study was to evaluate biochemical and clinical characteristics as well as bone health in a Danish cohort of adults with HPP. METHODS: We conducted a cross-sectional study assessing biochemical parameters, fracture prevalence, bone mineral density (BMD), bone turnover markers, physical performance and pain characteristics in 40 adults with HPP and 40 sex-, age-, BMI- and menopausal status-matched healthy controls. RESULTS: Patients with HPP had a significantly higher prevalence of non-vertebral, low-energy fractures (p = < 0.001). BMD at the lumbar spine, total hip, femoral neck, forearm and whole body did not differ between the groups. Low levels of the bone-specific alkaline phosphatase correlated significantly with higher BMD at the lumbar spine and femoral neck in both groups. The bone formation marker N-terminal propeptide of type 1 procollagen was significantly lower in patients with HPP than healthy controls (p = 0.006). Adults with HPP had significantly reduced walking capability (p = < 0.001) and lower body strength (p = < 0.001). Chronic pain was significantly more prevalent in adults with HPP than the control group (p = 0.029). CONCLUSIONS: The increased occurrence of low-energy fractures in adults with HPP is not explained by low BMD. Adults with HPP have reduced physical performance when compared with healthy controls.
Subject(s)
Hypophosphatasia , Humans , Absorptiometry, Photon , Alkaline Phosphatase/genetics , Bone Density , Cross-Sectional Studies , Femur Neck , Hypophosphatasia/complications , Hypophosphatasia/epidemiology , Hypophosphatasia/genetics , AdultABSTRACT
BACKGROUND: Hypophosphatasia (HPP) is an autosomal recessive or dominate disease affecting bone mineralization, and adults with HPP are in risk to develop metatarsal stress fractures and femoral pseudofractures. Given to the scarce data on the bone quality and its association to the fracture risk in adults with HPP, this study aimed to evaluate bone turnover, bone strength and structure in adults with HPP. METHODS: In this cross-sectional study, we included 14 adults with genetically verified HPP and 14 sex-, age-, BMI-, and menopausal status-matched reference individuals. We analyzed bone turnover markers, and measured bone material strength index (BMSi) by impact microindentation. Bone geometry, volumetric density and bone microarchitecture as well as failure load at the distal radius and tibia were evaluated using a second-generation high-resolution peripheral quantitative computed tomography system. RESULTS: Bone turnover markers did not differ between patients with HPP and reference individuals. BMSi did not differ between the groups (67.90 [63.75-76.00] vs 65.45 [58.43-69.55], p = 0.149). Parameters of bone geometry and volumetric density did not differ between adults with HPP and the reference group. Patients with HPP had a tendency toward higher trabecular separation (0.664 [0.613-0.724] mm vs 0.620 [0.578-0.659] mm, p = 0.054) and inhomogeneity of trabecular network (0.253 [0.235-0.283] mm vs 0.229 [0.208-0.252] mm, p = 0.056) as well as lower trabecular bone volume fraction (18.8 [16.4-22.7] % vs 22.8 [20.6-24.7] %, p = 0.054) at the distal radius. In addition, compound heterozygous adults with HPP had a significantly higher cortical porosity at the distal radius than reference individuals (1.5 [0.9-2.2] % vs 0.7 [0.6-0.7] %, p = 0.041). CONCLUSIONS: BMSi is not reduced in adults with HPP. Increased cortical porosity may contribute to the occurrence of femoral pseudofractures in compound heterozygous adults with HPP. However, further studies investigating larger cohorts of adults with HPP using methods of bone histomorphometry are recommended to adequately assess the bone quality in adults with HPP.
Subject(s)
Bone Diseases, Metabolic , Hypophosphatasia , Absorptiometry, Photon , Adult , Bone Density , Cross-Sectional Studies , Humans , Hypophosphatasia/diagnostic imaging , Hypophosphatasia/genetics , Radius/diagnostic imaging , Tibia/diagnostic imagingABSTRACT
BACKGROUND: Hypophosphatasia (HPP) is an inborn disease caused by pathogenic variants in ALPL. Low levels of alkaline phosphatase (ALP) are a biochemical hallmark of the disease. Scarce knowledge about the prevalence of HPP in Scandinavia exists, and the variable clinical presentations make diagnostics challenging. The aim of this study was to investigate the prevalence of ALPL variants as well as the clinical and biochemical features among adults with endocrinological diagnoses and persistent hypophosphatasaemia. METHODS: A biochemical database containing ALP measurements of 26,121 individuals was reviewed to identify adults above 18 years of age with persistently low levels of ALP beneath range (≤ 35 ± 2.7 U/L). ALPL genetic testing, biochemical evaluations and assessment of clinical features by a systematic questionnaire among included patients, were performed. RESULTS: Among 24 participants, thirteen subjects (54.2%) revealed a disease-causing variant in ALPL and reported mild clinical features of HPP, of which musculoskeletal pain was the most frequently reported (n = 9). The variant c. 571G > A; p.(Glu191Lys) was identified in six subjects, and an unreported missense variant (c.1019A > C; p.(His340Pro)) as well as a deletion of exon 2 were detected by genetic screening. Biochemical analyses showed no significant differences in ALP (p = 0.059), the bone specific alkaline phosphatase (BALP) (p = 0.056) and pyridoxal-5'-phosphate (PLP) (p = 0.085) between patients with an ALPL variant and negative genetic screening. Patients with a variant in ALPL had significantly higher PLP levels than healthy controls (p = 0.002). We observed normal ALP activity in some patients classified as mild HPP, and slightly increased levels of PLP in two subjects with normal genetic screening and four healthy controls. Among 51 patients with persistent hypophosphatasaemia, fifteen subjects (29.4%) received antiresorptive treatment. Two patients with unrecognized HPP were treated with bisphosphonates and did not show complications due to the treatment. CONCLUSIONS: Pathogenic variants in ALPL are common among patients with endocrinological diagnoses and low ALP. Regarding diagnostics, genetic testing is necessary to identify mild HPP due to fluctuating biochemical findings. Antiresorptive treatment is a frequent reason for hypophosphatasaemia and effects of these agents in adults with a variant in ALPL and osteoporosis remain unclear and require further studies.
ABSTRACT
Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are gut hormones secreted postprandially. In healthy humans, both hormones decrease bone resorption accompanied by a rapid reduction in parathyroid hormone (PTH). The aim of this study was to investigate whether the changes in bone turnover after meal intake and after GIP- and GLP-2 injections, respectively, are mediated via a reduction in PTH secretion. This was tested in female patients with hypoparathyroidism given a standardized liquid mixed-meal test (n = 7) followed by a peptide injection test (n = 4) using a randomized crossover design. We observed that the meal- and GIP- but not the GLP-2-induced changes in bone turnover markers were preserved in the patients with hypoparathyroidism. To understand the underlying mechanisms, we examined the expression of the GIP receptor (GIPR) and the GLP-2 receptor (GLP-2R) in human osteoblasts and osteoclasts as well as in parathyroid tissue. The GIPR was expressed in both human osteoclasts and osteoblasts, whereas the GLP-2R was absent or only weakly expressed in osteoclasts. Furthermore, both GIPR and GLP-2R were expressed in parathyroid tissue. Our findings suggest that the GIP-induced effect on bone turnover may be mediated directly via GIPR expressed in osteoblasts and osteoclasts and that this may occur independent of PTH. In contrast, the effect of GLP-2 on bone turnover seems to depend on changes in PTH and may be mediated through GLP-2R in the parathyroid gland. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Subject(s)
Hypoparathyroidism , Receptors, Gastrointestinal Hormone , Cross-Over Studies , Female , Glucagon-Like Peptide 2 , Humans , Hypoparathyroidism/drug therapyABSTRACT
Bariatric surgery induces significant and sustained weight loss and subsequently reduces obesity-related comorbidities. However, only a small percentage of patients with severe obesity undergo bariatric surgery in Denmark. There is limited knowledge about the experiences with and possible reservations to bariatric surgery among secondary healthcare providers. The aim of this cross-sectional study was to investigate referral patterns and knowledge regarding the criteria for bariatric surgery among Danish secondary healthcare providers, treating obesity-related diseases. A questionnaire regarding experiences with and reservations to referring patients for consideration for bariatric surgery, along with thoughts to specific patient cases were sent to several specialists: endocrinologists, obstetricians and gynaecologists, orthopaedic surgeons and otorhinolaryngologists. Most questions required responses on a 5-point Likert scale and frequency distributions were calculated. A total of 345 (44%) specialists responded to the questionnaires. Good knowledge of the criteria for referral to bariatric surgery varied among the specialist from 6% to 68%. One of the main issues was a concern about the medical and surgical postoperative complications, which was a barrier to and influenced referral decisions. Furthermore, specialists were more likely to refer patients to bariatric surgery when patients requested this. Except for endocrinologists, the Danish secondary healthcare specialists interviewed have limited knowledge about bariatric surgery, which results in a reluctance in referring patients. Our results indicate that there is a need to improve knowledge among specialists, regarding the indications, criteria and outcomes for bariatric surgery to establish a more pro-active, specialist led approach to referrals.
Subject(s)
Attitude of Health Personnel , Bariatric Surgery , Obesity, Morbid/surgery , Physicians , Cross-Sectional Studies , Denmark/epidemiology , Humans , Physicians/psychology , Physicians/statistics & numerical data , Practice Patterns, Physicians'/statistics & numerical data , Referral and Consultation/statistics & numerical dataABSTRACT
Pycnodysostosis (PYCD) is a rare recessive inherited skeletal disease, characterized by short stature, brittle bones, and recurrent fractures, caused by variants in the Cathepsin K encoding gene that leads to impaired osteoclast-mediated bone resorption. Hypophosphatasia (HPP) is a dominant or recessive inherited condition representing a heterogeneous phenotype with dental symptoms, recurrent fractures, and musculoskeletal problems. The disease results from mutation(s) in the tissue non-specific alkaline phosphate encoding gene with reduced activity of alkaline phosphatase and secondarily defective mineralization of bone and teeth. Here, we present the first report of a patient with the coexistence of PYCD and HPP. This patient presented typical clinical findings of PYCD, including short stature, maxillary hypoplasia, and sleep apnoea. However, the burden of disease was caused by over 30 fractures, whereupon most showed delayed healing and non-union. Biochemical analysis revealed suppressed bone resorption and low bone formation capacity. We suggest that the coexistence of impaired bone resorption and mineralization may explain the severe bone phenotype with poor fracture healing.
Subject(s)
Fractures, Multiple/genetics , Hypophosphatasia/genetics , Mutation/genetics , Pycnodysostosis/genetics , Alkaline Phosphatase/genetics , Bone and Bones/metabolism , Cathepsin K/genetics , Female , Fracture Healing/genetics , Fractures, Bone/complications , Fractures, Bone/genetics , Humans , Hypophosphatasia/complications , Male , Pycnodysostosis/complicationsABSTRACT
Hypophosphatasia (HPP) is a rare inborn, metabolic bone disorder caused by mutations in the tissue-nonspecific alkaline phosphatase-encoding gene: ALPL. The diagnosis is based on biochemical, clinical and genetic evaluation. Low levels of alkaline phosphatase is a hallmark in diagnosing HPP. Mild forms may present unspecific symptoms and be more frequent than previously assumed. Adults with HPP may present with low bone mass, however, bisphosphonates are contra-indicated for these patients. Finally, enzyme replacement therapy has opened new therapeutic perspectives regarding severe HPP.
Subject(s)
Hypophosphatasia , Adult , Child , Child, Preschool , Diagnosis, Differential , Humans , Hypophosphatasia/classification , Hypophosphatasia/diagnosis , Hypophosphatasia/drug therapy , Hypophosphatasia/therapy , Infant , Tooth Loss/etiologyABSTRACT
ATP-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit or evict nucleosomes or reposition them along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here, we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. The first assay senses various structural changes to a specific nucleosome within the nucleosomal array whereas the second assay is specific toward detecting repositioning of nucleosomes within the array. Both assays exploit changes to the accessibility of DNA to restriction enzymes during the remodeling reaction.
Subject(s)
Chromatin Assembly and Disassembly , Electrophoresis, Agar Gel/methods , Nucleosomes/metabolism , Animals , Drosophila , Histones/metabolism , Substrate SpecificityABSTRACT
BACKGROUND: Bariatric surgery is the most effective treatment for severe obesity. It results in significant and sustained weight loss and reduces obesity-related co-morbidities. Despite an increasing prevalence of severe obesity, the number of bariatric operations performed in Denmark has decreased during the past years. This is only partly explained by changes in the national guidelines for bariatric surgery. The purpose of the cross-sectional study is to investigate referral patterns and possible reservations regarding bariatric surgery among Danish primary care physicians (PCPs). SETTING: Primary care physicians in Denmark METHODS: A total of 300 Danish PCPs were invited to participate in a questionnaire survey regarding experiences with bariatric surgery, reservations about bariatric surgery, attitudes to specific patient cases, and the future treatment of severe obesity. Most questions required a response on a 5-point Likert scale (strongly disagree, disagree, neither agree nor disagree, agree, and strongly agree) and frequency distributions were calculated. RESULTS: 133 completed questionnaires (44%) were returned. Most physicians found that they had good knowledge about the national referral criteria for bariatric surgery. With respect to the specific patient cases, a remarkably smaller part of physicians would refer patients on their own initiative, compared with the patient's initiative. Fear of postoperative surgical complications and medical complications both influenced markedly the decision to refer patients for surgery. Only 9% of the respondents indicated that bariatric surgery should be the primary treatment option for severe obesity in the future. CONCLUSION: Danish PCPs express severe concerns about surgical and medical complications following bariatric surgery. This might, in part, result in a low rate of referral to bariatric surgery.