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Hypophosphatasia (HPP) is a rare, inherited, and systemic disorder characterized by impaired skeletal mineralization and low tissue nonspecific serum alkaline phosphatase (TNSALP) activity. It is caused by either autosomal recessive or dominant-negative mutations in the gene that encodes TNSALP. The phenotype of HPP is very broad including abnormal bone mineralization, disturbances of calcium and phosphate metabolism, pain, recurrent fracture, short stature, respiratory impairment, developmental delay, tooth loss, seizures, and premature death. Other than supportive care, there has been no disease-specific treatment available for those with HPP. Asfotase alfa is a fully humanized, recombinant enzyme replacement therapy for the management of HPP. It is available in several countries for the treatment of the more severe forms of HPP, namely perinatal and infantile HPP. This review will summarize the preclinical data on asfotase alfa and highlight the data from clinical trials and case reports. These data show the transformative nature of asfotase alfa when administered as part of an interdisciplinary treatment model.
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Neurofibromatosis type 1 (NF1) is a complex genetic disorder that affects a range of tissues including muscle and bone. Recent preclinical and clinical studies have shown that Nf1 deficiency in muscle causes metabolic changes resulting in intramyocellular lipid accumulation and muscle weakness. These can be subsequently rescued by dietary interventions aimed at modulating lipid availability and metabolism. It was speculated that the modified diet may rescue defects in cortical bone as NF1 deficiency has been reported to affect genes involved with lipid metabolism. Bone specimens were analyzed from wild type control mice as well as Nf1Prx1-/- (limb-targeted Nf1 knockout mice) fed standard chow versus a range of modified chows hypothesized to influence lipid metabolism. Mice were fed from 4 weeks to 12 weeks of age. MicroCT analysis was performed on the cortical bone to examine standard parameters (bone volume, tissue mineral density, cortical thickness) and specific porosity measures (closed pores corresponding to osteocyte lacunae, and larger open pores). Nf1Prx1-/- bones were found to have inferior bone properties to wild type bones, with a 4-fold increase in the porosity attributed to open pores. These measures were rescued by dietary interventions including a L-carnitine + medium-chain fatty acid supplemented chow previously shown to improve muscle histology function. Histological staining visualized these changes in bone porosity. These data support the concept that lipid metabolism may have a mechanistic impact on bone porosity and quality in NF1.
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Modelos Animais de Doenças , Camundongos Knockout , Neurofibromatose 1 , Animais , Neurofibromatose 1/dietoterapia , Neurofibromatose 1/patologia , Neurofibromatose 1/metabolismo , Neurofibromatose 1/genética , Camundongos , Fenótipo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Porosidade , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Metabolismo dos Lipídeos , Microtomografia por Raio-X , Masculino , Densidade Óssea , DietaRESUMO
OBJECTIVE: The prevalence of comorbid chronic kidney disease (CKD) and osteoarthritis (OA) is increasing globally. While sharing common risk factors, the mechanism and consequences of concurrent CKD-OA are unclear. The aims of the study were to develop a preclinical comorbid model, and to investigate the disease-modifying interactions. METHODS: Seventy (70) male 8-10 week-old C57BL/6 mice were subjected to 5/6 nephrectomy (5/6Nx)±destabilisation of medial meniscus (DMM) or sham surgery. OA pathology and CKD were assessed 12 weeks postinduction by blinded histology scoring, micro-CT, immunohistochemistry for osteoclast and matrix metalloproteinase (MMP)-13 activity, and serum analysis of bone metabolic markers. RESULTS: The 5/6Nx model recapitulated characteristic features of CKD, with renal fibrosis and deranged serum alkaline phosphatase, calcium and phosphate. There was no histological evidence of cartilage pathology induced by 5/6Nx alone, however, synovial MMP-13 expression and subchondral bone osteoclastic activity were increased (p<0.05), with accompanying reductions (p<0.05) in subchondral trabecular bone, bone volume and mineral density. DMM significantly (p<0.05) increased tibiofemoral cartilage damage, subchondral bone sclerosis, marginal osteophytes and synovitis, in association with increased cartilage and synovial MMP-13. DMM alone induced (p<0.05) renal fibrosis, proteinuria and increased (p<0.05) 5/6Nx-induced serum urea. However, DMM in 5/6Nx-mice resulted in significantly reduced (p<0.05) cartilage pathology and marginal osteophyte development, in association with reduced subchondral bone volume and density, and inhibition of 5/6Nx-induced subchondral bone osteoclast activation. CONCLUSION: This study assessed a world-first preclinical comorbid CKD-OA model. Our findings demonstrate significant bidirectional disease-modifying interaction between CKD and OA.
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Osteoartrite , Osteófito , Masculino , Camundongos , Humanos , Animais , Metaloproteinase 13 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Osteoartrite/patologia , Modelos Animais de Doenças , Osteófito/patologia , FibroseRESUMO
Senolytics are a category of drugs that reduce the impact of cellular senescence, an effect associated with a range of chronic and age-related diseases. Since the discovery of the first senolytics in 2015, the number of known senolytic agents has grown dramatically. This review discusses the broad categories of known senolytics-kinase inhibitors, Bcl-2 family protein inhibitors, naturally occurring polyphenols, heat shock protein inhibitors, BET family protein inhibitors, P53 stabilizers, repurposed anti-cancer drugs, cardiac steroids, PPAR-alpha agonists, and antibiotics. The approaches used to screen for new senolytics are articulated including a range of methods to induce senescence, different target cell types, various senolytic assays, and markers. The choice of methods can greatly influence the outcomes of a screen, with high-quality screens featuring robust systems, adequate controls, and extensive validation in alternate assays. Recent advances in single-cell analysis and computational methods for senolytic identification are also discussed. There is significant potential for further drug discovery, but this will require additional research into drug targets and mechanisms of actions and their subsequent rigorous evaluation in pre-clinical models and human trials.
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Antineoplásicos , Senoterapia , Humanos , Senescência Celular , Antineoplásicos/farmacologia , Descoberta de DrogasRESUMO
Introduction: Osteomyelitis remains a major clinical challenge. Many published rodent fracture infection models are costly compared with murine models for rapid screening and proof-of-concept studies. We aimed to develop a dependable and cost-effective murine bone infection model that mimics bacterial bone infections associated with biofilm and metal implants. Methods: Tibial drilled hole (TDH) and needle insertion surgery (NIS) infection models were compared in C57BL/6 mice (female, N = 150 ). Metal pins were inserted selectively into the medullary canal adjacent to the defect sites on the metaphysis. Free Staphylococcus aureus (ATCC 12600) or biofilm suspension (ATCC 25923) was locally inoculated. Animals were monitored for physiological or radiographic evidence of infection without prophylactic antibiotics for up to 14â¯d. At the end point, bone swabs, soft-tissue biopsies, and metal pins were taken for cultures. X-ray and micro-CT scans were performed along with histology analysis. Results: TDH and NIS both achieved a 100â¯% infection rate in tibiae when a metal implant was present with injection of free bacteria. In the absence of an implant, inoculation with a bacterial biofilm still induced a 40â¯%-50â¯% infection rate. In contrast, freely suspended bacteria and no implant consistently showed lower or negligible infection rates. Micro-CT analysis confirmed that biofilm infection caused local bone loss even without a metal implant as a nidus. Although a metal surface permissive for biofilm formation is impermeable to create progressive bone infections in animal models, the metal implant can be dismissed if a bacterial biofilm is used. Conclusion: These models have a high potential utility for modeling surgery-related osteomyelitis, with NIS being simpler to perform than TDH.
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PURPOSE: The creation of murine gene knockout models to study bone gene functions often requires the resource intensive crossbreeding of Cre transgenic and gene-floxed strains. The developmental versus postnatal roles of genes can be difficult to discern in such models. For example, embryonic deletion of the Sclerostin (Sost) gene establishes a high-bone mass phenotype in neonatal mice that may impact on future bone growth. To generate a postnatal skeletal knockout of Sost in adult mice, this study used a single injection of a bone-targeted recombinant adeno-associated virus (rAAV) vector. METHODS: 8-week-old Sostflox/flox mice were injected with saline (control) or a single injection containing 5 × 1011 vg AAV8-Sp7-Cre vector. Ai9 fluorescent Cre reporter mice were dosed in parallel to confirm targeting efficiency. After 6 weeks, detailed bone analysis was performed via microCT, biomechanical testing, and bone histology on vertebral and long bone specimens. RESULTS: The AAV8-Sp7-Cre vector induced widespread persistent recombination in the bone compartment. Regional microCT analyses revealed significant increases in bone with vector treatment. In the L3 vertebrae, Sostflox/flox:AAV-Cre showed a 22 % increase in bone volume and 21 % in trabecular bone fraction compared to controls; this translated to a 17 % increase in compressive strength. In the tibiae, Sostflox/flox:AAV-Cre led to small but statistically significant increases in cortical bone volume and thickness. These were consistent with a 25 % increase in mineral apposition rate, but this did not translate into increased four-point bending strength. Ploton silver nitrate stain on histological sections revealed an unexpected increase in canalicular density associated with Sost ablation. CONCLUSION: This report demonstrates a proof-of-concept that the AAV8-Sp7-Cre vector can efficiently produce postnatal skeletal knockout mice using gene-floxed strains. This technology has the potential for broad utility in the bone field with existing conditional lines. These data also confirm an important postnatal role for Sost in regulating bone homeostasis, consistent with prior studies using neutralizing Sclerostin antibodies, and highlights a novel role of Sost in canalicular remodeling.
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Proteínas Adaptadoras de Transdução de Sinal , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Animais , Proteínas Adaptadoras de Transdução de Sinal/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Osteogênese , Camundongos KnockoutRESUMO
Osteogenesis imperfecta (OI) is a genetic bone fragility disorder that features frequent fractures. Bone healing outcomes are contingent on a proper balance between bone formation and resorption, and drugs such as bone morphogenetic proteins (BMPs) and bisphosphonates (BPs) have shown to have utility in modulating fracture repair. While BPs are used for OI to increase BMD and reduce pain and fracture rates, there is little evidence for using BMPs as local agents for fracture healing (alone or with BPs). In this study, we examined wild-type and OI mice (Col1a2+/G610C ) in a murine tibial open fracture model with (i) surgery only/no treatment, (ii) local BMP-2 (10 µg), or (iii) local BMP-2 and postoperative zoledronic acid (ZA; 0.1 mg/kg total dose). Microcomputed tomography reconstructions of healing fractures indicated BMP-2 was less effective in an OI setting, however, BMP-2 +ZA led to considerable increases in bone volume (+193% WT, p < 0.001; +154% OI, p < 0.001) and polar moment of inertia (+125% WT, p < 0.01; +248% OI, p < 0.05). Tissue histology revealed a thinning of the neocortex of the callus in BMP-2 treated OI bone, but considerable retention of woven bone in the healing callus with BMP + ZA specimens. These data suggest a cautious approach may be warranted with the sole application of BMP-2 in an OI surgical setting as a bone graft substitute. However, this may be overcome by off-label BP administration.
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Fraturas Ósseas , Osteogênese Imperfeita , Camundongos , Animais , Osteogênese Imperfeita/tratamento farmacológico , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Consolidação da Fratura , Microtomografia por Raio-X , Densidade Óssea , Difosfonatos/farmacologia , Calo Ósseo/patologia , Proteínas Morfogenéticas Ósseas/uso terapêuticoRESUMO
Multiple myeloma is a hematological malignancy affecting the plasma cells. It is the second most common hematologic cancer in adults. Over 90% of patients develop local osteolytic lesions and skeletal-related events at some point during the progression of the disease. Bone lesions can induce severe pain and immobility and can also increase the risk of fractures and osteomyelitis. Skeletal complications are associated with poor clinical outcomes, affecting quality of life and mortality. Current standards of care for myeloma, e.g., autologous stem-cell transplantation (ASCT) and chemotherapy, do not lessen the risk of adverse events in bone. Once bone lesions are present, bone-targeted interventions are limited, with bone antiresorptive drugs being a mainstay of treatment. This review highlights the growing literature surrounding osteolytic lesions and bone infections associated with multiple myeloma and assesses current and emerging treatments. Emerging evidence from clinical trials suggests that denosumab can reduce skeletal-related events, and the potential application of bortezomib/1D11 can reduce bone destruction and pathological fractures in MM patients. Once established, bone lesions are prone to develop osteomyelitis - especially in immunocompromised individuals. Antibiotics and surgical interventions have been used to manage bone infections in most reported cases. As the bone infection risk associated with MM bone lesions become more evident, there is scope to improve patient management by mitigating this risk with prophylactic antimicrobial therapy.
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Herpes simplex virus (HSV) infections are a worldwide health problem in need of new effective treatments. Of particular interest is the identification of antiviral agents that act via different mechanisms compared to current drugs, as these could interact synergistically with first-line antiherpetic agents to accelerate the resolution of HSV-1-associated lesions. For this study, we applied a structure-based molecular docking approach targeting the nectin-1 and herpesvirus entry mediator (HVEM) binding interfaces of the viral glycoprotein D (gD). More than 527,000 natural compounds were virtually screened using Autodock Vina and then filtered for favorable ADMET profiles. Eight top hits were evaluated experimentally in African green monkey kidney cell line (VERO) cells, which yielded two compounds with potential antiherpetic activity. One active compound (1-(1-benzofuran-2-yl)-2-[(5Z)-2H,6H,7H,8H-[1,3] dioxolo[4,5-g]isoquinoline-5-ylidene]ethenone) showed weak but significant antiviral activity. Although less potent than antiherpetic agents, such as acyclovir, it acted at the viral inactivation stage in a dose-dependent manner, suggesting a novel mode of action. These results highlight the feasibility of in silico approaches for identifying new antiviral compounds, which may be further optimized by medicinal chemistry approaches.
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Osteogenesis imperfecta (OI) describes a series of genetic bone fragility disorders that can have a substantive impact on patient quality of life. The multidisciplinary approach to management of children and adults with OI primarily involves the administration of antiresorptive medication, allied health (physiotherapy and occupational therapy), and orthopedic surgery. However, advances in gene editing technology and gene therapy vectors bring with them the promise of gene-targeted interventions to provide an enduring or perhaps permanent cure for OI. This review describes emergent technologies for cell- and gene-targeted therapies, major hurdles to their implementation, and the prospects of their future success with a focus on bone disorders. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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Conservadores da Densidade Óssea , Osteogênese Imperfeita , Adulto , Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos , Criança , Terapia Genética , Humanos , Osteogênese , Osteogênese Imperfeita/tratamento farmacológico , Osteogênese Imperfeita/terapia , Qualidade de VidaRESUMO
INTRODUCTION: Osteogenesis imperfecta (OI) or brittle bone disease is a genetic disorder that results in bone fragility. Bisphosphonates such as zoledronic acid (ZA) are used clinically to increase bone mass and reduce fracture risk. Human growth hormone (hGH) has been used to promote long bone growth and forestall short stature in children with OI. The potential for hGH to improve bone quality, particularly in combination with ZA has not been robustly studied. METHODS: A preclinical study was performed using n = 80 mice split evenly by genotype (WT, Col1a2+/G610C). Groups of n = 10 were treated with +/-ZA and +/-hGH in a factorial design for each genotype. Outcome measures included bone length, isolated muscle mass, bone parameters assessed by microCT analysis, dynamic histomorphometry, and biomechanical testing. RESULTS: Treatment with hGH alone led to an increase in femur length in WT but not OI mice, however bone length was increased in both genotypes with the combination of hGH/ZA. MicroCT showed that hGH/ZA treatment increased cortical BV in both WT (+15%) and OI mice (+14.3%); hGH/ZA were also found to be synergistic in promoting cortical thickness in OI bone. ZA was found to have a considerably greater positive impact on trabecular bone than hGH. ZA was found to suppress bone turnover, and this was rescued by hGH treatment in terms of cortical periosteal perimeter, but not by dynamic bone remodeling. Statistically significant improvements in long bone by microCT did not translate into improvements in mechanical strength in a 4-point bending test, nor did vertebral strength improve in L4 compression testing in WT/OI bone. DISCUSSION/CONCLUSION: These data support hGH/ZA combination as a treatment for short stature, however the improvements granted by hGH alone and in combination with ZA on bone quality are modest. Increased periosteal perimeter does show promise in improving bone strength in OI, however a longer treatment time may be required to see effects on bone strength through mechanical testing.
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Hormônio do Crescimento Humano , Osteogênese Imperfeita , Animais , Densidade Óssea/fisiologia , Osso e Ossos , Modelos Animais de Doenças , Hormônio do Crescimento/uso terapêutico , Camundongos , Osteogênese Imperfeita/diagnóstico por imagem , Osteogênese Imperfeita/tratamento farmacológico , Osteogênese Imperfeita/genética , Ácido Zoledrônico/farmacologia , Ácido Zoledrônico/uso terapêuticoRESUMO
The potential health benefits of probiotics may not be realized because of the substantial reduction in their viability during food storage and gastrointestinal transit. Microencapsulation has been successfully utilized to improve the resistance of probiotics to critical conditions. Owing to the unique properties of biopolymers, they have been prevalently used for microencapsulation of probiotics. However, majority of microencapsulated products only contain a single layer of protection around probiotics, which is likely to be inferior to more sophisticated approaches. This review discusses emerging methods for the multilayer encapsulation of probiotic using biopolymers. Correlations are drawn between fabrication techniques and the resultant microparticle properties. Subsequently, multilayer microparticles are categorized based on their layer designs. Recent reports of specific biopolymeric formulations are examined regarding their physical and biological properties. In particular, animal models of gastrointestinal transit and disease are highlighted, with respect to trials of multilayer microencapsulated probiotics. To conclude, novel materials and approaches for fabrication of multilayer structures are highlighted.
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Probióticos , Animais , Biopolímeros , Colo , Composição de Medicamentos/métodos , Viabilidade MicrobianaRESUMO
The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a major public health burden and has resulted in millions of deaths worldwide. As effective treatments are limited, there is a significant requirement for high-throughput, low resource methods for the discovery of novel antivirals. The SARS-CoV-2 spike protein plays a key role in viral entry and has been identified as a therapeutic target. Using the available spike crystal structure, we performed a virtual screen with a library of 527 209 natural compounds against the receptor binding domain of this protein. Top hits from this screen were subjected to a second, more comprehensive molecular docking experiment and filtered for favourable ADMET properties. The in vitro activity of 10 highly ranked compounds was assessed using a virus neutralisation assay designed to facilitate viral entry in a physiologically relevant manner via the plasma membrane route. Subsequently, four compounds ZINC02111387, ZINC02122196, SN00074072 and ZINC04090608 were identified to possess antiviral activity in the µM range. These findings validate the virtual screening method as a tool for identifying novel antivirals and provide a basis for future drug development against SARS-CoV-2.
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Produtos Biológicos/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Antivirais/farmacologia , Produtos Biológicos/toxicidade , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Testes de Neutralização , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
The availability of donor human milk (DHM) is currently limited by the volumes that can be thermally pasteurized and kept in long-term cold storage. This study assesses the application of freeze-drying followed by low-dose gamma irradiation of DHM for simplified, safe long-term storage. Solid-phase microextraction (SPME) GC-MS, SDS and native PAGE gel electrophoresis demonstrated that the overall changes in volatile and protein profiles in Holder pasteurized and freeze-dried DHM was negligible compared to the natural variations in DHM. Freeze-dried DHM samples (moisture < 2.2 %) processed with 2 kGy gamma irradiation did not show any significant lipid oxidation end-products and variation in protein profile. Therefore, freeze-drying followed by in-packaging gamma irradiation could be a safe method for pasteurization, convenient storage and delivery of DHM at ambient temperature. These methods may generate a means to create a reserve stock of DHM for emergencies and humanitarian aid.
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Bancos de Leite Humano , Leite Humano , Liofilização , Humanos , PasteurizaçãoRESUMO
SIGNIFICANCE: Current methods for analyzing pathological muscle tissue are time consuming and rarely quantitative, and they involve invasive biopsies. Faster and less invasive diagnosis of muscle disease may be achievable using marker-free in vivo optical sensing methods. AIM: It was speculated that changes in the biochemical composition and structure of muscle associated with pathology could be measured quantitatively using visible wavelength optical spectroscopy techniques enabling automated classification. APPROACH: A fiber-optic autofluorescence (AF) and diffuse reflectance (DR) spectroscopy device was manufactured. The device and data processing techniques based on principal component analysis were validated using in situ measurements on healthy skeletal and cardiac muscle. These methods were then applied to two mouse models of genetic muscle disease: a type 1 neurofibromatosis (NF1) limb-mesenchyme knockout (Nf1Prx1 - / - ) and a muscular dystrophy mouse (mdx). RESULTS: Healthy skeletal and cardiac muscle specimens were separable using AF and DR with receiver operator curve areas (ROC-AUC) of >0.79. AF and DR analyses showed optically separable changes in Nf1Prx1 - / - quadriceps muscle (ROC-AUC >0.97) with no differences detected in the heart (ROC-AUC <0.67), which does not undergo gene deletion in this model. Changes in AF spectra in mdx muscle were seen between the 3 week and 10 week time points (ROC-AUC = 0.96) and were not seen in the wild-type controls (ROC-AUC = 0.58). CONCLUSION: These findings support the utility of in vivo fiber-optic AF and DR spectroscopy for the assessment of muscle tissue. This report highlights that there is considerable scope to develop this marker-free optical technology for preclinical muscle research and for diagnostic assessment of clinical myopathies and dystrophies.
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Tecnologia de Fibra Óptica , Músculos , Animais , Camundongos , Camundongos Endogâmicos mdx , Análise de Componente Principal , Análise EspectralRESUMO
Gut-on-a-chip microfluidic devices have emerged as versatile and practical systems for modeling the human intestine in vitro. Cells cultured under microfluidic conditions experience the effect of shear stress, used as a biomechanical cue to promote a faster cell polarization in Caco-2 cells when compared with static culture conditions. However, published systems to date have utilized a constant flow rate that fails to account for changes in cell shear stress ([Formula: see text]) resulting from changes in cell elongation that occur with differentiation. In this study, computational fluid dynamics (CFD) simulations predict that cells with villi-like morphology experience a [Formula: see text] higher than bulge-like cells at the initial growth stages. Therefore, we investigated the use of a dynamic flow rate to maintain a constant [Formula: see text] across the experiment. Microscopic assessment of cell morphology and dome formation confirmed the initiation of Caco-2 polarization within three days. Next, adopting our dynamic approach, we evaluated whether the following decreased flow could still contribute to complete cell differentiation if compared with the standard constant flow methodology. Caco-2 cells polarized under both conditions, secreted mucin-2 and villin and formed tight junctions and crypt-villi structures. Gene expression was not impacted using the dynamic flow rate. In conclusion, our dynamic flow approach still facilitates cell differentiation while enabling a reduced consumption of reagents.
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Dispositivos Lab-On-A-Chip , Microfluídica , Células CACO-2 , Humanos , Estresse Mecânico , Junções ÍntimasRESUMO
Fracture repair is a normal physiological response to bone injury. During the process of bony callus formation, a lacunocanalicular network (LCN) is formed de novo that evolves with callus remodeling. Our aim was the longitudinal assessment of the development and evolution of the LCN during fracture repair. To this end, 45 adult wild-type C57BL/6 mice underwent closed tibial fracture surgery. Fractured and intact contralateral tibias were harvested after 2, 3, and 6 weeks of bone healing (n = 15/group). High-resolution micro-computed tomography (µCT) and deconvolution microscopy (DV) approaches were applied to quantify lacunar number density from the calluses and intact bone. On histological sections, Goldner's trichrome staining was used to assess lacunar occupancy, fluorescein isothiocyanate staining to visualize the canalicular network, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining to examine osteocyte apoptosis. Analysis of µCT scans showed progressive decreases in mean lacuna volume over time (-27% 2-3 weeks; -13% 3-6 weeks). Lacunar number density increased considerably between 2 and 3 weeks (+156%). Correlation analysis was performed, showing a positive linear relationship between canalicular number density and trabecular thickness (R 2 = 0.56, p < 0.001) and an inverse relationship between mean lacuna volume and trabecular thickness (R 2 = 0.57, p < 0.001). Histology showed increases in canalicular number density over time (+22% 2-3 weeks, +51% 3-6 weeks). Lacunar occupancy in new bone of the callus was high (>90%), but the old cortical bone within the fracture site appeared necrotic as it underwent resorption. In conclusion, our data shows a progressive increase in the complexity of the LCN over time during fracture healing and demonstrates that this network is initiated during the early stages of repair. Further studies are needed to address the functional importance of osteocytes in bone healing, particularly in detecting and translating the effects of micromotion in the fracture. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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The most common pasteurisation method used by human milk banks is Holder pasteurisation. This involves thermal processing, which can denature important proteins and can potentially reduce the natural antimicrobial properties found in human milk. This study assesses the application of a hybrid method comprised of freeze-drying followed by low-dose gamma-irradiation for nonthermal donor human milk pasteurisation. Freeze-drying donor human milk followed by gamma-irradiation at 2 kGy was as efficient as Holder pasteurisation in the reduction of bacterial inoculants of Staphylococcus aureus (106 cfu/mL) and Salmonella typhimurium (106 cfu/mL) in growth inhibition assays. These assays also demonstrated that human milk naturally inhibits the growth of bacterial inoculants S. aureus, S. typhimurium, and Escherichia coli. Freeze drying (without gamma-irradiation) did not significantly reduce this natural growth inhibition. By contrast, Holder pasteurisation significantly reduced the milk's natural antimicrobial effect on S. aureus growth after 6 h (-19.8% p = 0.01). Freeze-dried and then gamma-irradiated donor human milk showed a strong antimicrobial effect across a dose range of 2-50 kGy, with only a minimal growth of S. aureus observed after 6 h incubation. Thus, a hybrid method of freeze-drying followed by 2 kGy of gamma-irradiation preserves antimicrobial properties and enables bulk pasteurisation within sealed packaging of powderised donor human milk. This work forwards a goal of increasing shelf life and simplifying storage and transportation, while also preserving functionality and antimicrobial properties.
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Reduced muscle tone, muscle weakness, and physical fatigue can impact considerably on quality of life for children with neurofibromatosis type 1 (NF1). Human muscle biopsies and mouse models of NF1 deficiency in muscle show intramyocellular lipid accumulation, and preclinical data have indicated that L-carnitine supplementation can ameliorate this phenotype. The aim of this study is to examine whether daily L-carnitine supplementation is safe and feasible, and will improve muscle strength and reduce fatigue in children with NF1. A 12-week Phase 2a trial was conducted using 1000 mg daily oral levocarnitine tartrate supplementation. Recruited children were between 8 and 12 years old with a clinical diagnosis of NF1, history of muscle weakness and fatigue, and naïve to L-carnitine. Primary outcomes were safety (self-reporting, biochemical testing) and compliance. Secondary outcomes included plasma acylcarnitine profiles, functional measures (muscle strength, long jump, handwriting speed, 6-minute-walk test [6MWT]), and parent-reported questionnaires (PedsQL™, CBCL/6-18). Six children completed the trial with no self-reported adverse events. Biochemical tests for kidney and liver function were normal, and the average compliance was 95%. Plasma acylcarnitine levels were low, but within a range not clinically linked to carnitine deficiency. For strength measures, there was a mean 53% increase in dorsiflexion strength (95% confidence interval [CI] 8.89-60.75; p = 0.02) and mean 66% increase in plantarflexion strength (95% CI 12.99-134.1; p = 0.03). In terms of muscle performance, there was a mean 10% increase in long jump distance (95% CI 2.97-16.03; p = 0.01) and 6MWT distance (95% CI 5.88-75.45; p = 0.03). Comparison with the 1000 Norms Project data showed a significant improvement in Z-score for all of these measures. Parent reports showed no negative impact on quality of life, and the perceived benefits led to the majority of individuals remaining on L-carnitine after the study. Twelve weeks of L-carnitine supplementation is safe and feasible in children with NF1, and a Phase 3 trial should confirm the efficacy of treatment.
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Carnitina/administração & dosagem , Fadiga/dietoterapia , Debilidade Muscular/dietoterapia , Neurofibromatose 1/dietoterapia , Cardiomiopatias/dietoterapia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Carnitina/efeitos adversos , Carnitina/deficiência , Carnitina/metabolismo , Criança , Suplementos Nutricionais/efeitos adversos , Fadiga/genética , Fadiga/patologia , Feminino , Humanos , Hiperamonemia/dietoterapia , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Masculino , Força Muscular/efeitos dos fármacos , Debilidade Muscular/metabolismo , Debilidade Muscular/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Doenças Musculares/dietoterapia , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Neurofibromatose 1/complicações , Neurofibromatose 1/metabolismo , Neurofibromatose 1/patologia , Qualidade de VidaRESUMO
Osteomyelitis and orthopedic infections are major clinical problems, limited by a lack of antibiotics specialized for such applications. In this paper, we describe the design and synthesis of a novel bone-binding antibiotic (BBA-1) and its subsequent structural and functional characterization. The synthesis of BBA-1 was the result of a two-step chemical conjugation of cationic selective antimicrobial-90 (CSA-90) and the bisphosphonate alendronate (ALN) via a heterobifunctional linker. This was analytically confirmed by HPLC, FT-IR, MS and NMR spectroscopy. BBA-1 showed rapid binding and high affinity to bone mineral in an in vitro hydroxyapatite binding assay. Kirby-Baur assays confirmed that BBA-1 shows a potent antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus comparable to CSA-90. Differentiation of cultured osteoblasts in media supplemented with BBA-1 led to increased alkaline phosphatase expression, which is consistent with the pro-osteogenic activity of CSA-90. Bisphosphonates, such as ALN, are inhibitors of protein prenylation, however, the amine conjugation of ALN to CSA-90 disrupted this activity in an in vitro protein prenylation assay. Overall, these findings support the antimicrobial, bone-binding, and pro-osteogenic activities of BBA-1. The compound and related agents have the potential to ensure lasting activity against osteomyelitis after systemic delivery.