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Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.
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Proteínas Recombinantes de Fusão , Humanos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/química , Superóxido Dismutase-1/isolamento & purificação , Superóxido Dismutase-1/metabolismo , Estabilidade Enzimática , Superóxido Dismutase/isolamento & purificação , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Escherichia coli/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Clonagem Molecular , Proteínas de InsetosRESUMO
Much progress has been made toward the development of wearable flexible strain sensors with high sensing performance to monitor human motion, but continuous function in harsh aqueous environments remains a significant challenge. A promising strategy has been the design of sensors with highly durable superhydrophobicity and maintenance of unique sensing properties. Herein, an extremely durable superhydrophobic strain sensor with an ultrawide sensing range was simply fabricated by directly brushing conductive carbon black nanoparticles (CBNPs) onto an elastic silicone rubber sheet (SS) with poly(dimethylsiloxane) (PDMS) coatings (i.e., SS/PDMS-CBNPs sensors). First, this method avoided the use of toxic solvents and a conventional prestretching treatment. Second, considering the easily destroyed rough structures and surface chemistry for conventional superhydrophobic sensors during practical applications, the prepared SS/PDMS-CBNP sensors showed excellent mechanical durability of both superhydrophobicity and sensing as examined by harsh abrasion (300 cycles), stretching (up to 200%), and ultrasonication (40 min) treatments. Third, the prepared superhydrophobic strain sensor exhibited high sensitivity (gauge factor of 101.75), high stretchability (0.015-460%), low hysteresis (83 ms), and long-term stability (10000 cycles). Fourth, the high biocompatibility of the SS/PDMS-CBNP sensor was demonstrated by rabbit skin irritation tests. Finally, the remarkable water-repellent and sensing properties of the SS/PDMS-CBNP sensor allowed its application to monitor a swimmer's real-time situation and send distress signals when needed.
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Dispositivos Eletrônicos Vestíveis , Animais , Humanos , Coelhos , Movimento (Física) , Água , Condutividade Elétrica , Interações Hidrofóbicas e HidrofílicasRESUMO
Graphite (Gr) has been considered as the most promising anode material for potassium-ion batteries (PIBs) commercialization due to its high theoretical specific capacity and low cost. However, Gr-based PIBs remain unfeasible at low temperature (LT), suffering from either poor kinetics based on conventional carbonate electrolytes or K+ -solvent co-intercalation issue based on typical ether electrolytes. Herein, a high-performance Gr-based LT rechargeable PIB is realized for the first time by electrolyte chemistry. Applying unidentate-ether-based molecule as the solvent dramatically weakens the K+ -solvent interactions and lowers corresponding K+ de-solvation kinetic barrier. Meanwhile, introduction of steric hindrance suppresses co-intercalation of K+ -solvent into Gr, greatly elevating operating voltage and cyclability of the full battery. Consequently, the as-prepared Gr||prepotassiated 3,4,9,10-perylene-tetracarboxylicacid-dianhydride (KPTCDA) full PIB can reversibly charge/discharge between -30 and 45 °C with a considerable energy density up to 197â Wh kgcathode -1 at -20 °C, hopefully facilitating the development of LT PIBs.
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BACKGROUND: Tubal obstructive infertility (TOI) is a challenging condition affecting many women worldwide. Acupuncture and herbal medicine have emerged as potential therapeutic options for enhancing fertility outcomes in these patients. However, the evidence regarding their efficacy remains inconclusive, necessitating a comprehensive systematic review and meta-analysis. METHOD: Computer searches were conducted in PubMed, Cochrane, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), VIP Information, Wanfang Database, and China Biology Medicine (CBM) databases to retrieve relevant literature on the efficacy and safety of acupuncture and related therapies for the treatment of tubal obstructive infertility. The search period extended from the inception of the databases to December 2022. Two researchers independently screened the literature based on strict inclusion criteria, extracted relevant data, and utilized Cochrane Collaboration tools and the Jadad scale to comprehensively assess the quality of the included studies. Subsequently, pairwise meta-analysis and network meta-analysis were performed using statistical software such as StataSE and Rstudio, and graphical representations were generated to present the results. RESULT: The network meta-analysis included 1580 articles, with 23 meeting the criteria. These studies involved 2355 patients and explored 13 intervention measures. Acupuncture-related therapies outperformed control interventions in improving pregnancy rates, tubal patency rates, and overall effectiveness while demonstrating a lower incidence of adverse events. EA+CHM was identified as the most effective for pregnancy rates, MOX for tubal patency rates, and MOX+AP for overall effectiveness. The safety profile of acupuncture-related interventions was acceptable. These findings support acupuncture-related therapies as effective and safe options for tubal obstructive infertility management. Further high-quality research is needed to validate and expand upon these results. CONCLUSION: These findings offer novel treatment strategies for acupuncture-related interventions, providing practitioners with evidence-based guidance. Addressing limitations through future research is crucial, including diverse literature, emphasizing higher-quality RCTs, and exploring a broader range of interventions with long-term follow-up data. Systematic assessment of adverse events, standardized techniques, and robust ranking methods should be considered.
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Terapia por Acupuntura , Infertilidade , Gravidez , Humanos , Feminino , Teorema de Bayes , Ensaios Clínicos Controlados Aleatórios como Assunto , Terapia por Acupuntura/métodos , Taxa de GravidezRESUMO
The aim of this study was to separate and purify recombinant ß-glucosidase (GLEGB) with elastin-like polypeptide (ELP) and graphene-binding peptide (GB) from cell lysis solution by foam separation and further purification. The study of foam property of GLEGB cell lysis solution indicated that it had excellent foaming property and foam stability, which was suitable for foam separation. This could be due to the GB tag with hydrophobicity, which made the recombinant ß-glucosidase with GB preferentially adsorb on the surface of bubbles. At optimum operating conditions of foam separation, the enzyme activity recovery of GLEGB could reach 95.63 ± 1.0%. The foam solution of GLEGB was further purified based on the thermally responsive property of the ELP tag, and the purification fold of GLEGB could reach 29.6 ± 0.5 at the optimum operating conditions. The prominent purification effect indicates that this technique is a simple and efficient technique for the separation and purification of recombinant enzymes.
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The development of a suitable immobilization strategy to improve the performance of immobilized glucose isomerase for the isomerization of glucose to fructose is crucial to promoting the industrial production of high-fructose syrup. In this work, a novel recyclable upper critical solution temperature (UCST)-type mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst (PVAA@GI) was prepared, and the immobilized glucose isomerase could capture the glucose substrate through the affinity of 4-vinylbenzeneboronic acid (4-VBA) and the glucose substrate, which led to the enhanced substrate affinity and catalytic efficiency of the PVAA@GI. The biocatalyst exhibited excellent stability in pH, thermal, storage, and recycling compared to the free enzyme. The mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst displayed reversibly soluble-insoluble characteristics with temperature change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the temperature after the reaction. The highest fructose production rate reached 62.79%, which would have potential application in the industrial production of high-fructose syrup.
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Aldose-Cetose Isomerases , Frutose , Glucose , Temperatura , Isomerismo , Enzimas Imobilizadas/metabolismo , Aldose-Cetose Isomerases/metabolismoRESUMO
In this study, the upper critical solution temperature (UCST)-responsive polymers poly (ethylene oxide) monomethyl ether-block-poly(acrylamide-co-acrylonitrile) (PEG-b-p(AAM-co-AN) were synthesized and successfully utilized to immobilize ß-glucosidase in crude enzyme solution. These UCST-responsive ß-glucosidase biocatalysts (PEG-b-p(AAM-co-AN@LytA-Glu) have specific UCST with tunable transition temperature, which could be tuned the separation temperature to the desired temperature range. The P2 @ LytA-Glu with an UCST of about 42.9 â was exploited by one-step covalent immobilization of ß-glucosidase in crude enzyme solution. The prepared P2 @ LytA-Glu exhibited significantly improved temperature, pH, storage, and operation stabilities compared with that of free enzyme. The catalytic rate of P2 @ Glu-LytA was 14.5% higher than that of P2-Glu (immobilized pure ß-glucosidase), which indicated that one-step immobilization of crude enzyme directly from crude enzyme solution was feasible, and it can greatly save the purification step and reduce the experimental cost. The engineered UCST-responsive immobilized enzymes are potentially useful for the practical green biocatalysis.
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Polímeros , beta-Glucosidase , Biocatálise , Enzimas Imobilizadas , Polietilenoglicóis , Temperatura , beta-Glucosidase/metabolismoRESUMO
Early Alzheimer's disease (AD) pathology can be found in cortical biopsies taken during shunt placement for Normal Pressure Hydrocephalus. This represents an opportunity to study early AD pathology in living patients. Here we report RNA-seq data on 106 cortical biopsies from this patient population. A restricted set of genes correlate with AD pathology in these biopsies, and co-expression network analysis demonstrates an evolution from microglial homeostasis to a disease-associated microglial phenotype in conjunction with increasing AD pathologic burden, along with a subset of additional astrocytic and neuronal genes that accompany these changes. Further analysis demonstrates that these correlations are driven by patients that report mild cognitive symptoms, despite similar levels of biopsy ß-amyloid and tau pathology in comparison to patients who report no cognitive symptoms. Taken together, these findings highlight a restricted set of microglial and non-microglial genes that correlate with early AD pathology in the setting of subjective cognitive decline.
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Doença de Alzheimer/complicações , Córtex Cerebral/patologia , Disfunção Cognitiva/imunologia , Redes Reguladoras de Genes/imunologia , Hidrocefalia de Pressão Normal/imunologia , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Astrócitos/imunologia , Astrócitos/patologia , Biópsia , Córtex Cerebral/citologia , Córtex Cerebral/imunologia , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Feminino , Humanos , Hidrocefalia de Pressão Normal/genética , Hidrocefalia de Pressão Normal/patologia , Hidrocefalia de Pressão Normal/cirurgia , Masculino , Microglia/imunologia , Microglia/patologia , Testes Neuropsicológicos , RNA-Seq , Estudos RetrospectivosRESUMO
Herein, a glucose oxidase@ZIF-8 composite (3-MPBA/GOx@ZIF-8) with enhanced enrichment was enabled the rapid encapsulation of glucose oxidase (GOx) into microporous zeolitic imidazolate framework-8 (ZIF-8) for the first time. The 3-MPBA/GOx@ZIF-8 not only has improved affinity and catalytic efficiency to the substrate but also can shorten the formation time. The optimum loading amount of GOx on ZIF-8 was determined to be 470â¯mg/g. The as-prepared 3-MPBA/GOx@ZIF-8 composite maintained the native conformation of the enzyme and showed excellent bioactivity, even in chemical agents or at high temperature. Furthermore, the 3-MPBA/GOx@ZIF-8 showed satisfactory reusability, preserving almost 80.8 % activity after 7 cycles. The Michaelis constant Km and specificity constant kcat/Km of the 3-MPBA/GOx@ZIF-8 were 0.03⯱â¯0.02â¯mM and 63.87⯱â¯1.96 s-1â¯mM-1, respectively, which were superior to corresponding values of free GOx. Therefore, the 3-MPBA/GOx@ZIF-8 displayed high catalytic efficiency, high loading efficiency and enhanced stability. Moreover, a new type of visual colorimetric sensor for screening of the diabetes was realized through the 3-MPBA/GOx@ZIF-8, which provided a new strategy for the analysis field of glucose.
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Glucose Oxidase , Zeolitas , Biomimética , Catálise , GlucoseRESUMO
To enhance microwave loss abilities, constructing composites with one-dimensional (1D) structure is an excellent scheme. In this work, a high-efficiency microwave absorber of MnO nanograins decorated vanadium nitride/carbon nanofibers (MnO-VN/C NFs) was successfully prepared for the first time via co-electrospinning technology and subsequent nitriding treatment. Studying in detail the specific relationship between nitriding time and the morphology of the as-prepared NFs, the precipitations of MnO nanoparticles with tailored structures were attached on the surface of VN/C NFs to optimize their electromagnetic parameters. When the nitriding time was 2.0 h at 600 °C, the MnO-VN/C NFs displayed good microwave absorption performances: the minimum reflection loss (RL) value was -63.2 dB at 8.8 GHz, and the bandwidth of RL < -10 dB was up to 6.4 GHz from 11.6 to 18 GHz at the thickness of 2.8 mm. Meanwhile, the absorption bandwidth (RL< -10 dB) could cover the whole X and Ku band by adjusting the thickness, respectively. The outstanding performances could be attributed to the good impedance matching and various loss pathways including conductive loss and interfacial and dipole polarizations. In these regards, MnO-VN/C NFs are likely to be utilized as a high-efficiency microwave absorber. And the strategy in this work can provide great help to design other 1D structural microwave absorbers with a broader absorbing band.
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Integrated hydrological and hydrodynamic modeling study has been conducted to investigate hurricane impact on Woonasquatucket River, Rhode Island, USA. Model simulation was conducted for the case study of 2010 storm event. The hydrological model simulates the runoff from the heavy rainstorm, while the river hydrodynamic model simulates the flood waves affected by the interactions of upstream rainfall runoff and downstream storm surge. Results indicate that the river floods was dominant by rainfall runoff in upper river reaches, but dominant by storm surge in the lower river area near the estuary.
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Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray, and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on aspartic acid-to-valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in non-phosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-associated virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with serine-to-aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV- vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC mutations-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation. KEY MESSAGES: HCM-D166V mice show decreased RLC phosphorylation and decompensated function. AAV9-S15D-RLC gene therapy in HCM-D166V mice, but not in WT-RLC, results in improved heart performance. Global longitudinal strain analysis shows enhanced contractility in AAV vs controls. Increased systolic and diastolic function is paralleled by higher contractile force. Phosphomimic S15D-RLC has a therapeutic potential for HCM.
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Cardiomiopatia Hipertrófica Familiar/terapia , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/uso terapêutico , Animais , Cardiomiopatia Hipertrófica Familiar/diagnóstico por imagem , Cardiomiopatia Hipertrófica Familiar/genética , Cardiomiopatia Hipertrófica Familiar/fisiopatologia , Modelos Animais de Doenças , Ecocardiografia , Feminino , Fibrose , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hemodinâmica , Humanos , Hidroxiprolina/metabolismo , Masculino , Camundongos , Contração Muscular , Músculos/metabolismoRESUMO
BACKGROUND/AIM: Crocetin is a readily bioavailable and bioactive compound extracted from Saffron. Previous studies indicated its various biomedical properties including antioxidant and anti-coagulation potencies. However, its effect on inflammation, notably within the cardiovascular system, has not been investigated yet. In the present study, we utilized human umbilical vein endothelial cell (HUVEC) to elucidate the effect of Crocetin on vascular inflammation. METHODS: Cell viability and toxicity were evaluated by MTT and Lactate dehydrogenase (LDH) assay, respectively. Pro-inflammatory chemokine
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Carotenoides/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/patologia , Inflamação/tratamento farmacológico , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Carotenoides/química , Carotenoides/farmacologia , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Interleucina-8/genética , Interleucina-8/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Células U937 , Vitamina A/análogos & derivadosRESUMO
The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1(+∕+)-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca(2+) sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and kinetics of the acto-myosin interaction. Specifically, slower myosin cross-bridge turnover rates and slower second-order MgATP binding rates of acto-myosin interactions were observed in IVS6-1 vs. WT reconstituted cardiac preparations. Our in vitro results suggest that when placed in vivo, IVS6-1 may lead to cardiomyopathy and early death of homozygous infants by severely compromising the ability of myosin to develop contractile force and maintain normal systolic and diastolic cardiac function.
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Microbiomes are ubiquitous and are found in the ocean, the soil, and in/on other living organisms. Changes in the microbiome can impact the health of the environmental niche in which they reside. In order to learn more about these communities, different approaches based on data from multiple omics have been pursued. Metagenomics produces a taxonomical profile of the sample, metatranscriptomics helps us to obtain a functional profile, and metabolomics completes the picture by determining which byproducts are being released into the environment. Although each approach provides valuable information separately, we show that, when combined, they paint a more comprehensive picture. We conclude with a review of network-based approaches as applied to integrative studies, which we believe holds the key to in-depth understanding of microbiomes.
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Using microarray and bioinformatics, we examined the gene expression profiles in transgenic mouse hearts expressing mutations in the myosin regulatory light chain shown to cause hypertrophic cardiomyopathy (HCM). We focused on two malignant RLC-mutations, Arginine 58âGlutamine (R58Q) and Aspartic Acid 166 â Valine (D166V), and one benign, Lysine 104 â Glutamic Acid (K104E)-mutation. Datasets of differentially expressed genes for each of three mutants were compared to those observed in wild-type (WT) hearts. The changes in the mutant vs. WT samples were shown as fold-change (FC), with stringency FC ≥ 2. Based on the gene profiles, we have identified the major signaling pathways that underlie the R58Q-, D166V- and K104E-HCM phenotypes. The correlations between different genotypes were also studied using network-based algorithms. Genes with strong correlations were clustered into one group and the central gene networks were identified for each HCM mutant. The overall gene expression patterns in all mutants were distinct from the WT profiles. Both malignant mutations shared certain classes of genes that were up or downregulated, but most similarities were noted between D166V and K104E mice, with R58Q hearts showing a distinct gene expression pattern. Our data suggest that all three HCM mice lead to cardiomyopathy in a mutation-specific manner and thus develop HCM through diverse mechanisms.
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Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Regulação da Expressão Gênica , Mutação , Cadeias Leves de Miosina/metabolismo , Algoritmos , Animais , Arginina/química , Biologia Computacional , Perfilação da Expressão Gênica , Ácido Glutâmico/química , Glutamina/química , Lisina/química , Camundongos , Camundongos Transgênicos , Família Multigênica , Miocárdio/metabolismo , Cadeias Leves de Miosina/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Análise de Componente Principal , Valina/químicaRESUMO
Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being "kicked" by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either "Standard" (Std) (decaying monotonically in time) or "Non-standard" (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.
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We discuss here the potential mechanisms of action associated with hypertrophic (HCM) or dilated (DCM) cardiomyopathy causing mutations in the myosin regulatory (RLC) and essential (ELC) light chains. Specifically, we focus on four HCM mutations: RLC-A13T, RLC-K104E, ELC-A57G and ELC-M173V, and one DCM RLC-D94A mutation shown by population studies to cause different cardiomyopathy phenotypes in humans. Our studies indicate that RLC and ELC mutations lead to heart disease through different mechanisms with RLC mutations triggering alterations of the secondary structure of the RLC which further affect the structure and function of the lever arm domain and impose changes in the cross bridge cycling rates and myosin force generation ability. The ELC mutations exert their detrimental effects through changes in the interaction of the N-terminus of ELC with actin altering the cross talk between the thick and thin filaments and ultimately resulting in an altered force-pCa relationship. We also discuss the effect of mutations on myosin light chain phosphorylation. Exogenous myosin light chain phosphorylation and/or pseudo-phosphorylation were explored as potential rescue tools to treat hypertrophy-related cardiac phenotypes.