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1.
J Biomed Sci ; 31(1): 8, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38229040

RESUMO

BACKGROUND: Chikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications. METHODS: In the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors. RESULTS: CHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood-brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedes albopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice. CONCLUSIONS: These findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.


Assuntos
Aedes , Vírus Chikungunya , Humanos , Camundongos , Animais , Vírus Chikungunya/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Aminoácidos , Mutação , Mamíferos
2.
Acta Pharm Sin B ; 13(5): 2039-2055, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37250169

RESUMO

Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available.

3.
Mech Ageing Dev ; 132(11-12): 552-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21982926

RESUMO

Sarcopenia and osteopenia are two common components of the frailty syndrome that may share a common underlying mechanism. Since frailty has been associated with increased fat infiltration in muscle and bone, we hypothesized that lamin A/C, a protein of the nuclear envelope that regulates adipose differentiation, could be associated with the pathophysiology of both osteo and sarcopenia in the frailty syndrome. Four-week-old lamin A/C null (Lmna(-/-)), heterozygous (Lmna(+/-)) and wild type (WT) mice were sacrificed and their mid-thigh analyzed for fat infiltration using invasive (histology) and non-invasive (µCT) methods. Lmna(-/-) mice showed a significant increase in inter- (~4-fold) and intra-myofiber (~2.5-fold) fat and marrow fat infiltration (~40-fold), with a significant decrease in muscle volume (-42.8%) and bone volume (-21.8%), as compared with WT controls. Furthermore, fat infiltration happened concomitantly with a significant decline in muscle and bone strength in Lmna(-/-) mice. From a mechanistic approach, high levels of pro-adipogenic factors PPARγ and C/EBPα were associated with a reduction in myogenic and osteogenic factors from the Wnt-10b/ß-catenin signalling pathway in Lmna(-/-) mice. In conclusion, lamin A/C could constitute the determinant factor in the pathogenesis and potential treatment of both sarcopenia and osteopenia, which are commonly observed in the frailty syndrome.


Assuntos
Adipócitos/metabolismo , Adipócitos/patologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Lamina Tipo A/deficiência , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Adipogenia , Idoso , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Modelos Animais de Doenças , Idoso Fragilizado , Humanos , Lamina Tipo A/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular , Osteogênese , PPAR gama/metabolismo , Sarcopenia/etiologia , Sarcopenia/genética , Sarcopenia/metabolismo , Sarcopenia/patologia , Transdução de Sinais
4.
PLoS One ; 6(4): e19313, 2011 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-21547077

RESUMO

Age-related bone loss is associated with changes in bone cellularity with characteristically low levels of osteoblastogenesis. The mechanisms that explain these changes remain unclear. Although recent in vitro evidence has suggested a new role for proteins of the nuclear envelope in osteoblastogenesis, the role of these proteins in bone cells differentiation and bone metabolism in vivo remains unknown. In this study, we used the lamin A/C null (Lmna⁻/⁻) mice to identify the role of lamin A/C in bone turnover and bone structure in vivo. At three weeks of age, histological and micro computed tomography measurements of femurs in Lmna⁻/⁻ mice revealed a significant decrease in bone mass and microarchitecture in Lmna⁻/⁻ mice as compared with their wild type littermates. Furthermore, quantification of cell numbers after normalization with bone surface revealed a significant reduction in osteoblast and osteocyte numbers in Lmna⁻/⁻ mice compared with their WT littermates. In addition, Lmna⁻/⁻ mice have significantly lower osteoclast number, which show aberrant changes in their shape and size. Finally, mechanistic analysis demonstrated that absence of lamin A/C is associated with increase expression of MAN-1 a protein of the nuclear envelope closely regulated by lamin A/C, which also colocalizes with Runx2 thus affecting its capacity as osteogenic transcription factor. In summary, these data clearly indicate that the presence of lamin A/C is necessary for normal bone turnover in vivo and that absence of lamin A/C induces low bone turnover osteopenia resembling the cellular changes of age-related bone loss.


Assuntos
Doenças Ósseas Metabólicas/genética , Lamina Tipo A/deficiência , Osteogênese/fisiologia , Animais , Western Blotting , Doenças Ósseas Metabólicas/fisiopatologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Lamina Tipo A/genética , Camundongos , Camundongos Knockout , Microscopia Confocal , Osteogênese/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Bone ; 49(3): 412-8, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21575749

RESUMO

The ability of exercise to decrease fat mass and increase bone mass occurs through mechanical biasing of mesenchymal stem cells away from adipogenesis and toward osteoblastogenesis. The mechanism explaining this effect remains poorly understood. Lamin A/C knockdown inhibits osteoblastogenesis while favors adipogenesis in vitro. In this study, we hypothesized that the presence of lamin A/C is required for the anabolic response of bone during exercise. Three-month-old female lamin A/C haploinsufficient (Lmna(+/-)) mice were exposed to strenuous maximal exercise protocol (2 sessions/week, 40 min/session) for 6 weeks. Wild type (WT) (exercise and sedentary) and sedentary Lmna(+/-) mice were used as controls. To determine changes in bone microarchitecture and cell numbers, distal femur was analyzed by microCT and histomorphometry respectively. Finally, levels of expression of nuclear ß-catenin and sclerostin, two proteins involved in the anabolic response to exercise, were determined by immunofluorescence. Histomorphometry analysis showed a significant increase in bone volume fraction (BV/TV) in exercised vs. sedentary WT mice. In contrast, exercised Lmna(+/-) mice showed a significant reduction in microarchitecture as compared with sedentary Lmna(+/-) controls including trabecular and cortical thinning. In addition, we found a significant increase in bone cells number in exercised vs. sedentary WT mice whereas exercised Lmna(+/-) mice showed a significant reduction in osteoblasts and osteocytes number as compared with sedentary Lmna(+/-) controls. Finally, levels of activated ß-catenin in osteoblasts and osteocytes were significantly decreased while sclerostin expression was increased in exercised Lmna(+/-) mice as compared with exercised WT controls. In summary, our data indicate that the presence of lamin A/C is required for the anabolic effect of exercise on bone thus suggesting a new important role of lamin A/C in bone biology.


Assuntos
Haploinsuficiência , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Fenômenos Biomecânicos , Densidade Óssea , Núcleo Celular/metabolismo , Feminino , Fêmur/metabolismo , Fêmur/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , beta Catenina/genética , beta Catenina/metabolismo
6.
J Mol Cell Cardiol ; 50(3): 479-86, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21156181

RESUMO

Lamin A/C mutations are the most common cause of familial dilated cardiomyopathy (DCM) but the pathogenetic mechanisms are incompletely understood. Nesprins are spectrin repeat-containing proteins that interact with lamin A/C and are components of the linker-of-nucleoskeleton-and-cytoskeleton (LINC) complex that connects the nuclear envelope to the actin cytoskeleton. Our aim was to determine whether changes in nesprin-1 and actin might contribute to DCM in homozygous Lmna knockout (Lmna(-/-)) mice. Here we find that Lmna(-/-) cardiomyocytes have altered nuclear envelope morphology, disorganization of nesprin-1 and heterogeneity in the distribution of nuclear and cytoskeletal actin. Functional interactions of nesprin-1 with nuclear G-actin and with the cytoskeletal γ-actin, α-cardiac actin and α-smooth muscle actin (α-SMA) isoforms were shown by immunoprecipitation and Western blotting. At 4-6 weeks of age, Lmna(-/-) mice had normal levels of γ-actin and α-cardiac actin, but α-SMA expression was increased by 50%. In contrast to the predominant vascular distribution of α-SMA in WT ventricular sections, α-SMA had a diffuse staining pattern in Lmna(-/-) sections. Osmotic swelling studies showed enhanced radial swelling in Lmna(-/-) cardiomyocytes indicative of cytoskeletal instability. The distensibility of Lmna(-/-) cardiomyocytes with osmotic stress was reduced by addition of α-SMA-specific fusion peptide. Our findings support a model in which uncoupling of the nucleus and cytoskeleton associated with disruption of the LINC complex promotes mechanical instability and defective force transmission in cardiomyocytes. Changes in the distribution and expression patterns of nuclear and cytoskeletal actin suggest that diverse transcriptional and structural defects may also contribute to DCM in Lmna(-/-) mice.


Assuntos
Actinas/metabolismo , Lamina Tipo A/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Actinas/genética , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Proteínas do Citoesqueleto , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Homozigoto , Lamina Tipo A/deficiência , Lamina Tipo A/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/patologia
7.
Circ Res ; 106(3): 573-82, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20019332

RESUMO

RATIONALE: Mutations in the LMNA gene, which encodes the nuclear lamina proteins lamin A and lamin C, are the most common cause of familial dilated cardiomyopathy (DCM). Mechanical stress-induced apoptosis has been proposed as the mechanism underpinning DCM in lamin A/C-deficient hearts, but supporting in vivo evidence has been lacking. OBJECTIVE: Our aim was to study interventions to modify mechanical stress in heterozygous Lmna knockout (Lmna(+/-)) mice. METHODS AND RESULTS: Cardiac structure and function were evaluated before and after exercise training, thoracic aortic constriction, and carvedilol treatment. Lmna(+/-) mice develop adult-onset DCM with relatively more severe disease in males. Lmna(+/-) cardiomyocytes show altered nuclear morphology and perinuclear desmin organization, with enhanced responses to hypo-osmotic stress indicative of cytoskeletal instability. Despite these structural defects that provide a template for mechanical stress-induced damage, young Lmna(+/-) mice subjected to 6 weeks of moderate or strenuous exercise training did not show induction of apoptosis or accelerated DCM. In contrast, regular moderate exercise attenuated DCM development in male Lmna(+/-) mice. Sustained pressure overload generated by thoracic aortic constriction depressed ventricular contraction in young wild-type and Lmna(+/-) mice with no sex or genotype differences in the time-course or severity of response. Treatment of male Lmna(+/-) mice from 12 to 40 weeks with the beta-blocker, carvedilol, prevented the dilatation and contractile dysfunction that was observed in placebo-treated mice. CONCLUSIONS: These data suggest that factors other than mechanical stress-induced apoptosis contribute to DCM and provide the first demonstration that regular moderate exercise and carvedilol can modify disease progression in lamin A/C-deficient hearts.


Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Carbazóis/uso terapêutico , Cardiomiopatia Dilatada/genética , Coração/fisiopatologia , Lamina Tipo A/deficiência , Miocárdio/patologia , Propanolaminas/uso terapêutico , Estresse Mecânico , Animais , Aorta Torácica , Apoptose , Cardiomiopatia Dilatada/tratamento farmacológico , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Carvedilol , Constrição , Desmina/análise , Feminino , Genótipo , Lamina Tipo A/genética , Masculino , Camundongos , Camundongos Knockout , Pressão Osmótica , Condicionamento Físico Animal , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia
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