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Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders. Thrombosis and inflammation coexist in many diseases, however, there is lack of treatments targeting both pathologies. This study reports a novel antibody against blood factor XII, which bears a promise to treat broad thrombo-inflammatory disorder. [ABSTRACT FROM AUTHOR]

Mitochondrial encephalomyopathies (ME) are frequently associated with mutations of mitochondrial DNA, but the pathogenesis of a subset of ME (sME) remains elusive. Here we report that haploinsufficiency of a mitochondrial inner membrane protein, Mic60, causes progressive neurological abnormalities with insulted mitochondrial structure and neuronal loss in mice. In addition, haploinsufficiency of Mic60 reduces mitochondrial membrane potential and cellular ATP production, increases reactive oxygen species, and alters mitochondrial oxidative phosphorylation complexes in neurons in an age‐dependent manner. Moreover, haploinsufficiency of Mic60 compromises brain glucose intake and oxygen consumption in mice, resembling human ME syndrome. We further discover that MIC60 protein expression declined significantly in human sME, implying that insufficient MIC60 may contribute for pathogenesis of human ME. Notably, systemic administration of antioxidant N‐acetylcysteine largely reverses mitochondrial dysfunctions and metabolic disorders in haplo‐insufficient Mic60 mice, also restores neurological abnormal symptom. These results reveal Mic60 is required in the maintenance of mitochondrial integrity and function, and likely a potential therapeutics target for mitochondrial encephalomyopathies. [ABSTRACT FROM AUTHOR]

Cellular senescence (CS), a state of permanent growth arrest, is intertwined with tumorigenesis. Due to the absence of specific markers, characterizing senescence levels and senescence-related phenotypes across cancer types remain unexplored. Here, we defined computational metrics of senescence levels as CS scores to delineate CS landscape across 33 cancer types and 29 normal tissues and explored CS-associated phenotypes by integrating multiplatform data from ~20 000 patients and ~212 000 single-cell profiles. CS scores showed cancer type-specific associations with genomic and immune characteristics and significantly predicted immunotherapy responses and patient prognosis in multiple cancers. Single-cell CS quantification revealed intra-tumor heterogeneity and activated immune microenvironment in senescent prostate cancer. Using machine learning algorithms, we identified three CS genes as potential prognostic predictors in prostate cancer and verified them by immunohistochemical assays in 72 patients. Our study provides a comprehensive framework for evaluating senescence levels and clinical relevance, gaining insights into CS roles in cancer- and senescence-related biomarker discovery. [ABSTRACT FROM AUTHOR]

Cell senescence, which is an irreversible state of cell proliferative arrest, has emerged as a potentially important contributor to tissue dysfunction and organismal ageing. Cell senescence is triggered by a variety of senescence stressors, which affect gene expression and multiple signalling pathways that give rise to various senescence phenotypes. Epigenetic mechanisms, as critical regulators of chromosomal architecture and gene expression, have added an extra dimension to the molecular mechanisms of cell senescence. Cell senescence is accompanied by changes in DNA methylation, histone-associated epigenetic processes, chromatin remodelling and ncRNA expression. Those senescence-associated epigenetic alterations interact with the senescence regulatory programme networks and lead to various cell senescence phenotypes. This review provides a comprehensive overview of epigenetic changes and their effects on cell senescence. The differences in epigenetic alterations among different types of senescence are also discussed. Furthermore, we summarise the interactions among different epigenetic mechanisms during cell senescence and analyse the possibility of using epigenetic signatures as biomarkers and therapeutic targets for the treatment of senescence-associated diseases. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase (PON) gene cluster (PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene (PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type (WT) control. The same protective tendency was also observed with an Apoe background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family. [ABSTRACT FROM AUTHOR]

Spatial expression patterns of homeobox (HOX) genes delineate positional identity of primary fibroblasts from different topographic sites. The molecular mechanism underlying the establishing or maintaining of HOX gene expression pattern remains an attractive developmental issue to be addressed. Our previous work suggested a critical role of CTCF/cohesin-mediated higher-order chromatin structure in RA-induced HOXA activation in human teratocarcinoma NT2/D1 cells. This study investigated the recruitment of CTCF and cohesin, and the higher-order chromatin structure of the HOXA locus in fetal lung and adult foreskin fibroblasts, which display complementary HOXA gene expression patterns. Chromatin contacts between the CTCF-binding sites were observed with lower frequency in human foreskin fibroblasts. This observation is consistent with the lower level of cohesin recruitment and 5' HOXA gene expression in the same cells. We also showed that CTCF-binding site A56 (CBSA56) related chromatin structures exhibit the most notable changes in between the two types of cell, and hence may stand for one of the key CTCF-binding sites for cell-type specific chromatin structure organization. Together, these results imply that CTCF/cohesin coordinates HOXA cluster higher-order chromatin structure and expression during development, and provide insight into the relationship between cell-type specific chromatin organization and the spatial collinearity. [ABSTRACT FROM AUTHOR]

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. Together with Oct4, Sox2 plays a major role as a master endogenous pluripotent genes trigger in reprogramming. It has been reported that Sirtuin 1 (Sirt1), a member of the Sirtuin family of NAD+-dependent protein deacetylases, is involved in embryonic stem cell antioxidation, differentiation, and individual development. However, as a deacetylation enzyme, whether Sirt1 influences reprogramming through its post-translational modification function remains unknown. In this study, we provide evidence that deacetylation of Sox2 by Sirt1 is required for reprogramming. We found that a low level of Sox2 acetylation could significantly increase reprogramming efficiency. Furthermore, we found that Sox2 can be deacetylated by Sirt1 in an Oct4-mediated manner. Compared with wild-type cells, Sirt1-null mouse embryonic fibroblasts exhibit decreased reprogramming efficiency, and overexpression of Sirt1 rescues this defect. In addition, Sirt1 functions in the regulation of reprogramming through deacetylating Sox2. Taken together, we have identified a new regulatory role of Sirt1 in reprogramming and provided a link between deacetylation events and somatic cell reprogramming. S tem C ells 2015;33:2135-2147 [ABSTRACT FROM AUTHOR]

The inactivation of plasminogen activator inhibitor-1 ( PAI-1) has been shown to exert beneficial effects in age-related vascular diseases. Limited information is available on the molecular mechanisms regarding the negatively regulated expression of PAI-1 in the vascular system. In this study, we observed an inverse correlation between SIRT1, a class III histone deacetylase, and PAI-1 expression in human atherosclerotic plaques and the aortas of old mice, suggesting that internal negative regulation exists between SIRT1 and PAI-1. SIRT1 overexpression reversed the increased PAI-1 expression in senescent human umbilical vein endothelial cells ( HUVECs) and aortas of old mice, accompanied by decreased SA-β-gal activity in vitro and improved endothelial function and reduced arterial stiffness in vivo. Moreover, the SIRT1-mediated inhibition of PAI-1 expression exerted an antisenescence effect in HUVECs. Furthermore, we demonstrated that SIRT1 is able to bind to the PAI-1 promoter, resulting in a decrease in the acetylation of histone H4 lysine 16 (H4K16) on the PAI-1 promoter region. Thus, our findings suggest that the SIRT1-mediated epigenetic inhibition of PAI-1 expression exerts a protective effect in vascular endothelial senescence. [ABSTRACT FROM AUTHOR]

SIRT1, a class III histone deacetylase, acts as a negative regulator for many transcription factors, and plays protective roles in inflammation and atherosclerosis. Transcription factor nuclear factor of activated T cells (NFAT) has been previously shown to play pro-inflammatory roles in endothelial cells. Inhibition of NFAT signaling may be an attractive target to regulate inflammation in atherosclerosis. However, whether NFAT transcriptional activity is suppressed by SIRT1 remains unknown. In this study, we found that SIRT1 suppressed NFAT-mediated transcriptional activity. SIRT1 interacted with NFAT, and the NHR and RHR domains of NFAT mediated the interaction with SIRT1. Moreover, we found that SIRT1 primarily deacetylated NFATc3. Adenoviral over-expression of SIRT1 suppressed PMA and calcium ionophore Ionomycin (PMA/Io)-induced COX-2 expression in human umbilical vein endothelial cells (HUVECs), while SIRT1 RNAi reversed the effects in HUVECs. Moreover, inhibition of COX-2 expression by SIRT1 in PMA/Io-treated HUVECs was largely abrogated by inhibiting NFAT activation. Furthermore, SIRT1 inhibited NFAT-induced COX-2 promoter activity, and reduced NFAT binding to the COX-2 promoter in PMA/Io-treated HUVECs. These results suggest that suppression of NFAT transcriptional activity is involved in SIRT1-mediated inhibition of COX-2 expression induced by PMA/Io, and that the negative regulatory mechanisms of NFAT by SIRT1 may contribute to its anti-inflammatory effects in atherosclerosis. [ABSTRACT FROM AUTHOR]

Angiotensin II (AngII) induces the development of vascular hypertrophy and hypertension. We have shown previously that overexpression of class III deacetylase SIRT1 inhibits AngII-induced hypertrophy in vascular smooth muscle cells (VSMCs). However, the direct role of SIRT1 in VSMCs in response to AngII infusion in vivo remains unclear. Here, we found that the expression and activity of SIRT1 in mouse aortas was decreased significantly by AngII infusion. VSMC-specific SIRT1 transgene (SV-Tg) prevented the increase in systolic blood pressure (SBP) caused by AngII infusion without affecting heart function in mice. SIRT1 overexpression alleviated vascular remodeling in mouse thoracic and renal aortas induced by AngII infusion, and significantly inhibited reactive oxygen species (ROS) generation, vascular inflammation, and collagen synthesis in arterial walls. Reduced expression of transforming growth factor-β 1 (TGF-β1) was also observed in the aortas of AngII-infused SV-Tg mice. Moreover, SIRT1 overexpression decreased AngII-increased binding of nuclear factor-κB on its specific binding sites on TGF-β1 promoter. Taken together, these data demonstrate that SIRT1 overexpression in VSMCs reduces SBP and inhibits AngII-induced vascular remodeling in mice. The inhibition of vascular remodeling contributes, at least in part, to the antihypertensive effect of SIRT1. Key message: [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) can be a consequence of diabetes, hypertension, immunologic disorders, and other exposures, as well as genetic factors that are still largely unknown. Glomerular filtration rate (GFR), which is widely used to measure kidney function, has a heritability ranging from 25% to 75%, but only 1.5% of this heritability is explained by genetic loci that have been identified to date. In this study we tested for associations between GFR and 234 SNPs in 26 genes from pathways of blood pressure regulation in 3,025 rural Chinese participants of the “Genetic Epidemiology Network of Salt Sensitivity” (GenSalt) study. We estimated GFR (eGFR) using baseline serum creatinine measurements obtained prior to dietary intervention. We identified significant associations between eGFR and 12 SNPs in 6 genes (ACE, ADD1, AGT, GRK4, HSD11B1, and SCNN1G). The cumulative effect of the protective alleles was an increase in mean eGFR of 4 mL/min per 1.73 m2, while the cumulative effect of the risk alleles was a decrease in mean eGFR of 3 mL/min per 1.73 m2. In addition, we identified a significant interaction between SNPs in CYP11B1 and ADRB2. We have identified common variants in genes from pathways that regulate blood pressure and influence kidney function as measured by eGFR, providing new insights into the genetic determinants of kidney function. Complex genetic effects on kidney function likely involve interactions among genes as we observed for CYP11B1 and ADRB2. [ABSTRACT FROM AUTHOR]

Objective To identify chromosomal regions harboring quantitative trait loci for waist circumference (WC) and body mass index (BMI). Design and Methods A genome-wide linkage scan and regional association study WC and BMI among 633 Chinese families was conducted. Results A significant linkage signal for WC was observed at 22q13.31-22q13.33 in the overall analysis (LOD = 3.13). Follow-up association study of 22q13.31-13.33 revealed an association between the TBC1D22A gene marker rs16996195 and WC (false discovery rate [FDR]-Q < 0.05). In gender-stratified analysis, suggestive linkage signals were attained for WC at 2p24.3-2q12.2 and 22q13.33 among females (LOD = 2.54 and 2.15, respectively). Among males, 6q12-6q13 was suggestively linked to BMI (LOD = 2.03). Single marker association analyses at these regions identified male-specific relationships of six single nucleotide polymorphisms (SNPs) at 2p24.3-2q12.2 (rs100955, rs13020676, rs13014034, rs12990515, rs17024325, and rs2192712) and five SNPs at 6q12-6q13 (rs7747318, rs7767301, rs12197115, rs12203049, and rs9454847) with the obesity-related phenotypes (all FDR-Q < 0.05). At chromosome 6q12-6q13, markers rs7755450 and rs11758293 predicted BMI in females (both FDR-Q < 0.05). Conclusions Genomic regions on chromosomes 2, 6, and 22 which may harbor important obesity-susceptibility loci were described. Follow-up study of these regions revealed several novel variants associated with obesity related traits. Future work to confirm these promising findings is warranted. [ABSTRACT FROM AUTHOR]

Nkx2.5 plays protective roles in cardiac homeostasis and survival in the postnatal hearts. However, the underlying molecular mechanisms that mediate the protective functions of Nkx2.5 remain unknown. Here, we showed that Nkx2.5 was downregulated in response to various stresses and was required for protection against the stress-induced apoptosis of cardiomyocytes. SIRT1, a member of the sirtuin family of proteins, was found to be a direct transcriptional target of Nkx2.5 and was required for the Nkx2.5-mediated protection of cardiomyocytes from doxorubicin (DOX)-induced apoptosis. Moreover, using chromatin immunoprecipitation assays, we found that Nkx2.5 was able to bind to the SIRT1 promoter and that this binding was significantly decreased in DOX-treated mouse hearts. Furthermore, the cardiac-specific overexpression of SIRT1 decreased the DOX-induced apoptosis of cardiomyocytes in SIRT1 transgenic mouse hearts compared with the hearts of their wild-type littermates. These findings demonstrate that SIRT1 acts as a direct transcriptional target of Nkx2.5 that maintains cardiomyocyte homeostasis and survival. [ABSTRACT FROM AUTHOR]

BACKGROUND We examined the association between 14 endothelial system genes and salt-sensitivity of blood pressure (BP). METHODS After a 3-day baseline examination, during which time the usual diet was consumed, 1,906 Chinese participants received a 7-day low-sodium diet (51.3 mmol of sodium/day) followed by a 7-day high-sodium diet (307.8 mmol of sodium/day). BP measurements were obtained at baseline and at the end of each intervention using a random-zero sphygmomanometer. RESULTS The DDAH1 rs11161637 variant was associated with reduced BP salt sensitivity, conferring attenuated systolic BP (SBP) and mean arterial pressure (MAP) decreases from baseline to the low-sodium intervention (both P = 2×10−4). Examination of genotype–sex interactions revealed that this relation was driven by the strong associations observed in men (P for interactions = 1.10×10−4 and 0.008, respectively). When switching from the low- to high-sodium intervention, increases in diastolic BP (DBP) and MAP were attenuated by the COL18A1 rs2838944 minor A allele (P = 1.41×10−4 and 1.55×10−4, respectively). Conversely, the VWF rs2239153 C variant was associated with increased salt sensitivity, conferring larger DBP and MAP reductions during low-sodium intervention (P = 1.22×10−4 and 4.44×10−5, respectively). Ten variants from 3 independent SELE loci displayed significant genotype–sex interactions on DBP and MAP responses to low-sodium (P for interaction = 1.56×10−3 to 1.00×10−4). Among men, minor alleles of 4 correlated markers attenuated BP responses to low-sodium intake, whereas minor alleles of another 4 correlated markers increased BP responses. No associations were observed in women for these variants. Further, qualitative interactions were shown for 2 correlated SELE markers. CONCLUSIONS These data support a role for the endothelial system genes in salt sensitivity. [ABSTRACT FROM AUTHOR]

BackgroundIncreased blood pressure (BP) reactivity to cold pressor test (CPT) is a risk factor for hypertension. Genetic factors may influence the variation of BP response to CPT among individuals. We explored the association of genetic variants in the apelin system genes (APLN, APLNR and ACE2) and BP response to CPT in a Chinese population.MethodsA total of 1,998 Han Chinese participants from the Genetic Epidemiology Network of Salt Sensitivity completed a CPT. The percentage changes of BP right after the end of ice-water immersion were used as the measurement of BP responses to CPT. Twenty-two single nucleotide polymorphisms (SNPs) were selected and genotyped, including both tag and potential functional SNPs of the APLN, APLNR, and ACE2 genes. A mixed-effect linear model was used to assess the association between SNPs and BP responses to CPT.ResultsIn women, three SNPs (rs1514283, rs4646176, and rs879922) of the ACE2 gene were significantly associated with the diastolic BP (DBP) response to CPT in the general and recessive genetic models after adjustment for multiple testing (all false discovery rate q < 0.05). There were no significant associations of polymorphisms in APLN and APLNR genes with BP responses to CPT.ConclusionsOur study identified genetic variants in the ACE2 gene that were significantly associated with DBP responses to cold stress in the Chinese female population. Future studies are warranted to confirm these findings.American Journal of Hypertension 2012; doi:10.1038/ajh.2012.61 [ABSTRACT FROM AUTHOR]

BackgroundBlood pressure (BP) homeostasis involves complex interactions among genetic and nongenetic factors, providing major challenges to dissection of the genetic components that influence BP and hypertension. In this study, we examine the effects of interaction of genetic variants with physical activity on BP in a relatively genetically homogenous cohort of rural Chinese villagers.MethodsGeneralized estimating equations analysis was used to test for associations of systolic blood pressure (SBP) and diastolic blood pressure (DBP) with variants in 24 genes in BP pathways (196 single-nucleotide polymorphisms (SNPs)) among 3,142 Chinese participants divided according to physical activity (active vs. inactive groups).ResultsIn the physically active group, two SNPs in NR3C2 were significantly associated with lower SBP, and a SNP in SCNN1B was significantly associated with lower SBP and DBP. In the physically inactive group, a SNP in APLNR was associated with lower SBP, a SNP in GNB3 (guanine nucleotide binding protein, β polypeptide 3) was associated with higher SBP and DBP, and a SNP in BDKRB2 (bradykinin receptor B2) was associated with lower DBP. Cumulative effects in carriers of minor alleles of these SNPs showed reductions of SBP and DBP as large as 8 and 5 mm Hg, respectively, in the active individuals compared to inactive individuals carrying the same number of minor alleles.ConclusionsWe found that physical activity modifies the effects of genetic variants on BP. However, our results also show that active individuals with specific genotypes always have lower BP than inactive individuals with the same genotypes, demonstrating the overall beneficial effects of physical activity on BP.American Journal of Hypertension (2011). doi:10.1038/ajh.2011.97 [ABSTRACT FROM AUTHOR]

Dietary potassium-supplementation has been associated with a decreased risk of hypertension and other cardiovascular outcomes. However, blood pressure (BP) responses to potassium supplementation vary among individuals. This study was designed to examine the association between 12 single nucleotide polymorphisms (SNPs) in the adducin 1 alpha (ADD1) and guanine nucleotide binding protein (G protein) beta polypeptide 3 (GNB3) genes and systolic BP (SBP), diastolic BP (DBP), and mean arterial pressure (MAP) responses to potassium-supplementation. We conducted a 7-day high-sodium intervention (307.8 mmol sodium/day) followed by a 7-day high-sodium with potassium-supplementation (60 mmol potassium/day) among 1906 Han Chinese participants from rural north China. BP measurements were obtained at the end of each intervention period using a random-zero sphygmomanometer. We identified significant associations between ADD1 variant rs17833172 and SBP, DBP, and MAP responses to potassium-supplementation (all P < 0.0001) that remained significant after adjustment for multiple comparisons. In participants that were heterozygous or homozygous for the G allele of this marker, SBP, DBP, and MAP response to potassium-supplementation were − 3.52 ( − 3.82, − 3.21), − 1.41 ( − 1.66, − 1.15) and − 2.12 ( − 2.37, − 1.87), respectively, as compared to the corresponding responses of 1.99 (0.25, 3.73), − 0.65 ( − 0.10, − 0.21), and − 0.23 ( − 0.37, 0.83), respectively, for those who were homozygous for A allele. In addition, participants with at least one copy of the G allele of rs12503220 of the ADD1 gene had significantly increased DBP and MAP response to potassium-supplementation ( P = 0.0041 and 0.01, respectively), which was also significant after correction for multiple testing. DBP and MAP responses to potassium-supplementation were − 1.36 ( − 1.63, − 1.10) and − 2.07 ( − 2.32, − 1.82) for those with at least G allele compared to corresponding responses of 0.86 ( − 0.68, 2.40) and − 0.45 ( − 1.74, 0.84) for those who were homozygous for A allele. In summary, our study identified novel associations between genetic variants of the ADD1 gene and BP response to potassium-supplementation, which could have important clinical and public health implications. Future studies aimed at replicating these novel findings are warranted. [ABSTRACT FROM AUTHOR]

Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA frament containing the inverted human α-globin genes (ϑ, α1, α2, and ζ) from BAC191K2 and the locus control region (LCR) of human β-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments. [ABSTRACT FROM AUTHOR]

Small RNAs, including short interfering RNAs (siRNAs) and microRNAs (miRNAs), are ubiquitous, versatile repressors of gene expression in plants, animals, and many fungi. They can trigger destruction of homologous mRNA or inhibition of cognate mRNA translation and play an important role in maintaining the stable state of chromosome structure and regulating the expression of protein-coding genes. Furthermore, the recent research showed that there exists close relationship between small RNAs and human diseases. Several human diseases have surfaced in which miRNAs or their machinery might be implicated, such as some neurological diseases and cancers. The specificity and potency of small RNAs suggest that they might be promising as therapeutic agents. This article will review the role of small RNAs in some human diseases etiology and investigations of taking siRNAs as therapeutic tools for treating viral infection, cancer, and other diseases. We also discuss the potential of miRNAs in gene therapy. © 2004 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Targeted gene modification mediated by single-stranded oligonucleotides (SSOs) holds great potential for widespread use in a number of biological and biomedical fields, including functional genomics and gene therapy. By using this approach, specific genetic changes have been created in a number of prokaryotic and eukaryotic systems. In mammalian cells, the precise mechanism of 550-mediated chromosome alteration remains to be established, and there have been problems in obtaining reproducible targeting efficiencies. It has previously been suggested that the chromatin structure, which changes throughout the cell cycle, may be a key factor underlying these variations in efficiency. This hypothesis prompted us to systematically investigate SSO-mediated gene repair at various phases of the cell cycle in a mammalian cell line. We found that the efficiency of SSO-mediated gene repair was elevated by &ap;10-foId in thymidine-treated S-phase cells. The increase in repair frequency correlated positively with the duration of SSO/thymidine coincubation with host cells after transfection. We supply evidence suggesting that these increased repair frequencies arise from a thymidine-induced slowdown of replication fork progression. Our studies provide fresh insight into the mechanism of SSO-mediated gene repair in mammalian cells and demonstrate how its efficiency may be reliably and substantially increased. [ABSTRACT FROM AUTHOR]

The host immune responses, including T lymphocytes mediated immune response and humoral immune responses are the important parts of the challenges in gene therapy. There are some potential immunostimulants in gene delivery systems, such as viral and non-viral vectors. Viral gene products, transgene products, viral proteins derived from viral particles required by dead-end infection, and CpG DNA in plasmid may play important roles in inducing the host immune responses when foreign genes are transferred into the targeted tissues. The immune responses should lead to many problems in gene therapy: transient expression of therapeutic gene, non-efficient re-administration of the same vectors, and severe side-effects in clinical trials. Although RNAi may act as gene therapeutic agent for suppression of specific gene expression, little attention has been given to the potential non-specific effects that might be induced. It was reported that small interfering RNAs (siRNAs) can induce the host interferon response following transfected to mammalian cells. Facing these challenges, a number of studies have been focused on taking measures to solve them, such as immunosuppression, selection of different administration routes and dose of the vectors, using the tissue-specific promoters and modifying the vectors. © 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 6, 748-761, 2004 [ABSTRACT FROM AUTHOR]

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Trials of retroviral vector-mediated human β-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human β-globin gene expression cassette for gene therapy of β-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3′-enhancer, and derivatives from the β-locus control region or α-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8×104 cfu/mL and 1.0×106 cfu/mL. We found that proviral DNA was intact in most G418-resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human β-globin gene expression was analyzed with RNase protection assay. The percentage of human β-globin transcript relative to endogenous murine α-globin transcript were 101.8±64.3% ( n=10), 40.1±28.7% ( n=4), 31.1±31.9% ( n=12), 52.4±11.2% ( n=12), and 53.6±8.6% ( n=12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for β-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C→T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human β-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA. [ABSTRACT FROM AUTHOR]

Special oligonucleotides for targeted gene correction have attracted increasing attention recently, one of which is the chimeric RNA·DNA oligonucleotide (RDO) system. RDOs for targeted gene correction were first designed in 1996, and are typically 68 nucleotides in length including continuous RNA and DNA sequences (RNA is 2′-O-methyl-modified). They have a 25 bp double stranded region homologous to the targeted gene, two hairpin ends of T loop and a 5 bp GC clamp, that give the molecule much greater stability [ Fig. 1]. One mismatch site in the middle of the double-stranded region is designed for targeted gene therapy. RDOs have been used recently for targeted gene correction of point mutations both in vitro and in vivo , but many problems must be solved before clinical application. One of the solutions is to optimize the delivery vectors for RDOs. To date, few RDO delivery systems have been used. Therefore, new vectors should be tried for RDO transfer, such as the use of nanoparticles. Additionally, different kinds of modifications should be applied to RDO carrier systems to increase the total correction efficiency in vivo . Only with the development of delivery systems can RDOs be used for gene therapy, and successfully applied to functional genomics. [ABSTRACT FROM AUTHOR]

Expression regulation of the β-globin gene cluster is a result of synergistic interactions between cis-elements and trans-acting factors. Previous studies usually concentrated on the core sequence of each hypersensitive site in the locus control region of the β-globin gene cluster. But more and more evidence illustrates that the flanking regions are indispensable also. Using electrophoretic mobility shift assay and solid-phase DNase I footprinting methods, we identified a small nuclear protein from K562 cells that binds specifically to the first AT-rich region flanking the hypersensitive site 2 core sequence of the human β-globin gene locus control region. N-terminal sequencing of the enriched protein proved that it is a member of the high-mobility group protein 2 family. This indicates that the AT-rich region in human hypersensitive site 2 may take part in the regulation of the β-globin gene cluster by facilitating DNA bending, which is a prerequisite for the looping mechanism in this region.Key words: β-globin gene, locus control region, hypersensitive site 2, AT-rich region, high-mobility group protein 2.La régulation de l'expression du groupe des gènes de la β-globine résulte d'interactions synergiques entre des éléments cis- et des facteurs trans-régulateurs. Les études antérieures ont surtout porté sur la séquence centrale de chaque site hypersensible dans la région de régulation du locus du groupe des gènes de la β-globine. Mais de plus en plus de données montrent que les régions flanquantes sont également indispensables. En utilisant le test de retardement sur gel et la méthode d'empreinte à la DNase I en phase solide, nous avons identifié une petite protéine nucléaire dans les cellules K562 qui se lie spécifiquement à la première région riche en AT flanquante de la séquence centrale du site hypersensible 2 de la région de régulation du locus des gènes de la β-globine humaine. Le séquençage de la partie N-terminale de la protéine partiellement purifiée démontre qu'elle fait partie de la famille des protéines HMG-2. Cela indique que la région riche en AT dans le site hypersensible 2 humain pourrait intervenir dans la régulation du groupe des gènes de la β-globine en facilitant la courbure de l'ADN, pré-requise pour le mécanisme de formation d'une boucle dans cette région.Mots clés : gène de la globine β, région de régulation du locus, site hypersensible 2, région riche en AT, protéine HMG-2.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Mutations in gene sequence can cause many genetic disorders, and researchers have attempted to develop treatments or cures at the DNA level for these diseases. Several strategies including triple-helix-forming oligonucleotides (TFOs), chimeric RNA/DNA oligonucleotide (RDO), and short single-stranded oligodeoxynucleotide (ODN) have been used to correct the dysfunctional genes in situ in the chromosome. Experimental data from cells and animal models suggest that all these strategies can repair the mutations in situ at DNA level. More effective structures of oligonucleotide, efficient delivery systems, and gene correction efficiency should be improved. Development of these strategies holds great potentials for treatments of genetic defects and other disorders. [ABSTRACT FROM AUTHOR]

The human β-globin locus is a classic model of the eukaryotic multigene family with tissue- and temporally specific expression. Over the past few years, great advances have been achieved in studies of β-globin locus regulation. The dominant role of the β-globin locus control region (LCR) in chromatin opening and developmental switching has been challenged, and elements beyond the LCR have been studied in depth. More recently, the fields of research have been expanded to intergenic transcription, nuclear localization and histone modification. Several models have been proposed to elucidate the regulation mechanism; among them, the looping and linking models are the most prevalent. Different models are the summarization of the observations made at different times and a persuasive model must be based on a systematic understanding of the numerous observations. The objective of this review is to provide an overview of progress in the area of β-globin regulation and then to discuss models for it. [ABSTRACT FROM AUTHOR]

In the forthcoming postgenomic era, identification of regulatory DNA sequences is becoming increasingly important for characterizing DNA-binding proteins and for elucidating the regulatory mechanisms of gene expression. Presently, there lack efficient methods to broadly screen and identify DNA regulatory elements on a large scale. We established herein an efficient strategy to screen regulatory sequences from bacterial artificial chromosome (BAC) DNAs containing human alpha- and beta-globin gene clusters based on polymerase chain reaction and electrophoretic mobility shift assay (EMSA) techniques without purified transcription factors. Twenty-three subclones derived from alpha-BAC DNA by bulk EMSA selection retained the ability to bind nuclear proteins of K562 cells when retested by EMSA. In 19 clones sequenced, 14 are identical to those registered in GenBank and five have one base difference. All of the 24 randomly picked beta-BAC clones showed specific binding with nuclear proteins of K562 cells. In 11 clones sequenced, eight are identical to those registered in GenBank and three have one base difference. This approach could be particularly powerful if combined with other systematic methods for identifying cis-regulatory DNA elements.Key words: gene expression regulation, bacterial artificial chromosome, alpha- and beta-globin gene clusters.À l'ère post-génomique qui débute, l'identification des séquences régulatrices dans l'ADN est de plus en plus importante pour caractériser les protéines se liant à l'ADN et pour élucider les mécanismes de régulation de l'expression des gènes. Actuellement, il n'y a pas de méthodes efficaces pour cribler et identifier des éléments régulateurs dans l'ADN à grande échelle. Nous avons mis au point une stratégie efficace pour cribler des séquences régulatrices dans l'ADN de chromosomes artificiels bactériens (BAC) contenant les groupes de gènes de la globine alpha et de la globine beta. Cette stratégie est basée sur la réaction de polymérisation en chaîne (PCR) et des tests de retardement sur gel qui ne nécessitent pas de facteurs de transcription purifiés. Vingt-trois sous-clones du BAC alpha sélectionnés par des tests de retardement sur gel conservent la capacité de lier des protéines nucléaires de cellules K562 lors de nouveaux tests de retardement sur gel. Parmi les 19 clones séquencés, 14 sont identiques à ceux déjà inscrits dans la GenBank et les 5 autres diffèrent seulement par une base. Tous les 24 clones du BAC beta choisis au hasard lient spécifiquement des protéines nucléaires de cellules K562. Parmi les 11 clones séquencés, 8 sont identiques à ceux déjà inscrits dans la GenBank et les 3 autres diffèrent seulement par une base. Cette approche pourrait être particulièrement puissante si elle était combinée avec d'autres méthodes systématiques d'identification d'éléments cis-régulateurs de l'ADN.Mots clés : régulation expression de gènes, chromosome artificiel bactérien, groupes de gènes de la globine de l'alpha et de la globine beta.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) is a promising viral vector in treating many kinds of hereditary diseases. The broad host range, low level of immune response, and longevity of gene expression observed with this vector have enabled the initiation of a number of clinical trials using this gene delivery system. Another potential benefit of AAV vectors is their ability to integrate site-specifically in the presence of Rep proteins. However, this virus is not well characterized. To obtain high level, persistent expression of the foreign gene, some problems should be solved. In this article, we will describe the advances in some fields of recombinant AAV technology that overcome certain limitations of the vector as a gene delivery system, such as the transduction efficiency, the production, the package capacity, and elimination of immune responses, as well as the applications involving these recombinant vectors for the treatment of some diseases. [ABSTRACT FROM AUTHOR]

Using ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the status of DNA–protein interactions at hypersensitive site 3.5 (HS3.5) of the locus control region in K562 and HEL cells, we found that there was protein occupancy in vivo at HS3.5 in both cell lines and the status of DNA–protein interaction was different between K562 and HEL. These data provide direct evidence that specific nuclear factor – DNA complexes form in vivo at functionally important sequence motifs of the HS3.5 in erythroid cells. This indicates that HS3.5 may play an important role in the regulation of the β-globin gene cluster. K562 is a human erythroleukemia cell line in which the embryonic ℇ-globin gene is predominantly expressed, while the HEL cell line expresses predominantly the fetal β-globin genes. Thus, HS3.5 might also be involved in the regulation of developmental stage-specific expression of β-globin genes. Our results are also consistent with the model that each hypersensitive site acts as a functional unit and HS3.5 may facilitate the formation of the HS3 functional unit.Key words: β-globin gene, hypersensitive site, phylogenetic footprint, differential phylogenetic footprint, in vivo footprinting, developmental regulation.L'amplification en chaîne par polymérase et la méthode d'empreinte sur l'ADN in vivo ont été utilisées pour étudier les interactions entre des protéines et l'ADN au site hypersensible 3.5 (HS3.5) de la région de régulation du locus dans des cellules K562 et HEL. Les résultats montrent que des protéines sont liées au site HS3.5 in vivo dans les deux lignées cellulaires et que l'interaction des protéines avec l'ADN diffère entre les cellules K562 et HEL. Ces résultats sont une preuve directe que des complexes entre des facteurs nucléaires spécifiques et l'ADN se forment in vivo sur des motifs de la séquence du site HS3.5 ayant une importance fonctionnelle dans les cellules érythrocytaires. Cela indique que le site HS3.5 jouerait un rôle important dans la régulation du groupe de gènes de la globine β. La lignée K562 de cellules érythroleucémiques humaines exprime principalement le gène de la globine ℇ embryonnaire, tandis que la lignée de cellules HEL exprime surtout les gènes de la globine γ foetale. Le site HS3.5 pourrait également intervenir dans la régulation de l'expression des gènes de la globine β au cours d'étapes spécifiques du développement. Nos résultats sont également en accord avec le modèle selon lequel chaque site hypersensible est une unité fonctionnelle et que le site HS3.5 faciliterait la formation de l'unité fonctionnelle HS3.Mots clés : gène, globine β, site hypersensible, empreinte phylogénétique, empreinte phylogénétique différentielle, empreinte sur l'ADN in vivo, régulation du développement.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

The human globin gene cluster, which represents a prototypical eukaryotic multigene locus, has been investigated for more than two decades and is classic model for coordinate control of tissue-specific gene expression. It is well known that globin gene expression is restricted to specific tissues and that globin genes are sequentially switched on during development. What intricate regulatory mechanisms account for tissue-specific transcriptional control of globin gene expression? Previous studies have focused on the interactions of trans-acting factors and cis-acting elements including the locus control region (LCR), which is considered a potent enhancer in globin gene switching. More recent studies have not only focused on the local DNA regulatory elements but also on remodelling of chromatin and transcription at the globin gene cluster within the native genomic context. Moreover, several studies have presented extensive data that address whether the LCR is required to open the chromatin. Although there is increased insight into the regulation of the β-globin gene switching, many aspects relating to the developmental activation of distinct globin genes remain elusive. [ABSTRACT FROM AUTHOR]

Tc1/mariner transposons are widespread in the eukaryotes. In vitro transposition test indicated that the transposase is the only protein that is needed in transpositions. It was shown later that the reconstructed Tc1-like transposon, “sleeping beauty” in fish, and the Tc1 transposon in C. elegans jumps in human cells. This discovery indicates that the Tc1/mariner transposon may be engineered as a somatic gene therapy vector if coupled with an efficient gene delivery system. We introduced the Tc1 transposon from C. elegans into different mammalian cell lines and detected the transposition events, indicating that Tc1 transposon functions in different mammalian cells. Interestingly, a high inversion frequency of the transposon was also detected, suggesting that this type of transposon may add variations to host genome when it is horizontally transferred into a new species. [ABSTRACT FROM AUTHOR]