Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open-reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were performed with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with MRI (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a three-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.
Duchenne muscular dystrophy; rAAV; exon-skipping; cardiomyopathy
Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×109 Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.
Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) has been recently identified as a marker of aggressive behavior in several types of tumors. The aim of the present study was to detect the expression of the IMP3 protein in colorectal adenocarcinoma (CRA) and to identify a correlation with the clinicopathological features of the disease. IMP3 was evaluated in 186 samples of CRA using immunohistochemical methods. The correlation between IMP3 expression and the clinicopathological features of colorectal cancer was evaluated by the χ2 and Fisher’s exact tests. Survival rates were calculated using the Kaplan-Meier method and the correlation between IMP3 protein expression and the prognosis of patients with CRA was analyzed using Cox analysis. Of the 186 adjacent normal mucosa (ANM) cases, the 22 that exhibited dysplasia demonstrated weak IMP3 expression and the 164 without dysplasia showed no expression. Of the 186 CRA cases, immunohistochemical staining for IMP3 was observed in 143 cases (76.9%). A comparison of IMP3 expression between the CRA and ANM samples revealed stronger immunohistochemical reactivity in the CRA tissues (P<0.01). High IMP3 expression was associated with differentiation, lymphoid metastasis, TNM stage, Ki-67 labeling index and a poor patient outcome (P<0.05). In the multivariate analysis, IMP3 emerged as an independent predictor of survival. The present study demonstrated that IMP3 is able to promote the aggressiveness of cancer behavior, resulting in a poor prognosis for patients with CRA. Consequently, IMP3 may be regarded as a novel proliferation and prognostic indicator for patients with CRA.
colorectal adenocarcinoma; insulin-like growth factor-II mRNA-binding protein 3; immunohistochemistry; prognosis
One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.
Activating transcription factor-2 (ATF2) is associated with tumor progression, but is not well-studied in head and neck squamous cell carcinoma (HNSCC). Its effects in stress and its importance in other survival mechanisms were studied.
ATF2 expression and nuclear activation were confirmed in HNSCC. Following modulation of ATF2, in vitro effects on proliferation and chemosensitivity were studied. Effects on in vivo tumor growth and Interleukin 8 (IL-8) expression were determined. Tumor Necrosis Factor (TNF) alpha treatment was used to further evaluate cytokine production and chemosensitivity.
Reductions of ATF2 resulted in significant nuclear p-ATF2 activation, cisplatin resistance and augmented IL-8 expression without affecting in vivo tumor growth. In this setting, TNF increases p-p38 phosphorylation and chemosensitivity while further enhancing IL-8 production.
Our data suggest regulatory roles for ATF2 in TNF-related mechanisms of HNSCC. Its perturbation and nuclear activation are associated with significant effects on survival and cytokine production.
apoptosis; chemoresistance; IL-8; ATF2; cisplatin
Apoptosis may play an important role in the mechanism underlying the GJB2 gene conditional knockout (cCx26) mice cochlear cell death. The objective of this study was to explore the the damage mode of the outer hair cells (OHCs) and its real time point of apoptosis and provide information to further explore the role of apoptosis in the happening of hearing loss in cCx26 mice.
Cochleae from mice at various developmental stages (P8, P12, and P21) were dissected out and first used to be observed under the scanning electron microscope (SEM). Basilar membranes from mice at P8, P14, P18, and P21 were stained by fluorescein isothiocyanate-conjugated phalloidin and propidium iodide (PI) and examined under confocal microscope.
The loss of OHCs of cCx26 knockout mice was first set between P12 and P21 under SEM. Whole mount phalloidin and PI staining revealed that obvious apoptotic appearance of the OHCs surface morphology was observed at P18.
Typical apoptotic morphology was found in the OHCs in the organ of Corti of the cCx26 mice at P18. This may provide information to further study the role of apoptosis in the occurrence of hearing loss of cCx26 mice.
Apoptosis; Outer auditory hair cells; Connexin 26; Sensorineural hearing loss
Huntingtin interacting protein 1 (HIP1) is a 116-kDa endocytic protein, which is necessary for the maintenance of several tissues in vivo as its deficiency leads to degenerative adult phenotypes. HIP1 deficiency also inhibits prostate tumor progression in mice. To better understand how deficiency of HIP1 leads to such phenotypes, we analyzed tumorigenic potential in mice homozygous for a Hip1 mutant allele, designated Hip1Δ3-5, which is predicted to result in a frame-shifted, nonsense mutation in the NH2 terminus of HIP1. In contrast to our previous studies using the Hip1 null allele, an inhibition of tumorigenesis was not observed as a result of the homozygosity of the nonsense Δ3-5 allele. To further examine the contrasting results from the prior Hip1 mutant mice, we cultured tumor cells from homozygous Δ3-5 allele–bearing mice and discovered the presence of a 110-kDa form of HIP1 in tumor cells. Upon sequencing of Hip1 DNA and message from these tumors, we determined that this 110-kDa form of HIP1 is the product of splicing of a cryptic U12-type AT-AC intron. This event results in the insertion of an AG dinucleotide between exons 2 and 6 and restoration of the reading frame. Remarkably, this mutant protein retains its capacity to bind lipids, clathrin, AP2, and epidermal growth factor receptor providing a possible explanation for why tumorigenesis was not altered after this knockout mutation. Our data show how knowledge of the transcript that is produced by a knockout allele can lead to discovery of novel types of molecular compensation at the level of splicing.
The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells. We have demonstrated that deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin at Ser-724, (corresponding to Ser-726 in human erythrocytes), a domain-target of protein kinase C (PKC). PKC phosphorylates adducin and leads to decreased F-actin capping and dissociation of spectrin from actin, implicating a significant role of such phosphorylation in cytoskeletal remodeling. We evaluated adducin phosphorylation in erythrocytes from patients with sickle cell disease and found it consistently increased at Ser-726. In addition, ROS concentration is elevated in sickle erythrocytes by 150–250% compared to erythrocytes from normal control individuals. Here, we review previous studies demonstrating that altered phosphorylation of erythrocyte cytoskeletal proteins and increased ROS production result in disruption of cytoskeleton stability in healthy and sickle cell erythrocytes. We discuss in particular the known and potential roles of protein kinase C and the Rac GTPases in these two processes.
Pandemic influenza represents a major threat to global health. Vaccination is the most economic and effective strategy to control influenza pandemic. Conventional vaccine approach, despite being effective, has a number of major deficiencies including limited range of protection, total dependence on embryonated eggs for production, and time consuming for vaccine production. There is an urgent need to develop novel vaccine strategies to overcome these deficiencies.
The major objective of this work was to develop a novel vaccine strategy combining recombinant haemagglutinin (HA) protein and a master cell (MC) activator C48/80 for intranasal immunization. We demonstrated in BALB/c mice that MC activator C48/80 had strong adjuvant activity when co-administered with recombinant HA protein intranasally. Vaccination with C48/80 significantly increased the serum IgG and mucosal surface IgA antibody responses against HA protein. Such increases correlated with stronger and durable neutralizing antibody activities, offering protection to vaccinated animals from disease progression after challenge with lethal dose of A/California/04/2009 live virus. Furthermore, protected animals demonstrated significant reduction in lung virus titers, minimal structural alteration in lung tissues as well as higher and balanced production of Th1 and Th2 cytokines in the stimulated splenocytes when compared to those without C48/80.
The present study demonstrates that the novel vaccine approach of combining recombinant HA and mucosal adjuvant C48/80 is safe and effective in eliciting protective immunity in mice. Future studies on the mechanism of action of C48/80 and potential combination with other vaccine strategies such as prime and boost approach may help to induce even more potent and broad immune responses against viruses from various clades.
Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/ remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals creation of an acidic environment favors growth of acid enduring and acid generating species, which causes further reduction in the plaque pH. In this study we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype “acid-activated peptides” (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries.
Targeted antimicrobial therapy; pH dependent antimicrobial activity; biofilm; Streptococcus mutans
Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus (S. mutans) can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent S. mutans colonization.
Controlled amounts of S. mutans were mixed with S. mutans-free saliva, grown into biofilms and visualized by antibody staining and cfu quantization. Two specifically-targeted antimicrobial peptides (STAMPs) against S. mutans were tested for their ability to reduce S. mutans biofilm incorporation upon treatment of the inocula. The resulting biofilms were also evaluated for their ability to resist subsequent exogenous S. mutans colonization.
S. mutans colonization was considerably reduced (9 ± 0.4 fold reduction, p=0.01) when the surface was preoccupied with saliva-derived biofilms. Furthermore, treatment with S. mutans-specific STAMPs yielded S. mutans-deficient biofilms with very significant protection against further S. mutans colonization (5min treatment: 38 ± 13 fold reduction p=0.01; 16 hr treatment: 96 ± 28 fold reduction p=0.07).
S. mutans infection is reduced by the presence of existing biofilms. Thus maintaining a healthy or “normal” biofilm through targeted antimicrobial therapy (such as the STAMPs) could represent an effective strategy for the treatment and prevention of S. mutans colonization in the oral cavity and caries progression.
Targeted antimicrobial therapy; antimicrobial peptide; biofilm; Streptococcus mutans; protective colonization; caries
Despite remarkable responses to the tyrosine kinase inhibitor imatinib, CML patients are rarely cured by this therapy perhaps due to imatinib-refractoriness of leukemia-initiating cells (LICs). Evidence for this is limited because of poor engraftment of human CMLLICs in NOD-SCID mice and non-physiologic expression of oncogenes in retroviral transduction mouse models. To address these challenges, we generated mice bearing conditional knockin alleles of two human oncogenes: HIP1/PDGFβR (H/P) and AML1-ETO (A/E). Unlike retroviral transduction, physiologic expression of H/P or A/E individually failed to induce disease, but co-expression of both H/P and A/E led to rapid onset of a fully penetrant, myeloproliferative disorder (MPD), indicating cooperativity between these two alleles. Although imatinib dramatically decreased disease burden, LICs persisted, demonstrating imatinib-refractoriness of LICs.
RcaC is a large, complex response regulator that controls transcriptional responses to changes in ambient light color in the cyanobacterium Fremyella diplosiphon. The regulation of RcaC activity has been shown previously to require aspartate 51 and histidine 316, which appear to be phosphorylation sites that control the DNA binding activity of RcaC. All available data suggest that during growth in red light, RcaC is phosphorylated and has relatively high DNA binding activity, while during growth in green light RcaC is not phosphorylated and has less DNA binding activity. RcaC has also been found to be approximately sixfold more abundant in red light than in green light. Here we demonstrate that the light-controlled abundance changes of RcaC are necessary, but not sufficient, to direct normal light color responses. RcaC abundance changes are regulated at both the RNA and protein levels. The RcaC protein is significantly less stable in green light than in red light, suggesting that the abundance of this response regulator is controlled at least in part by light color-dependent proteolysis. We provide evidence that the regulation of RcaC abundance does not depend on any RcaC-controlled process but rather depends on the presence of the aspartate 51 and histidine 316 residues that have previously been shown to control the activity of this protein. We propose that the combination of RcaC abundance changes and modification of RcaC by phosphorylation may be necessary to provide the dynamic range required for transcriptional control of RcaC-regulated genes.
Objectives. To find potential diagnostic markers or therapeutic targets, we used differential display technique to identify genes that are over or under expressed in human ovarian cancer.
Methods. Genes were initially identified by differential display between two human ovarian surface epithelium cultures and two ovarian cancer cell lines, A2780 and Caov-3. Genes were validated by relative quantitative RT-PCR and RNA in situ hybridization.
Results. Twenty-eight non-redundant sequences were expressed differentially in the normal ovarian epithelium and ovarian cancer cell lines. Seven of the 28 sequences showed differential expression between normal ovary and ovarian cancer tissue by RT-PCR. USP36 was over-expressed in ovarian cancer cell lines and tissues by RT-PCR and RNA in situ hybridization. Northern blot analysis and RT-PCR revealed two transcripts for USP36 in ovarian tissue. The major transcript was more specific for ovarian cancer and was detected by RT-PCR in 9/9 ovarian cancer tissues, 3/3 cancerous ascites, 5/14 (36%) sera from patients with ovarian cancer, and 0/7 sera from women without ovarian cancer.
Conclusion. USP36 is overexpressed in ovarian cancer compared to normal ovary and its transcripts were identified in ascites and serum of ovarian cancer patients.
ovarian cancer; biological markers; ubiquitin specific peptidase 36 (USP36); deubiquitinating enzyme
In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.
Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase involved in reducing mutagenic effects of 7,8-dihydro-8-oxo-guanine (8-oxoG). The C-terminal domain of MutY is required for 8-oxoG recognition and is critical for mutation avoidance of oxidative damage. To determine which residues of this domain are involved in 8-oxoG recognition, we constructed four MutY mutants based on similarities to MutT, which hydrolyzes specifically 8-oxo-dGTP to 8-oxo-dGMP. F294A-MutY has a slightly reduced binding affinity to A/G mismatch but has a severe defect in A/8-oxoG binding at 20°C. The catalytic activity of F294A-MutY is much weaker than that of the wild-type MutY. The DNA binding activity of R249A-MutY is comparable to that of the wild-type enzyme but the catalytic activity is reduced with both A/G and A/8-oxoG mismatches. The biochemical activities of F261A-MutY are nearly similar to those of the wild-type enzyme. The solubility of P262A-MutY was improved as a fusion protein containing streptococcal protein G (GB1 domain) at its N-terminus. The binding of GB1-P262A-MutY with both A/G and A/8-oxoG mismatches are slightly weaker than those of the wild-type protein. The catalytic activity of GB1-P262A-MutY is weaker than that of the wild-type enzyme at lower enzyme concentrations. Importantly, all four mutants can complement mutY mutants in vivo when expressed at high levels; however, F294A, R249A and P262A, but not F261A, are partially defective in vivo when they are expressed at low levels. These results strongly support that the C-terminal domain of MutY is involved not only in 8-oxoG recognition, but also affects the binding and catalytic activities toward A/G mismatches.
Two strains of obligately barophilic bacteria were isolated from a sample of the world’s deepest sediment, which was obtained by the unmanned deep-sea submersible Kaiko in the Mariana Trench, Challenger Deep, at a depth of 10,898 m. From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to Shewanella benthica and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus Moritella. The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain. This is the first evidence of the existence of an extreme-barophile bacterium of the genus Moritella isolated from the deep-sea environment.