Draft Genome Sequence of Candida saopaulonensis from a Very Premature Infant with Sepsis.

The rare fungus Candida saopaulonensis has never been reported to be associated with human infection. We report the draft genome sequence of the first clinical isolate of C. saopaulonensis, which was isolated from a very premature infant with sepsis. This is the first genome assembly reaching the near-complete chromosomal level with structural annotation for this species, opening up avenues for exploring evolutionary patterns and genetic mechanisms of pathogenesis.

Emergence of multiple fluconazole-resistant Candida parapsilosis sensu stricto clones with persistence and transmission in China.

OBJECTIVES: We explored the epidemiological and molecular characteristics of Candida parapsilosis sensu stricto isolates in China, and their mechanisms of azole resistance. METHODS: Azole susceptibilities of 2318 non-duplicate isolates were determined using CLSI broth microdilution. Isolates were genotyped by a microsatellite typing method. Molecular resistance mechanisms were also studied and functionally validated by CRISPR/Cas9-based genetic alterations. RESULTS: Fluconazole resistance occurred in 2.4% (n = 56) of isolates, and these isolates showed a higher frequency of distribution in ICU inpatients compared with susceptible isolates (48.2%, n = 27/56 versus 27.8%, 613/2208; P = 0.019). Microsatellite-genotyping analysis yielded 29 genotypes among 56 fluconazole-resistant isolates, of which 10 genotypes, including 37 isolates, belonged to clusters, persisting and transmitting in Chinese hospitals for 1-29 months. Clusters harbouring Erg11Y132F (5/10; 50%) were predominant in China. Among these, the second most dominant cluster MT07, including seven isolates, characteristically harbouring Erg11Y132F and Mrr1Q625K, lent its carriage to being one of the strongest associations with cross-resistance and high MICs of fluconazole (>256 mg/L) and voriconazole (2-8 mg/L), causing transmission across two hospitals. Among mutations tested, Mrr1Q625K led to the highest-level increase of fluconazole MIC (32-fold), while mutations located within or near the predicted transcription factor domain of Tac1 (D440Y, T492M and L518F) conferred cross-resistance to azoles. CONCLUSIONS: This study is the first Chinese report of persistence and transmissions of multiple fluconazole-resistant C. parapsilosis sensu stricto clones harbouring Erg11Y132F, and the first demonstration of the mutations Erg11G307A, Mrr1Q625K, Tac1L263S, Tac1D440Y and Tac1T492M as conferring resistance to azoles.

The first established microsatellite markers to distinguish Candida orthopsilosis isolates and detection of a nosocomial outbreak in China.

The infection proportion of Candida orthopsilosis, a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C. orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1-12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission.

Performance of two egg parasitoids of brown marmorated stink bug before and after cold storage.

Introduction: The genus Trissolcus includes a number of egg parasitoids that are known to contribute to the control of Halyomorpha halys. The number of progenies, particularly females, is important for the efficient mass rearing of species used in augmentative biological control programs. Cold storage is an important technique for extending the shelf life of natural enemies used in such programs. Methods: We assessed how fecundity, sex ratio, lifespan, and the number of hosts parasitized within 24 h were affected by host density for T. japonicus and T. cultratus when offered fresh H. halys eggs and how these parameters were affected if adult parasitoids were first placed in cold storage (11°C in the dark) for 19 weeks before being used for propagation. Results: The fecundity were 110.2 and 84.2 offspring emerged at 25°C, for parasitoids not placed in cold storage; among the offspring that emerged, 82.6% and 85.6% were female for T. japonicus and T. cultratus, respectively. If first placed in cold storage, T. japonicus and T. cultratus produced 35.1 and 24.6 offspring per female, respectively, although cold storage significantly extended the shelf life. The survival rates of parasitoids that were placed in cold storage were 90.3% and 81.3% for females, and 3.2% and 0.9% for males of T. japonicus and T. cultratus, respectively. The number of hosts parasitized within 24 h was not shown to be density dependent, but it was significantly lower after cold storage. Discussion: This information can be used to estimate the likely production for augmented rearing colonies for use in biological control programs.

Decreased echinocandin susceptibility in Candida parapsilosis causing candidemia and emergence of a pan-echinocandin resistant case in China.

Candida parapsilosis is becoming a predominant non-albicans cause of invasive candidiasis (IC). Echinocandins are the preferred choice for IC treatment and prophylaxis. Resistance to echinocandins in C. parapsilosis has emerged in several countries, but little is known about the susceptibility profile in China or about mechanisms of resistance. Here, we investigated the echinocandin susceptibilities of 2523 C. parapsilosis isolates collected from China and further explored the resistance mechanism among echinocandin-resistant isolates. Anidulafungin exhibited the highest MICs (MIC50/90, 1 and 2 µg/mL; GM, 0.948 µg/mL), while caspofungin showed better activity (0.5 and 1 µg/mL; 0.498 µg/mL). Significantly higher echinocandin MICs were observed among blood-derived isolates compared to others, especially for caspofungin (GM, 1.348 µg/mL vs 0.478 µg/mL). Isolates from ICU and surgical wards also showed higher MICs. Twenty isolates showed intermediate phenotypes for at least one echinocandin. One was resistant to all three echinocandins, fluconazole and voriconazole, which caused breakthrough IC during long-term exposure to micafungin. WGS revealed this isolate carried a mutation S656P in hotspot1 region of Fks1. Bioinformatics analyses suggested that this mutation might lead to an altered protein conformation. CRISPR Cas9-mediated introduction of this mutation into a susceptible reference C. parapsilosis strain increased MICs of all echinocandins 64-fold, with similar results found in the subspecies, C. orthopsilosis and C. metapsilosis. This is the first report of a multi-azole resistant and pan-echinocandin resistant C. parapsilosis isolate, and the identification of a FKS1S656P conferring pan-echinocandin resistance. Our study underscores the necessity of rigorous management of antifungal use and of monitoring for antifungal susceptibility.

Enhanced biodegradation of oil-contaminated soil oil in shale gas exploitation by biochar immobilization.

The enhanced biodegradation of oil-contaminated soil by fixing microorganisms with corn cob biochar was investigated. It was found that the components of oil in the test soil were mainly straight-chain alkanes and branched alkanes. When using corn cob biochar as a carrier to immobilize microorganisms, the best particle size of corn cob biochar as an immobilization carrier was 0.08 mm, and the best immobilization time was 18 h. SEM analysis confirmed that the microorganisms were immobilized on the corn cob biochar. Immobilized microorganisms exhibited high biodegradability under stress to high concentrations of petroleum pollutants, heavy metals, and organic pollutants. Infrared spectroscopy analysis showed that oxygen-containing groups such as hydroxyl, carboxyl, and methoxy on the surface of biochar were involved in the complexation of heavy metals. The mechanism of immobilization promoted microbial degradation of oil contamination was explained by gas chromatography mass. First, alkanes and aromatics were adsorbed by corn cob biochar and passed to immobilized microorganisms to promote their degradation. Their bioavailability increased, especially for aromatics. Second, biochar provided a more suitable environment for microorganisms to degrade. Third, the conversion of ketones to acids was accelerated during the biodegradation of alkanes, and the biodegradation of alkanes was accelerated by immobilization. The biodegradable efficiency of oil by immobilized microorganisms in soil was 70.10% within 60 days, 28.80% higher than that of free microorganisms. The degradation of immobilized microorganisms was highly correlated with the activities of catalase, urease, and polyphenol oxidase.

NicE-C efficiently reveals open chromatin-associated chromosome interactions at high resolution.

Enhancer-promoter communication is known to regulate spatiotemporal dynamics of gene expression. Several methods are available to capture enhancer-promoter interactions, but they either require large amounts of starting materials and are costly, or provide a relative low resolution in chromatin contact maps. Here, we present nicking enzyme-assisted open chromatin interaction capture (NicE-C), a method that leverages nicking enzyme-mediated open chromatin profiling and chromosome conformation capture to enable robust and cost-effective detection of open chromatin interactions at high resolution, especially enhancer-promoter interactions. Using TNF stimulation and mouse kidney aging as models, we applied NicE-C to reveal characteristics of dynamic enhancer-promoter interactions.

Study on the Effect of Petroleum Components on the Elution of Oily Sludge by a Compound Biosurfactant.

Currently, research on oily sludge treatment mainly focuses on optimizing the deoiling effect and research on the deoiling mechanism, and the influence of petroleum components on the properties and treatment of oily sludge is rarely considered. Therefore, in this study, petroleum substances in three types of oil sludge were eluted using the biosurfactant cleaning technology, and the influence of petroleum components on the cleaning process was explored. The results showed that the biosurfactants rhamnolipid and sophorolipid had a synergistic effect, and the oil-removal rate was as high as 92.2% when the SL mass fraction was 0.4 in the compound biosurfactant. Three types of oily sludge, wellsite-landing sludge, pipeline-landing sludge, and tank-bottom sludge, were cleaned by the compound biosurfactant; the results showed that the residual petroleum substance in liquid and solid phases, the turbidity value, and the zeta-potential value of the supernatant of oil sludge samples after cleaning increased with the increase in the heavy components of the oily sludge, and the oil-removal rate decreased gradually. After cleaning, the average relative molecular weight of the three oil phases increased with the heavy components, which was increased by 1.83, 4.83, and 10.72%, respectively, and the increase in molecular weight increased the difficulty of cleaning. After cleaning, the retention time and peak intensity of the oil sample changed significantly, and it had a stronger elution effect on low-molecular-weight alkanes. It was found that the compound biosurfactant had a good elution effect on polycyclic aromatic hydrocarbons, but the increase in the content of heavy components and the increase in aromatic rings increased the difficulty of cleaning. Moreover, it was found that the compound biosurfactant could not completely elute the petroleum substances on the surface of solid particles, and the asphaltene components in the oil phase were more difficult to elute than other components.

Engineered Capsids for Efficient Gene Delivery to the Retina and Cornea.

Adeno-associated viral (AAV) vectors represent an ideal vehicle for human gene transfer. One advantage to the AAV vector system is the availability of multiple naturally occurring serotypes that provide selective tropisms for various target cells. Strategies to enhance the properties of the natural AAV isolates have been developed and can be divided into two approaches, rational design or directed evolution. The rational design approach utilizes knowledge of AAV capsids to make targeted changes to the capsid to alter transduction efficiency or specificity, while the directed evolution approach does not require a priori knowledge of capsid structure and includes random mutagenesis, capsid shuffling, or random peptide insertion. In this study, we describe the generation of novel variants for both AAV2 and AAV5 using a rational design approach and knowledge of AAV receptor binding, surface charge, and AAV capsid protein posttranslational modifications. The novel AAV2 and AAV5 variants demonstrate improved transduction properties in both the mouse retina and cornea. The translational fidelity of the novel AAV2 variant was confirmed in the context of the nonhuman primate (NHP) retina, whereas a NHP tissue explant model was established to allow the rapid assessment of translational fidelity between species for the AAV5 variants. The capsid-modified AAV2 and AAV5 variants described in this study have novel attributes that will add to the efficacy and specificity of their potential use in gene therapy for a range of human ocular diseases.

Reorganized 3D Genome Structures Support Transcriptional Regulation in Mouse Spermatogenesis.

Three-dimensional chromatin structures undergo dynamic reorganization during mammalian spermatogenesis; however, their impacts on gene regulation remain unclear. Here, we focused on understanding the structure-function regulation of meiotic chromosomes by Hi-C and other omics techniques in mouse spermatogenesis across five stages. Beyond confirming recent reports regarding changes in compartmentalization and reorganization of topologically associating domains (TADs), we further demonstrated that chromatin loops are present prior to and after, but not at, the pachytene stage. By integrating Hi-C and RNA-seq data, we showed that the switching of A/B compartments between spermatogenic stages is tightly associated with meiosis-specific mRNAs and piRNAs expression. Moreover, our ATAC-seq data indicated that chromatin accessibility per se is not responsible for the TAD and loop diminishment at pachytene. Additionally, our ChIP-seq data demonstrated that CTCF and cohesin remain bound at TAD boundary regions throughout meiosis, suggesting that dynamic reorganization of TADs does not require CTCF and cohesin clearance.

Rearrangement of macronucleus chromosomes correspond to TAD-like structures of micronucleus chromosomes in Tetrahymena thermophila.

The somatic macronucleus (MAC) and germline micronucleus (MIC) of Tetrahymena thermophila differ in chromosome numbers, sizes, functions, transcriptional activities, and cohesin complex location. However, the higher-order chromatin organization in T. thermophila is still largely unknown. Here, we explored the higher-order chromatin organization in the two distinct nuclei of T. thermophila using the Hi-C and HiChIP methods. We found that the meiotic crescent MIC has a specific chromosome interaction pattern, with all the telomeres or centromeres on the five MIC chromosomes clustering together, respectively, which is also helpful to identify the midpoints of centromeres in the MIC. We revealed that the MAC chromosomes lack A/B compartments, topologically associating domains (TADs), and chromatin loops. The MIC chromosomes have TAD-like structures but not A/B compartments and chromatin loops. The boundaries of the TAD-like structures in the MIC are highly consistent with the chromatin breakage sequence (CBS) sites, suggesting that each TAD-like structure of the MIC chromosomes develops into one MAC chromosome during MAC development, which provides a mechanism of the formation of MAC chromosomes during conjugation. Overall, we demonstrated the distinct higher-order chromatin organization in the two nuclei of the T. thermophila and suggest that the higher-order chromatin structures may play important roles during the development of the MAC chromosomes.

biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

FTY720 Attenuates Angiotensin II-Induced Podocyte Damage via Inhibiting Inflammatory Cytokines.

FTY720, a new chemical substance derived from the ascomycete Isaria sinclairii, is used for treating multiple sclerosis, renal cancer, and asthma. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite and exists in red blood cells. FTY720 is a synthetic S1P analog which can block S1P evoking physiological effects. Recently studies show that S1P was participating in activated inflammation cells induced renal injury. The objective of this study was to assess the protective effect of FTY720 on kidney damage and the potential mechanism of FTY720 which alleviate podocyte injury in chronic kidney disease. In this study, we selected 40 patients with IgA nephropathy and examined their clinical characteristics. Ang II-infusion rat renal injury model was established to evaluate the glomeruli and tubulointerstitial lesion. The result showed that the concentration of S1P in serum and urine was positively correlated with IgA nephropathy patients' renal injury. FTY720 could reduce renal histological lesions induced by Ang II-infusion in rats. Moreover, FTY720 decreased S1P synthesis in Ang II-infusion rats via downregulation of inflammatory cytokines including TNF-α and IL-6. In addition, FTY720 alleviated exogenous S1P-induced podocyte damage. In conclusion, FTY720 is able to attenuate S1P-induced podocyte damage via reducing inflammatory cytokines.

Three-dimensional regulation of transcription.

Cells can adapt to environment and development by reconstructing their transcriptional networks to regulate diverse cellular processes without altering the underlying DNA sequences. These alterations, namely epigenetic changes, occur during cell division, differentiation and cell death. Numerous evidences demonstrate that epigenetic changes are governed by various types of determinants, including DNA methylation patterns, histone posttranslational modification signatures, histone variants, chromatin remodeling, and recently discovered chromosome conformation characteristics and non-coding RNAs (ncRNAs). Here, we highlight recent efforts on how the two latter epigenetic factors participate in the sophisticated transcriptional process and describe emerging techniques which permit us to uncover and gain insights into the fascinating genomic regulation.

Adeno-associated virus-mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E(-/-) mice.

BACKGROUND: The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis. METHODS: Apolipoprotein E deficient (ApoE(-/-) ) mice were injected intravenously with a recombinant adeno-associated virus (AAV8-ASM) that constitutively expressed high levels of human ASM in liver and plasma. RESULTS: Plasma sphingomyelin levels were reduced at early but not later time points after the administration of AAV8-ASM despite persistently elevated circulating ASM. No change in serum lipoprotein levels was observed. Thirteen or 17 weeks after the administration of AAV8-ASM, the amount of plaque formation in the aortic sinus was comparable to that of mice treated with a control AAV. CONCLUSIONS: Unexpectedly, the lesion area of the entire aorta was reduced significantly in the AAV8-ASM virus-treated group. Hepatic expression and secretion of ASM into the circulation did not accelerate or exacerbate, but rather decreased, lesion formation in ApoE(-/-) mice. Thus, plasma ASM activity does not appear to be rate limiting for plaque formation during atherogenesis.

A hypoxic inducible factor-1 alpha hybrid enhances collateral development and reduces vascular leakage in diabetic rats.

BACKGROUND: Diabetes mellitus is a common comorbidity of atherosclerosis. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of the angiogenic response to hypoxia. METHODS: We studied the effects of adenoviral vectors expressing a constitutively active HIF-1 alpha hybrid (Ad2/HIF-1 alpha/VP16) or vascular endothelial growth factor (Ad2/VEGF) on collateral development and vascular leakiness in a diabetic rat model of hindlimb ischemia. RESULTS: After the removal of the right femoral artery, the mRNA levels of VEGF, angiopoietin-1 and angiopietin-4 in the calf muscles, as measured by Taqman reverse transcriptase-polymerase chain reaction, were transiently elevated in Zucker lean (ZL) but not Zucker diabetic fatty (ZDF) rats. The angiographic score, as determined by post-mortem angiography, was significantly lower in ZDF animals 35 days after surgery compared to their ZL counterparts. In separate animals, intramuscular injection of Ad2/HIF-1a/VP16 and Ad/2VEGF into the thigh muscles significantly increased the angiographic score and capillary density 21 and 35 days after the injection compared to Ad2/CMVEV (a vector expressing no transgene) or vehicle. After the injection of Ad2/CMVEV or vehicle, the Evans-blue dye content in the thigh muscles was significantly higher in ZDF rats than their ZL counterparts. Ad2/HIF-1 alpha/VP16 but not Ad2/VEGF reduced tissue Evans blue dye content. CONCLUSIONS: The endogenous angiogenic response to ischemia was impaired in ZDF rats, possibly due to down-regulation of angiogenic factors. Ad2/HIF-1 alpha/VP16 enhanced collateral development and reduced vascular leakage in the ischemic hindlimb of ZDF rats indicating that hybrid HIF-1 alpha angiogenic therapy may be efficacious for peripheral vascular disease with a diabetic comorbidity.

Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor alpha/retinoid X receptor.

In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid beta-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid beta-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1alpha/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for beta-oxidation. Furthermore, adenovirus-mediated expression of HIF-1alpha/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor alpha (PPARalpha)/retinoid X receptor (RXR), in the presence or absence of a PPARalpha ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPARalpha/RXR.

Adenovirus-mediated expression of beta-adrenergic receptor kinase C-terminus reduces intimal hyperplasia and luminal stenosis of arteriovenous polytetrafluoroethylene grafts in pigs.

BACKGROUND: Hemodialysis vascular access dysfunction is the single most important cause of morbidity in kidney hemodialysis patients. Failure of an arteriovenous polytetrafluoroethylene (PTFE) graft, the most common form of hemodialysis access, is primarily due to intimal hyperplasia and thrombosis at the venous anastomosis. METHODS AND RESULTS: This study was aimed at evaluating the efficacy and safety of an adenoviral vector (Ad2/betaARKct) encoding the carboxyl terminus of beta-adrenergic receptor kinase (betaARKct) in a pig model of arteriovenous PTFE graft failure. Transduction of the external jugular vein with Ad2/betaARKct (5E9, 5E10, or 5E11 particles per vein) did not result in systemic toxicity, as measured by clinical and pathological assessments. Ad2/betaARKct significantly reduced neointimal hyperplasia in the graft/vein anastomosis. It also improved the graft patency rate and angiographic score, as measured histologically and angiographically, compared with vehicle or empty viral vector controls. CONCLUSIONS: Our results suggest that local administration of adenoviral vectors encoding betaARKct into the jugular vein represents a viable strategy to treat AV graft hemodialysis vascular access failure.

Expression of constitutively stable hybrid hypoxia-inducible factor-1alpha protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury.

Preconditioning in cultured cardiomyocytes elevates the expression of several protective genes including Glut-4 and heat shock protein (HSP)70. Hypoxia-inducible factor-1 (HIF-1) is known to mediate the transcriptional activation of hypoxia-responsive genes. In this study, we examined the effect of adenovirus-mediated expression of constitutively stable hybrid forms of HIF-1alpha on cardiomyocyte viability and gene expression. Cultured neonatal rat cardiomyocytes were subjected to simulated ischemia-reperfusion with or without preinfection with recombinant adenoviral vectors [Ad2/HIF-1alpha/herpes simplex virus protein VP16 and Ad2/HIF-1alpha/nuclear factor-kappaB (NF-kappaB)]. Cellular viability and mRNA levels of several cardioprotective genes were measured. We demonstrated that infection with Ad2/HIF-1alpha/VP16 and Ad2/HIF-1alpha/NF-kappaB mimicked the upregulation of the mRNA levels of vascular endothelial growth factor (VEGF), Glut-1, Glut-4, HSP70, and inducible NO synthase (iNOS) and the protection of cultured neonatal rat cardiomyocytes by late-phase preconditioning against simulated ischemia-reperfusion. The same dose of a control viral vector expressing no transgene had no effect. Preconditioning also elevated HIF-1alpha protein levels. These results suggest that adenovirus-mediated expression of HIF-1alpha/VP16 or HIF-1alpha/NF-kappaB, a constitutively stable hybrid transcriptional factor, protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes.

Catheter-mediated delivery of adenoviral vectors expressing beta-adrenergic receptor kinase C-terminus inhibits intimal hyperplasia and luminal stenosis in rabbit iliac arteries.

BACKGROUND: Previous studies have shown that incubation of balloon-injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the beta-adrenergic receptor kinase (Ad2/betaARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of betaARKct could be achieved using a catheter-based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis. METHODS: In this study, Ad2/betaARKct was infused into the balloon-injured site of rabbit iliac arteries using a porous infusion catheter over 2 min. Twenty-eight days after gene transfer, angiographic and histological assessments were performed. RESULTS: Angiographic and histological assessments indicate significant (p < 0.05) inhibition of iliac artery neointima formation and lumen stenosis by Ad2/betaARKct. Our studies demonstrate that an inhibitory effect of Ad2/betaARKct on neointima formation is achievable using a catheter-based vector delivery system and that the inhibition of neointima formation translates into a gain in the vessel minimal luminal diameter. The extent of inhibition (35%) was comparable to that observed with adenoviral-mediated expression of thymidine kinase plus ganciclovir treatment, a cytotoxic gene therapy approach for restenosis. CONCLUSIONS: These results suggest that adenoviral-mediated gene transfer of betaARKct is a clinically viable cytostatic gene therapy strategy for the treatment of restenosis.