Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

Genetic therapies for cystic fibrosis lung disease.

The aim of gene therapy for cystic fibrosis (CF) lung disease is to efficiently and safely express the CF transmembrane conductance regulator (CFTR) in the appropriate pulmonary cell types. Although CF patients experience multi-organ disease, the chronic bacterial lung infections and associated inflammation are the primary cause of shortened life expectancy. Gene transfer-based therapeutic approaches are feasible, in part, because the airway epithelium is directly accessible by aerosol delivery or instillation. Improvements in standard delivery vectors and the development of novel vectors, as well as emerging technologies and new animal models, are propelling exciting new research forward. Here, we review recent developments that are advancing this field of investigation.

Acceptance of a Novel, Highly Palatable, Calorically Dense, and Nutritionally Complete Diet in Dogs with Benign and Malignant Tumors.

Diminished appetite and poor eating behavior accompanied by weight loss or cachexia are often reported in dogs living with cancer. This study was conducted to determine the acceptance and eating enthusiasm in dogs with cancer for a new therapeutic, nutritionally balanced, and calorically dense food designed for dogs with cancer. Adult dogs with diagnosis of cancer were recruited from general and oncology practices and were fed the study food for 28 days. Evaluations included physical examination, body weight, food intake, caloric intake, hematology and serum biochemistry, and owner assessments, namely food evaluation, quality of life, and stool scores. The dogs transitioned smoothly and tolerated the food very well. The results showed high food acceptance within the first day, with continued eating enthusiasm over the 28 days. Significant increases in food and caloric intake were observed, with the study food having a positive impact on body weight in dogs that were losing weight and helping to maintain a high quality of life. Blood laboratory parameters remained within reference ranges. Thus, the therapeutic study food was well accepted and efficacious in supporting continued eating and required caloric intake, promoting a healthy weight gain and maintaining a high quality of life in dogs with cancer.

Comparison of two software programs used to determine the relative supersaturation of urine ions.

Introduction: Relative supersaturation (RSS) values for urine crystals are a measure of the risk of urinary stone formation and have been shown to be lowered in foods shown to aid in the management of urolithiasis. In order to calculate RSS in pets, computer programs have been developed to calculate RSS and aid in the understanding of stone formation in veterinary medicine. However, some older programs have not been updated for use in animals, and the specific coefficients used are not publically available. One of the first RSS programs was developed in BASIC computer language and published in 1985 which was called EQUIL2. The EQUIL2 program was updated to a compiled version compatible with a PC platform. However, the formulas could not be read or altered. Methods: This study evaluates a new program with known coefficients to the original EQUIL2 program. The RSS values of the two programs were compared through a t-test, calculating the r2 from correlation analysis, Lin's concordance correlation coefficient, and by a Bland-Altman analysis of outputs from the two programs using urine samples from healthy dogs and cats. Results and Discussion: Our results show that for both magnesium ammonium phosphate (struvite) and calcium oxalate, the RSS values of the original program could be calculated from the new programs RSS values. Although the actual RSS values were different (as might be expected through the use of the updated coefficients and different thermodynamic stability constants in the calculations) the results were highly correlated, finding elevations and reductions in RSS proportionally in the same urine samples. The current work creates a foundation for using the modernized program to calculate RSS and provides a shared method for understanding the risk of struvite and calcium oxalate stone formation.

Alpha-Lipoic Acid as a Nutritive Supplement for Humans and Animals: An Overview of Its Use in Dog Food.

Alpha-lipoic acid (a-LA) is used as a nutritive additive in dog food. Therefore, we performed a systematic review of studies published to date in PubMed, Google Scholar, Cochrane Library and MedlinePlus involving alpha-lipoic acid supplementation, which included human clinical trials as well as animal studies, to evaluate its utility as a supplement in foods for healthy, adult dogs. While an upper limit of alpha-lipoic acid intake in humans has not been conclusively determined, the levels for oral intake of a-LA have been better defined in animals, and distinct differences based on species have been described. The maximum tolerated oral dose of a-LA in dogs has been reported as 126 mg/kg body weight and the LD50 as 400 to 500 mg/kg body weight. The antioxidant, anti-inflammatory and neuro-protective benefits of alpha-lipoic acid in dogs were observed at concentrations much lower than the maximum tolerated dose or proposed LD50. At concentrations of 2.7-4.94 mg/kg body weight/day, alpha-lipoic acid is well tolerated and posed no health risks to dogs while providing improved antioxidant capacity. This review thereby supports the utility of alpha-lipoic acid as an effective nutritive additive in dog food.

Alpha-Lipoic Acid Is an Effective Nutritive Antioxidant for Healthy Adult Dogs.

This study was designed to determine the effect of alpha-lipoic acid on the glutathione status in healthy adult dogs. Following a 15 month baseline period during which dogs were fed a food containing no alpha-lipoic acid, dogs were randomly allocated into four groups. Groups were then fed a nutritionally complete and balanced food with either 0, 75, 150 or 300 ppm of alpha-lipoic acid added for 6 months. Evaluations included physical examination, body weight, food intake, hematology, serum biochemistry profile and measurements of glutathione in plasma and erythrocyte lysates. Throughout, blood parameters remained within reference ranges, dogs were healthy and body weight did not change significantly. A significant increase of 0.05 ng/mL of total glutathione in red blood cell (RBC) lysate for each 1 mg/kg bodyweight/day increase in a-LA intake was observed. In addition, a significant increase was observed for GSH, GSSG and total glutathione in RBC lysate at Month 6. We conclude that alpha-lipoic acid, as part of a complete and balanced food, was associated with increasing glutathione activity in healthy adult dogs.

In vitro synthesis of long DNA products in reactions with HIV-RT and nucleocapsid protein.

In vitro reaction conditions using HIV reverse transcriptase (RT) and nucleocapsid protein (NC) that allowed efficient synthesis of single-stranded DNA products over a thousand nucleotides in length from genomic HIV RNA were characterized. Consistent with previous reports, the reactions required high concentrations of NC and RT. Long products were produced as a result of frequent strand transfer between RNA templates, averaging at least one transfer per 300 nucleotides synthesized. No change in RT processivity was observed in the reactions in the presence versus absence of NC. Synthesis of long products required formation of a high molecular mass aggregate between NC and nucleic acids. The aggregate formed rapidly and pelleted with low speed centrifugation. The aggregate was accessible to RT as pre-formed aggregates synthesized long products when RT was added. NC finger mutants lacking either finger one or two or with the finger positions switched were all effective in promoting long products. This suggests that the aggregation/condensation but not helix-destabilizing activity of NC was required. We propose that these high molecular mass aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC to close proximity, thereby mimicking the role of the capsid environment within the host cell.

Influence of sequence identity and unique breakpoints on the frequency of intersubtype HIV-1 recombination.

BACKGROUND: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population. RESULTS: Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies. CONCLUSION: Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A/D HIV-1 recombinants, generated with each round of infection, are not replication-competent and do not survive in the multiple-cycle system. Ability of one HIV-1 isolate to outgrow the other leads to reduced co-infections, heterozygous virus production, and recombination frequencies.