Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat

Background

Adeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV’s ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye.

Methodology/Principle Findings

AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient.

Conclusions/Significance

This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.     

Low levels of taurine introgression in the current Brazilian Nelore and Gir indicine cattle populations

Background

Nelore and Gir are the two most important indicine cattle breeds for production of beef and milk in Brazil. Historical records state that these breeds were introduced in Brazil from the Indian subcontinent, crossed to local taurine cattle in order to quickly increase the population size, and then backcrossed to the original breeds to recover indicine adaptive and productive traits. Previous investigations based on sparse DNA markers detected taurine admixture in these breeds. High-density genome-wide analyses can provide high-resolution information on the genetic composition of current Nelore and Gir populations, estimate more precisely the levels and nature of taurine introgression, and shed light on their history and the strategies that were used to expand these breeds.

Results

We used the high-density Illumina BovineHD BeadChip with more than 777 K single nucleotide polymorphisms (SNPs) that were reduced to 697 115 after quality control filtering to investigate the structure of Nelore and Gir populations and seven other worldwide populations for comparison. Multidimensional scaling and model-based ancestry estimation clearly separated the indicine, European taurine and African taurine ancestries. The average level of taurine introgression in the autosomal genome of Nelore and Gir breeds was less than 1% but was 9% for the Brahman breed. Analyses based on the mitochondrial SNPs present in the Illumina BovineHD BeadChip did not clearly differentiate taurine and indicine haplotype groupings.

Conclusions

The low level of taurine ancestry observed for both Nelore and Gir breeds confirms the historical records of crossbreeding and supports a strong directional selection against taurine haplotypes via backcrossing. Random sampling in production herds across the country and subsequent genotyping would be useful for a more complete view of the admixture levels in the commercial Nelore and Gir populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0109-5) contains supplementary material, which is available to authorized users.     

Effect of non-lytic concentrations of Brij series detergents on the metabolism-independent ion permeability properties of human erythrocytes.

Subcritical micellar concentrations (sub-CMC) of Brij-series detergents alter ion movements between human erythrocytes and their environment when metabolism has been slowed down by incubation at zero degrees centigrade. The effect of nonhemolytic concentrations of detergents on the erythrocyte K+ and Na+ movements is described. Results indicate a significant difference in monovalent cation movements, depending on the number of hydrophilic polyoxyethylene units (n). There is an increasing loss of K+ and gain of Na+ as n increases from 4 to 20. Where n > or = 21, ion movements are not significantly different from those found in erythrocytes not exposed to detergents. The carbon chain length of the detergent fatty acid residue (10-18 carbons) appears to be relatively unimportant, but detergents with unsaturated (oleic acid) hydrophobic regions potentiate K+ release and Na+ uptake when compared to the corresponding saturated fatty acid (stearic acid). The erythrocyte stabilizing effect of detergents against hypo-osmotic shock correlates well with the increase of monovalent ion traffic and the mobility of membrane lipids revealed by fluorescence anisotropy measurements.     

Interactions of Divalent Cations in Their Action on ATPases from Cucumber Roots

A microsomal fraction (10,000–30.000 g) was prepared from roots of Cucumis satirus L. (cv. Bestseller Fl) grown in solution culture. The ATPase activity was stimulated by Mg and Mn with optima between pH 6 and 7. Stimulation by Ca was obtained only above pH 7. Activations by Mg and Mn were inhibited by Ca, Mn and Mg interacted, so that Mn appeared strongly inhibitory for activation by Mg and Mg weakly inhibitory for activation by Mn: the simplest interpretation for this would be two separate enzymes. Cucumber accumulates and deposits Ca contrary to wheat and oat, which contain Ca-activated ATPase in the pH region 6 to 7. The Ca data for cucumber are compared with earlier findings from wheat and oat and are tentatively related to known physiological differences.     

Steady-state volumes and metabolism-independent osmotic adaptation in mammalian erythrocytes

Erythrocytes of various mammalian species -- including human -- maintain osmotic balance with the blood plasma (osmotic activity 270-310 mosmol). However, their intracellular levels of osmotically active ions (potassium, sodium, chloride, and hydrogencarbonate), water content and osmotic resistance deviate significantly. In the present report we study the relationship among intracellular water, potassium and sodium levels of the erythrocytes of various mammalian species and in the developing calf. In addition, the osmotic resistance, K(+) (Rb(+)) uptake and the DPH fluorescence anisotropy of various erythrocytes and erythrocyte ghost membranes were correlated. The results show no statistically significant relationship between erythrocyte water content and [K(+)+Na(+)] levels or K(+)/Na(+) ratios. The reversal of erythrocyte K(+)/Na(+) ratios coincides with the decrease of steady-state ATP levels in the developing calf. The mobility of lipids within the hydrophobic inner layer of the plasma membrane relates closely to passive K(+) (Rb(+)) uptake, and plays a significant role in regulatory volume changes.     

Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovirus

Human cytomegalovirus (HCMV) terminase is composed of subunits pUL56 (130 kDa) and pUL89 (~75 kDa), encoded by the UL56 and UL89 genes. In a recent investigation, we demonstrated that the main ATPase activity is associated with the large terminase subunit pUL56. The protein has two putative ATP-binding sites, which were suggested to be composed of the sequence (amino acids 463–470) for ATP-binding site 1 and YNETFGKQ (amino acids 709–716) for the second site. We now demonstrate using a 1.5 kb fragment encoding the C-terminal half of pUL56 that ATP-binding site 1 is not critical for the function, whereas ATP-binding site 2 is required for the enzymatic activity. Mutation G714A in this protein reduced the ATPase activity to ~65% and the double mutation G714A/K715N showed a reduction up to 75%. However, the substitution of E711A revoked the effect of the substitutions. The functional character of the ATP-binding site was demonstrated by transfer of YNETFGKQLSIACLR (709–723) to glutathione-S-transferase (GST). Interestingly, vanadate, an ATPase inhibitor, has the ability to block the ATPase activity of pUL56 as well as of Apyrase, while the antitumor ATP-mimetic agent geldanamycin, did not affect the ATP-binding of pUL56. Furthermore, in contrast to an inactive control compound, the specific HCMV terminase inhibitor BDCRB showed a partial inhibition of the pUL56-specific ATPase activity. Our results clearly demonstrated that (i) the enzymatic activity of the terminase subunit pUL56 could be inhibited by vanadate, (ii) only the ATP-binding site 2 is critical for the pUL56 function and (iii) glycine G714 is an invariant amino acid.     

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.