Evaluating the Functionality of Conjunctiva Using a Rabbit Dry Eye Model

Purpose. To assess the conjunctival functionality in a rabbit dry eye (DE) model. Methods. Nictitating membrane, lacrimal and Harderian glands were surgically excised from male New Zealand white rabbits using minimally invasive surgery. Fluorescein/rose Bengal staining of ocular surface (OS) and Schirmer test were done before (BE) and after excision (AE). The expression of interleukin- (IL-) 1β, tumor necrosis factor- (TNF-) α, and MUC5AC proteins were estimated by immunoblotting from conjunctival impression cytology specimens. MUC5AC mRNA was quantified as well. The effect of epithelial sodium channel (ENaC) blockers on tear production and potential differences (PD) of OS were assessed under anesthesia in rabbits with and without surgery. Results. Increase in corneal and conjunctival staining was observed 1 month AE compared to BE. Schirmer tests failed to show decrease in tear production. Elevated IL-1β, and TNF-α, 1 month AE indicated inflammation. MUC5AC expression was elevated 1 month AE. ENaC blockers did not improve tear production in rabbit eyes AE but characteristic changes in PD were observed in rabbits with surgery. Conclusions. DE biomarkers are important tools for OS assessment and MUC5AC expression is elevated in rabbit DE. PD measurement revealed significant electrophysiological changes in rabbits with surgery

Degradation of Polycyclic Aromatic Hydrocarbons by a Newly Discovered Enteric Bacterium, Leclercia adecarboxylata

A bacterial strain, PS4040, capable of degrading polycyclic aromatic hydrocarbons for use as the sole carbon source was isolated from oily-sludge-contaminated soil. The 16S rRNA gene showed 98.8% homology to that of Leclercia adecarboxylata. Comparative molecular typing with the clinical strain of L. adecarboxylata revealed that there were few comigrating and few distinct amplimers among them

Coding and Noncoding Genomic Regions of Entamoeba histolytica Have Significantly Different Rates of Sequence Polymorphisms: Implications for Epidemiological Studies

To evaluate genetic variability among Entamoeba histolytica strains, we sequenced 9,077 bp from each of 14 isolates. The polymorphism rates from coding and noncoding regions were significantly different (0.07% and 0.37%, respectively), indicating that these regions are subject to different selection pressures. Additionally, single nucleotide polymorphisms (SNPs) potentially associated with specific clinical outcomes were identified

Expression patterns of conjunctival mucin 5AC and aquaporin 5 in response to acute dry eye stress

The relationship between aquaporin (AQP) 5 and mucin (MUC) 5AC in the conjunctiva was investigated in response to acute dry eye (DE) stress. A mixed-mechanism rabbit DE model, in which the main lacrimal gland, Harderian gland, and nictitating membrane were resected, was further explored in this study. Conjunctival impression cytology specimens were harvested before excision (BE) and up to 3 months after excision (AE) in 8 (16 eyes) male New Zealand White rabbits, and immunoblotting was employed to assess the expression of AQP5 and MUC5AC. It was observed that AQP5 and MUC5AC showed a positive, synchronous expression pattern with progressive upregulation at protein level up to 2 months AE. At 3 months, the expression of both proteins decreased, but was still higher than that of BE. Such a synchronous relationship was further observed in mouse conjunctiva epithelium primary cells under hyperosmotic condition. Moreover, the co-immunoprecipitation of AQP5 and MUC5AC suggested a possible physical interaction between the two molecules. Our data indicates that conjunctival AQP5 and MUC5AC act synchronously in response to acute DE stress.Arizona Biomedical Research Commission Research [ADHS14-082988]; Department of Ophthalmology and Vision Science at University of ArizonaOpen access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing

Here we report a novel finding of an antisense oligonucleotide (ASO) microwalk in which we examined the position-specific role of intronic residues downstream from the 5′ splice site (5′ ss) of SMN2 exon 7, skipping of which is associated with spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Our results revealed the inhibitory role of a cytosine residue at the 10th intronic position (10C), which is neither conserved nor associated with any known splicing motif. Significance of 10C emerged from the splicing pattern of SMN2 exon 7 in presence of a 14-mer ASO (L14) that sequestered two adjacent hnRNP A1 motifs downstream from 10C and yet promoted SMN2 exon 7 skipping. Another 14-mer ASO (F14) that sequestered both, 10C and adjacent hnRNP A1 motifs, led to a strong stimulation of SMN2 exon 7 inclusion. The inhibitory role of 10C was found to be tightly linked to its unpaired status and specific positioning immediately upstream of a RNA:RNA helix formed between the targeting ASO and its intronic target. Employing a heterologous context as well as changed contexts of SMN2 intron 7, we show that the inhibitory effect of unpaired 10C is dependent upon a long-distance interaction involving downstream intronic sequences. Our report furnishes one of the rare examples in which an ASO-based approach could be applied to unravel the critical role of an intronic position that may not belong to a linear motif and yet play significant role through long-distance interactions

An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing

Here we report a novel finding of an antisense oligonucleotide (ASO) microwalk in which we examined the position-specific role of intronic residues downstream from the 5′ splice site (5′ ss) of SMN2 exon 7, skipping of which is associated with spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Our results revealed the inhibitory role of a cytosine residue at the 10th intronic position (10C), which is neither conserved nor associated with any known splicing motif. Significance of 10C emerged from the splicing pattern of SMN2 exon 7 in presence of a 14-mer ASO (L14) that sequestered two adjacent hnRNP A1 motifs downstream from 10C and yet promoted SMN2 exon 7 skipping. Another 14-mer ASO (F14) that sequestered both, 10C and adjacent hnRNP A1 motifs, led to a strong stimulation of SMN2 exon 7 inclusion. The inhibitory role of 10C was found to be tightly linked to its unpaired status and specific positioning immediately upstream of a RNA:RNA helix formed between the targeting ASO and its intronic target. Employing a heterologous context as well as changed contexts of SMN2 intron 7, we show that the inhibitory effect of unpaired 10C is dependent upon a long-distance interaction involving downstream intronic sequences. Our report furnishes one of the rare examples in which an ASO-based approach could be applied to unravel the critical role of an intronic position that may not belong to a linear motif and yet play significant role through long-distance interactions.This is an article from RNA 16 (2010): 1167, doi:10.1261/rna.2154310. Posted with permission.</p

Increase in conjunctival AQP5 and MUC5AC expression in rabbits after surgery.

<p>In comparison to before excision (BE), the AQP5 protein increased significantly at 1, and 2 months after excision (AE), and then decreased slightly at the end of 3 months AE. The MUC5AC protein expression followed similar pattern of upregulation, demonstrating a synchronous relationship with AQP5. The protein (immunoblot) signals of AQP5 and MUC5AC were presented relative to the internal control “Gapdh” signal.</p

Characterization and hyperosmotic treatment of the mouse conjunctival epithelium primary cells.

<p>The confirmation of the origin of primary epithelial cells was done by assessing the levels of cytokeratin 4(K4) and cytokeratin 12(K12) by RT-qPCR (Fig 3A). Under hyperosmotic conditions (350 mOsm and 400 mOsm) for 24h, significant increase in the mRNA expression of proinflammatory cytokine markers, TNF-α and IL-1β, was noted (Fig 3B).</p

Co-immunoprecipitation (CoIP) of AQP5 and MUC5AC.

<p>(2A) The detection of MUC5AC in Ab-Ag complex by immunoprecipitation of AQP5 from rabbit conjunctival tissue protein lysate with AQP5 antibody bound to Dynabead^® Magnetic beads coupled Protein A. Input: The normal rabbit conjunctival tissue protein lysate (Positive control for MUC5AC); Co-IP: MUC5AC protein signal detected in the Co-IP Ab-Ag complex by AQP5 antibody; Negative controls: Beads+AQP5 (magnetic beads incubated with only the AQP5 antibody); Beads + rabbit IgG (magnetic beads incubated with only the IgG antibody); Beads only (magnetic beads alone incubated in the reaction buffer). (2B) The CoIP of AQP5 by immunoprecipitation of MUC5AC from rabbit conjunctival tissue protein lysate with MUC5AC antibody bound to Dynabead^® Magnetic beads coupled Protein A. Input: The normal rabbit conjunctival tissue protein lysate (Positive control for AQP5); Co-IP: AQP5 protein signal detected in the Co-IP Ab-Ag complex by MUC5AC antibody; Negative controls: Beads+ MUC5AC: Beads + rabbit IgG; Beads only.</p

Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis