ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease.

Cellular senescence is a state of stable cell cycle arrest triggered by diverse stresses. Establishment of senescence occurs in conjunction with a multitude of chromatin changes, which are just beginning to be studied. These chromatin changes are hypothesized to be causative for senescence. Currently, a preferred method to study such changes is chromatin immunoprecipitation followed by sequencing (ChIP-Seq). This is usually done by cross-linking the cells with formaldehyde and then generating chromatin fragments between 150 and 300bp by sonication. The DNA replication-independent histone chaperone HIRA plays an important role in control of chromatin in nonproliferating senescent cells. While investigating the role of HIRA in senescence, we found conventional ChIP protocols to be problematic, routinely yielding too low amounts of DNA for sequencing. To overcome these problems we adapted and optimized an alternative ChIP method that does not rely on cross-linking and sonication for chromatin fragmentation, and is able to easily isolate chromatin from senescent cells ready for immunoprecipitation. This method uses Benzonase endonuclease for solubilization of uncross-linked chromatin by digestion of DNA and RNA, in the absence of proteolytic activity. Using this protocol, we were easily able to immunoprecipitate HIRA with sufficient DNA for Illumina sequencing.

Comparison of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis.

OBJECTIVES: To identify an appropriate sampling technique(s) to accurately detect the bacteria causing urinary tract infections in dogs with urolithiasis. METHODS: Twenty-one dogs with urolithiasis were included in the study. Three types of samples were taken from each dog. Urine was collected by cystocentesis, and a urinary bladder mucosal biopsy and urolith were retrieved during cystotomy. The samples were then cultured on blood agar and MacConkey's agar to identify the bacteria associated with urinary tract infections. RESULTS: Bacterial urinary tract infection was found in 16 cases (76.19 per cent). The most prevalent bacteria found to cause urinary tract infection were Escherichia coli (n=7), followed by coagulase-positive Staphylococcus species (n=4), Klebsiella pneumoniae (n=2), Pseudomonas aeruginosa (n=2) and Proteus mirabilis (n=1). In the case of a positive urine culture, the same bacteria were also cultured from the urinary bladder mucosal biopsy alone or from both the urinary bladder mucosal biopsy and urolith. However, in the case of a negative urine culture, bacteria were found to be present in the urinary bladder mucosal biopsy or urolith cultures in 23.81 per cent of dogs. The uroliths that gave positive culture results were either infection-induced uroliths composed of struvite and calcium carbonate phosphate, ammonium acid urate only or metabolic uroliths composed of calcium oxalate and calcium phosphate, or calcium phosphate only. All the uroliths that gave negative culture results were metabolic uroliths composed of calcium oxalate and/or calcium phosphate, and uric acid and calcium phosphate. CLINICAL SIGNIFICANCE: When the culture from the urine obtained by cystocentesis is negative, cultures of urinary bladder mucosal biopsy and urolith are recommended in dogs with urolithiasis in order to accurately assess the microbiological status of the urinary tract.