Increasing age and severe intraoperative hypotension associated with nonsurvival in dogs with gallbladder mucocele undergoing cholecystectomy.

OBJECTIVE: To identify prognostic indicators and inflammatory markers associated with nonsurvival in dogs with gallbladder mucoceles (GBMs) following cholecystectomy and to evaluate C-reactive protein (CRP) and haptoglobin concentrations in dogs with GBMs compared to healthy controls. ANIMALS: 25 dogs that underwent cholecystectomy for removal of GBM and 20 healthy control dogs. METHODS: A prospective, multicenter cohort study. Survival outcomes to hospital discharge and 2 weeks postdischarge were recorded from medical records. Laboratory variables, inflammatory markers (CRP and haptoglobin), and 25-hydroxyvitamin(OH) D (25[OH]D) concentrations were measured preoperatively. Associations between signalment, clinicopathologic variables, acute patient physiologic and laboratory evaluation (APPLEFAST) scores, inflammatory markers, 25(OH)D concentration, and survival were analyzed using logistic regression. RESULTS: 76% (19/25) and 68% (17/25) of dogs survived to hospital discharge and 2 weeks postdischarge, respectively. For each additional year of age, the odds of nonsurvival in hospital and 2 weeks postdischarge increased by 2.2 (P = .01; 95% CI, 1.2 to 5.0) and 1.7 (P = .04; 95% CI, 1.0 to 3.2), respectively. Intraoperative systolic blood pressure ≤ 65 mm Hg increased the probability of nonsurvival in hospital (P < .04). Gallbladder perforation, APPLEFAST scores, and preoperative serum concentrations of CRP, haptoglobin, and 25(OH)D were not associated with survival. Serum CRP and haptoglobin concentrations were greater in dogs with GBM compared to controls (P < .001). CLINICAL RELEVANCE: Increasing age and intraoperative systolic blood pressure ≤ 65 mm Hg were associated with nonsurvival in dogs with GBM undergoing cholecystectomy. Serum CRP, haptoglobin, and 25(OH)D were not associated with nonsurvival postcholecystectomy in this sample population.

Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.

The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of m5C calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.

Correction: Serum 25-hydroxyvitamin D concentrations in dogs with gallbladder mucocele.

[This corrects the article DOI: 10.1371/journal.pone.0244102.].

Serum 25-hydroxyvitamin D concentrations in dogs with gallbladder mucocele.

Gallbladder mucocele (GBM) is a common biliary disorder in dogs. Gallbladder hypokinesia has been proposed to contribute to its formation and progression. The specific cause of gallbladder stasis in dogs with GBM as well as viable treatment options to resolve dysmotility remains unknown. Vitamin D deficiency is one of the many potential causes of gallbladder hypokinesia in humans and repletion results in complete resolution of stasis. Improving our understanding of the relationship between serum vitamin D and GBM could help identify dogs as a model for humans with gallbladder hypokinesia. Furthermore, this relationship could provide insight into the pathogenesis of GBM and support the need for future studies to investigate vitamin D as a novel treatment target. Therefore, goals of this study were i) to determine if serum 25-hydroxyvitamin(OH)D concentrations were decreased in dogs with GBM, ii) if serum 25(OH)D concentrations were different in clinical versus dogs subclinical for GBM, and iii) to determine if serum 25(OH)D concentrations could predict the ultrasonographic type of GBM. Sixty-two dogs (clinical, n = 26; subclinical, n = 36) with GBM and 20 healthy control dogs were included in this prospective observational study. Serum 25(OH)D concentrations were measured with a competitive chemiluminescence immunoassay. Overall, dogs with GBM had lower serum 25(OH)D concentrations than control dogs (P = 0.004). Subsequent subgroup analysis indicated that this difference was only significant in the subclinical group compared to the control dogs (P = 0.008), and serum 25(OH)D concentrations did not significantly differ between dogs clinical for GBM versus subclinical or control dogs, indicating that inflammatory state in clinical dogs was not the major constituent of the observed findings. Decreasing serum 25(OH)D concentrations, but not clinical status, was associated with a more advanced developmental stage of GBM type determined by ultrasonography. Our results indicate that vitamin D has a role in dogs with GBM. Additional studies are needed to assess if reduced vitamin D in dogs with GBM is a cause or effect of their biliary disease and to investigate if vitamin D supplementation could be beneficial for dogs with GBM.