Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System

We present a simple and rapid method to isolate extracellular vesicles (EVs) by using a polyethylene glycol/dextran aqueous two-phase system (ATPS). This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs.     

Association between Hypertension and the Prevalence of Low Back Pain and Osteoarthritis in Koreans: A Cross-Sectional Study

Background

Hypertension and musculoskeletal disorders are highly prevalent in adult populations. The objective of this study was to investigate the association between hypertension and prevalence of low back pain (LBP) and osteoarthritis in Koreans.

Methods

A total 17,128 participants (age ≥20 years) who answered low back pain and osteoarthritis items in the 4th Korean National Health and Nutrition Examination Survey (2007–2009) were analyzed. Odds ratios were calculated using logistic regression and were adjusted for age, sex, income level, education, occupation, BMI, smoking status, alcohol consumption, and physical activity.

Results

Lifetime prevalence of LBP in hypertensive subjects was 34.4%, and that of osteoarthritis 26.2%. LBP prevalence was significantly lower in hypertensives (fully adjusted OR 0.79; 95% CI 0.70–0.90), and both LBP and osteoarthritis prevalence was significantly lower in participants with systolic blood pressure ≥140mmHg than those with <120mmHg (fully adjusted OR 0.81; 95% CI 0.70–0.94, and 0.81; 95% CI 0.68–0.96, respectively). Prevalence of LBP in subjects with diastolic blood pressure ≥90mmHg was also significantly lower than those with <80mmHg (fully adjusted OR 0.73; 95% CI 0.63–0.85). LBP and osteoarthritis prevalence did not differ by systolic or diastolic blood pressure interval in respondents taking antihypertensive medication. LBP and osteoarthritis prevalence increased with longer hypertension duration (fully adjusted p for trend 0.028, and 0.0008, respectively).

Conclusions

Hypertension showed an inverse relationship with LBP and osteoarthritis prevalence, which may be ascribed to hypertension-associated hypalgesia, and antihypertensive medication intake and longer hypertension duration attenuated this association.     

The Role of ARF6 in Biliary Atresia

Background & Aims

Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods

To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results

Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10^-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10^-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10^-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10^-7), ERK/MAPK and CREB canonical pathways (p<1 x10^-34), and functional networks for cellular development and proliferation (p<1 x10^-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions

The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.     

Recovery of Corneal Endothelial Cells from Periphery after Injury

Background

Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.

Aim

To investigate the recovery process of corneal endothelial cells (CECs) from corneal endothelial injury.

Methods

Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group). Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.

Results

Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.

Conclusions

CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.     

Factors Associated with Mortality and Graft Failure in Liver Transplants: A Hierarchical Approach

Background

Liver transplantation has received increased attention in the medical field since the 1980s following the introduction of new immunosuppressants and improved surgical techniques. Currently, transplantation is the treatment of choice for patients with end-stage liver disease, and it has been expanded for other indications. Liver transplantation outcomes depend on donor factors, operating conditions, and the disease stage of the recipient. A retrospective cohort was studied to identify mortality and graft failure rates and their associated factors. All adult liver transplants performed in the state of São Paulo, Brazil, between 2006 and 2012 were studied.

Methods and Findings

A hierarchical Poisson multiple regression model was used to analyze factors related to mortality and graft failure in liver transplants. A total of 2,666 patients, 18 years or older, (1,482 males; 1,184 females) were investigated. Outcome variables included mortality and graft failure rates, which were grouped into a single binary variable called negative outcome rate. Additionally, donor clinical, laboratory, intensive care, and organ characteristics and recipient clinical data were analyzed. The mortality rate was 16.2 per 100 person-years (py) (95% CI: 15.1–17.3), and the graft failure rate was 1.8 per 100 py (95% CI: 1.5–2.2). Thus, the negative outcome rate was 18.0 per 100 py (95% CI: 16.9–19.2). The best risk model demonstrated that recipient creatinine ≥ 2.11 mg/dl [RR = 1.80 (95% CI: 1.56–2.08)], total bilirubin ≥ 2.11 mg/dl [RR = 1.48 (95% CI: 1.27–1.72)], Na⁺ ≥ 141.01 mg/dl [RR = 1.70 (95% CI: 1.47–1.97)], RNI ≥ 2.71 [RR = 1.64 (95% CI: 1.41–1.90)], body surface ≥ 1.98 [RR = 0.81 (95% CI: 0.68–0.97)] and donor age ≥ 54 years [RR = 1.28 (95% CI: 1.11–1.48)], male gender [RR = 1.19(95% CI: 1.03–1.37)], dobutamine use [RR = 0.54 (95% CI: 0.36–0.82)] and intubation ≥ 6 days [RR = 1.16 (95% CI: 1.10–1.34)] affected the negative outcome rate.

Conclusions

The current study confirms that both donor and recipient characteristics must be considered in post-transplant outcomes and prognostic scores. Our data demonstrated that recipient characteristics have a greater impact on post-transplant outcomes than donor characteristics. This new concept makes liver transplant teams to rethink about the limits in a MELD allocation system, with many teams competing with each other. The results suggest that although we have some concerns about the donors features, the recipient factors were heaviest predictors for bad outcomes.     

Pancreatic polypeptide inhibits somatostatin secretion

Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin-containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c-Fos immunoreactivity in somatostatin-containing cells in the hippocampus compared with PBS-injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.     

NMR analyses on the interactions of the yeast Tim50 C-terminal region with the presequence and Tim50 core domain

The mitochondrial targeting signal in the presequence of mitochondrial precursor proteins is recognized by Tom20 and subsequently by Tim50 in mitochondria. Yeast Tim50 contains two presequence binding sites in the conserved core domain and in the fungi-specific C-terminal presequence binding domain (PBD). We report the NMR analyses on interactions of a shorter variant of PBD (sPBD), a shorter variant of PBD, with presequences. The presequence is recognized by sPBD in a similar manner to Tom20. sPBD can also bind to the core domain of Tim50 through the presequence binding region, which could promote transfer of the presequence from sPBD to the core domain in Tim50.