Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into a variety of cell types, which could be used for cell transplantation therapies as well as drug discovery studies. However, the large-scale culture of undifferentiated human ES cells is currently limited by their dependency on mouse embryonic fibroblast feeder layers. The proteomics approach was employed to characterize the environment that supports the growth of undifferentiated human ES cells and to identify factors critical for their independent growth. Conditioned medium from mouse embryonic fibroblast feeder layers, STO cell line, was concentrated and subjected to analyses by two-dimensional electrophoresis mass spectrometry. In total, 136 unique protein species were identified which included some that are known to participate in cell growth and differentiation, extracellular matrix formation and remodeling, in addition to the unexpected but interesting finding of many nominally intracellular proteins. This approach has thus revealed the complexity of the environment provided by the feeder cells and provides a useful starting point for future studies. Moreover, candidates from the initial list of identified proteins can be further investigated for their effects on the growth and differentiation of human ES cells in a defined culture environment.     

Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p

The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins. The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN. This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner. In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated. CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside. Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p. We propose that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins.     

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.     

High Performance Flexible Supercapacitor Electrodes Composed of Ultralarge Graphene Sheets and Vanadium Dioxide

Little is known regarding the effect of the graphene lateral size on the electrochemical performance of hybrid graphene electrode. This work examines the electrochemical performance of a flexible hybrid supercapacitor electrode composed of ultralarge graphene oxide (UGO; mean lateral size of 47 ± 22 μm) and vanadium dioxide (VO₂) nanobelts, referring to a reference electrode composed of small scale graphene oxide (SGO; mean lateral size of 0.8 ± 0.5 μm) and VO₂.Thermal treatment converts UGO/VO₂ and SGO/VO₂ to URGO/VO₂ (denoted VURGO) and SRGO/VO₂ (denoted VSRGO) electrodes, respectively. The sheet resistance of the VURGO film (0.57 ± 0.03 kΩ sq.^–1) was two orders of magnitude lower than that of the VSRGO (55.74 ± 9.35 kΩ sq.^–1). The VURGO hybrid electrode showed a specific capacitance of 769 F g^?1, which was significantly better than the corresponding values for the VSRGO electrode (385 F/g). These results support the notion that the use of ultralarge graphene sheets (≈22 500 μm²) lowers the intersheet resistance due to the presence of fewer intersheet tunneling barriers. This article highlights the potential utility of URGO (as a conductive support) in hybrid electrode containing VO₂ nanobelts for high performance flexible hybrid supercapacitor.     

The Effect of Online Hemodiafiltration on Infections: Results from the CONvective TRAnsport STudy

Background

Hemodialysis (HD) patients have a high risk of infections. The uremic milieu has a negative impact on several immune responses. Online hemodiafiltration (HDF) may reduce the risk of infections by ameliorating the uremic milieu through enhanced clearance of middle molecules. Since there are few data on infectious outcomes in HDF, we compared the effects of HDF with low-flux HD on the incidence and type of infections.

Patients and Methods

We used data of the 714 HD patients (age 64 ±14, 62% men, 25% Diabetes Mellitus, 7% catheters) participating in the CONvective TRAnsport STudy (CONTRAST), a randomized controlled trial evaluating the effect of HDF as compared to low-flux HD. The events were adjudicated by an independent event committee. The risk of infectious events was compared with Cox regression for repeated events and Cox proportional hazard models. The distributions of types of infection were compared between the groups.

Results

Thirty one percent of the patients suffered from one or more infections leading to hospitalization during the study (median follow-up 1.96 years). The risk for infections during the entire follow-up did not differ significantly between treatment arms (HDF 198 and HD 169 infections in 800 and 798 person-years respectively, hazard ratio HDF vs. HD 1.09 (0.88–1.34), P = 0.42. No difference was found in the occurrence of the first infectious event (either fatal, non-fatal or type specific). Of all infections, respiratory infections (25% in HDF, 28% in HD) were most common, followed by skin/musculoskeletal infections (21% in HDF, 13% in HD).

Conclusions

HDF as compared to HD did not result in a reduced risk of infections, larger studies are needed to confirm our findings.

Trial Registration

ClinicalTrials.gov NCT00205556     

Retinal Microvascular Abnormalities and Risk of Renal Failure in Asian Populations

BackgroundRetinal microvascular signs may provide insights into the structure and function of small vessels that are associated with renal disease. We examined the relationship of retinal microvascular signs with both prevalent and incident end-stage renal disease (ESRD) in a multi-ethnic Asian population.MethodsA total of 5763 subjects (aged ≥40 years) from two prospective population-based studies (the Singapore Malay Eye Study and the Singapore Prospective Study) were included for the current analysis. Retinopathy was graded using the modified Airlie House classification system. Retinal vascular parameters were measured using computer-assisted programs to quantify the retinal vessel widths (arteriolar and venular caliber) and retinal vascular network (fractal dimension). Data on ESRD was obtained by record linkage with the ESRD cases registered by National Registry of Diseases Office, Singapore. Multi-variable adjusted regression analyses were performed to assess the associations of baseline retinal vascular parameters and prevalent and incident ESRD.ResultsAt baseline, 21(0.36%) persons had prevalent ESRD. During a median follow-up of 4.3 years, 33 (0.57%) subjects developed ESRD. In our analyses, retinopathy was associated with prevalent ESRD (multi-variable adjusted odds ratio [OR], 3.21, 95% confidence interval [CI]: 1.28–8.05) and incident ESRD (multi-variable adjusted hazard ratio [HR], 2.51, 95%CI: 1.14–5.54). This association was largely seen in person with diabetes (HR, 2.60, 95%CI: 1.01–6.66) and not present in persons without diabetes (HR, 1.65, 95%CI: 0.14–18.98). Retinal arteriolar caliber, retinal venular caliber and retinal vascular fractal dimension were not associated with ESRD.ConclusionRetinopathy signs in persons with diabetes are related to an increased risk of ESRD; however, other microvascular changes in the retina are not associated with ESRD.     

Psychometric Properties of the Problem Areas in Diabetes (PAID) Instrument in Singapore

Background

Emotional distress is an important dimension in diabetes, and several instruments have been developed to measure this aspect. The Problem Areas in Diabetes (PAID) scale is one such instrument which has demonstrated validity and reliability in Western populations, but its psychometric properties in Asian populations have not been examined.

Methods

This was a secondary analysis of data from patients with Type 2 diabetes mellitus recruited through convenience sampling from a diabetes specialist outpatient clinic in Singapore. The following psychometric properties were assessed: Construct validity through confirmatory factor analysis (CFA) and Rasch analysis, concurrent validity through correlation with related scales (Kessler Psychological Distress Scale, Diabetes Health Profile—psychological distress, Audit of Diabetes Dependent Quality of Life), reliability through assessment of internal consistency and floor and ceiling effects, and sensitivity by estimating effect sizes for known clinical and social functioning groups.

Results

203 patients with mean age of 45±12 years were analysed. None of the previously published model structures achieved a good fit on CFA. On Rasch analysis, four items showed poor fit and were removed. The abridged 16-item PAID mapped to a single latent trait, with a high degree of internal consistency (Cronbach ɑ 0.95), but significant floor effect (24.6% scoring at floor). Both 20-item and 16-item PAID scores were moderately correlated with scores of related scales, and sensitive to differences in clinical and social functioning groups, with large effect sizes for glycemic control and diabetes related complications, nephropathy and neuropathy.

Conclusion

The abridged 16-item PAID measures a single latent trait of emotional distress due to diabetes whereas the 20-item PAID appears to measures more than one latent trait. However, both the 16-item and 20-item PAID versions are valid, reliable and sensitive for use among Singaporean patients with diabetes.     

Serum Magnesium and Sudden Death in European Hemodialysis Patients

Despite suggestions that higher serum magnesium (Mg) levels are associated with improved outcome, the association with mortality in European hemodialysis (HD) patients has only scarcely been investigated. Furthermore, data on the association between serum Mg and sudden death in this patient group is limited. Therefore, we evaluated Mg in a post-hoc analysis using pooled data from the CONvective TRAnsport STudy (CONTRAST, NCT00205556), a randomized controlled trial (RCT) evaluating the survival risk in dialysis patients on hemodiafiltration (HDF) compared to HD with a mean follow-up of 3.1 years. Serum Mg was measured at baseline and 6, 12, 24 and 36 months thereafter. Cox proportional hazards models, adjusted for confounders using inverse probability weighting, were used to estimate hazard ratios (HRs) of baseline serum Mg on all-cause mortality, cardiovascular mortality, non-cardiovascular mortality and sudden death. A generalized linear mixed model was used to investigate Mg levels over time. Out of 714 randomized patients, a representative subset of 365 (51%) were analyzed in the present study. For every increase in baseline serum Mg of 0.1 mmol/L, the HR for all-cause mortality was 0.85 (95% CI 0.77–94), the HR for cardiovascular mortality 0.73 (95% CI 0.62–0.85) and for sudden death 0.76 (95% CI 0.62–0.93). These findings did not alter after extensive correction for potential confounders, including treatment modality. Importantly, no interaction was found between serum phosphate and serum Mg. Baseline serum Mg was not related to non-cardiovascular mortality. Mg decreased slightly but statistically significant over time (Δ -0.011 mmol/L/year, 95% CI -0.017 to -0.009, p = 0.03). In short, serum Mg has a strong, independent association with all-cause mortality, cardiovascular mortality and sudden death in European HD patients. Serum Mg levels decrease slightly over time.     

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.