Identification and characterization of FAM124B as a novel component of a CHD7 and CHD8 containing complex

BACKGROUND: Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry. PRINCIPLE FINDINGS: The hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues. CONCLUSION: Our results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders

Lipid Interference in the Determination of the Concentration of Haemoglobin in Plasma Using the ACA SX Analyzer

Peer Reviewe

Spectroscopic characterization of nonnative conformational states of cytochrome c

Protein and heme structural changes of ferric and ferrous cytochrome c (Cyt-c) that are induced by electrostatic binding (e.g., liposomes, electrodes), by hydrophobic interactions (e.g., monomeric sodium dodecyl sulfate), by guanidium hydrochloride (GuHCl), and at low pH and high temperature were studied by UV-vis absorption, circular dichroism (CD), electron paramagnetic resonance (EPR), and (surface-enhanced) resonance Raman [(SE)RR] spectroscopy. In a global spectral analysis, all species that differ with respect to the heme structure were identified and characterized in terms of the spin and ligation state of the heme as well as of protein secondary and tertiary structure changes. The results indicate that the upper part of the heme pocket including the Met-80 ligand is the most labile protein region such that this ligand is dissociated from the heme iron in all normative Cyt-c states. Among these states, there are two six-coordinated low-spin (LS) configurations with H2O or His-33 serving as the sixth (axial) ligand. Whereas the ferric H2O/His-18-ligated low-spin species is only formed in the A state at low pH and high ionic strength, the His-33/His- 18-ligation pattern corresponds to a stable ferric configuration inasmuch as it can be induced by electrostatic and hydrophobic interactions and under nondenaturing and denaturing conditions, that is, nearly independent of the secondary structure. Conversely, the heme pocket on the opposite side of the heme remains largely preserved except for ferric Cyt-c at very low pH and high GuHCl concentrations as indicated by the replacement of His-18 by a water molecule. Structural changes that are localized in the heme pocket and lead to a ferric bis-His-coordinated LS, a ferric water/His and mono-His high-spin (HS), and a ferrous mono-His HS configuration may be induced by hydrophobic or electrostatic interactions with the front surface of Cyt-c. The present study contributes to a consistent description of the conformational manifold of Cyt-c, which is essential for elucidating the role of conformational transitions during the natural functions of Cyt-c in energy transduction and apoptosis

Optical coherence tomography, Scheimpflug imaging, and slit-lamp biomicroscopy in the early detection of graft detachment after Descemet membrane endothelial keratoplasty

Purpose: To evaluate the efficacy of anterior segment optical coherence tomography (OCT), Scheimpflug imaging, and slit-lamp biomicroscopy in the early detection of a (partial) graft detachment after Descemet membrane endothelial keratoplasty (DMEK). Methods: Anterior segment OCT, Scheimpflug imaging, and slit-lamp biomicroscopy were performed in 120 eyes of 110 patients after DMEK. Results: Seventy-eight eyes showed a normal corneal clearance, and the attached Descemet grafts could not be identified with any of the imaging techniques. Forty-two eyes showed persistent stromal edema in the first postoperative month. In transplanted corneas that (partially) did not clear in the early postoperative period, OCT had an added diagnostic value in 36% of cases (15 of 42 eyes) in visualizing whether the graft was detached and, in particular, to discriminate between a "flat" graft detachment and delayed corneal clearance. In contrast, in the presence of corneal edema, Scheimpflug imaging did not provide more information than slit-lamp biomicroscopy in the detection of a graft detachment. Conclusions: Anterior segment OCT may be an effective tool in the detection of an early graft detachment after DMEK, to determine if secondary surgical intervention is indicated or is to be avoided.7 page(s

Improving endothelial explant tissue culture by novel thermoresponsive cell culture system

Aim Studying cell migration of corneal endothelial cellsin vitrois challenging because the capacity for cell migration needs to be maintained while at the same time the tissue must remain fixed on a rigid substrate. In this study, we report a thermoresponsive culture technique designed to maintain cellular viability, and to reduce tissue handling in order to analyzein vitroendothelial cell migration from corneal grafts. Materials and Methods As a test tissue, fifteen Quarter-Descemet membrane endothelial keratoplasty (Q-DMEK) grafts were used that were embedded in a three-dimensional culture system using a temperature-reversible hydrogel and cultured over 2-3 weeks in a humidified atmosphere at 37 degrees C and 5% CO2. Results All grafts could be successfully cultured inside the thermoresponsive polymer solution for periods of up to 21 days. Using this system, cell migration could be assessed by light microscopy at fixed time intervals. At the end of the culture period, the gel could be removed from all grafts and immunohistochemistry analysis showed that endothelial cells were able to maintain confluence, viability, and junctional integrity. Some problems were encountered when using the thermoresponsive cell culture system. These were mostly structural inconsistencies during the sol-to-gel transition phase that resulted in the formation of tiny bubbles in the matrix. Additionally, areas with different viscosity resulted in optical distortions showing up as folds throughout the matrix which can persist even after several cycles of culture medium exchange. These effects had impact on the imaging quality but did not affect the viability of the explant tissue. Conclusion This study proves that temperature-reversible hydrogel is a very useful matrix for studyingin vitrocorneal endothelial cell migration from explant grafts and allows for subsequent biological investigation after gel removal.Ophthalmic researc

Phacoemulsification after descemet membrane endothelial keratoplasty : incidence and influence on endothelial cell density

PURPOSE: To analyze the incidence of cataract extraction after Descemet membrane endothelial keratoplasty (DMEK) in phakic eyes and to evaluate the effect of phacoemutsification after DMEK on the donor endothelial cell density (ECD).METHODS: The clinical data of phakic patients with DMEK were examined. From this cohort, all patients who subsequently underwent phacoemutsification after DMEK were reviewed. Data from a prospectively collected dataset were analyzed, including demographic profile, ECD, corrected distance visual acuity (CDVA), central corneal thickness (CCT), and complications.RESULTS: From a series of 261 phakic patients with DMEK, 35 eyes (13.4%) required cataract surgery within the mean follow-up period of 54.2 +/- 28 months. The mean time from DMEK to phacoemutsification was 18 +/- 13 months (range: 3 69 months). The probability of cataract extraction after DMEK was 0.06 (95% CI: 0.03 to 0.09) at 1 year and 0.17 (95% CI: 0.12 to 0.22) at 10 years, respectively. ECD decreased from 1,314 +/- 524 cells/mm(2) before phacoemulsification to 1,167 +/- 443 cells/mm(2) (-11%) at 1 to 6 months postoperatively (P = .333). CDVA improved from 0.27 +/- 0.13 tog MAR preoperatively to 0.07 +/- 0.12 logMAR at 1 to 6 months postoperatively. CCT before phacoemulsification was 532 +/- 46 mu m and remained stable at 539 +/- 56 mu m at 1 to 6 months after phacoemulsification. Phacoemulsification did not elicit DMEK graft detachment in any of the eyes studied.CONCLUSIONS: The incidence and 10-year projection of cataract extraction in phakic eyes with DMEK was relatively tow. Phacoemulsification after DMEK provided excellent CDVA outcomes, did not induce graft detachment, and was associated with an acceptable decrease in ECD.Ophthalmic researc