Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene

Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "D?hle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.     

RNA-Seq Signatures Normalized by mRNA Abundance Allow Absolute Deconvolution of Human Immune Cell Types

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3D Evaluation of the Lamina Cribrosa with Swept-Source Optical Coherence Tomography in Normal Tension Glaucoma

Purpose

Although the lamina cribrosa (LC) is the primary site of axonal damage in glaucoma, adequate methods to image and measure it are currently lacking. Here, we describe a noninvasive, in vivo method of evaluating the LC, based on swept-source optical coherence tomography (SS-OCT), and determine this method’s ability to quantify LC thickness.

Methods

This study comprised 54 eyes, including normal (n = 18), preperimetric glaucoma (PPG; n = 18), and normal tension glaucoma (NTG; n = 18) eyes. We used SS-OCT to obtain 3 x 3 mm cube scans of an area centered on the optic disc, and then synchronized reconstructed B- and en-face images from this data. We identified the LC in these B-scan images by marking the visible borders of the LC pores. We marked points on the anterior and posterior borders of the LC in 12 B-scan images in order to create a skeleton model of the LC. Finally, we used B-spline interpolation to form a 3D model of the LC, including only reliably measured scan areas. We calculated the average LC thickness (avgLCT) in this model and used Spearman''s rank correlation coefficient to compare it with circumpapillary retinal nerve fiber layer thickness (cpRNFLT).

Results

We found that the correlation coefficient of avgLCT and cpRNFLT was 0.64 (p < 0.01). The coefficient of variation for avgLCT was 5.1%. AvgLCT differed significantly in the groups (normal: 282.6 ± 20.6 μm, PPG: 261.4 ± 15.8 μm, NTG: 232.6 ± 33.3 μm). The normal, PPG and NTG groups did not significantly differ in age, sex, refractive error or intraocular pressure (IOP), although the normal and NTG groups differed significantly in cpRNFLT and Humphrey field analyzer measurements of mean deviation.

Conclusion

Thus, our results indicate that the parameters of our newly developed method of measuring LC thickness with SS-OCT may provide useful and important data for glaucoma diagnosis and research.     

The Vitamin D analogue TX 527 blocks NF-kappaB activation in peripheral blood mononuclear cells of patients with Crohn's disease

Crohn's disease (CD) is an inflammatory disease characterized by the activation of the immune system in the gut. Since tumor necrosis factor (TNF-alpha) plays an important role in the initiation and perpetuation of intestinal inflammation in CD, we investigated whether TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)], a Vitamin D analogue, could affect peripheral blood mononuclear cells (PBMC) proliferation and exert an immunosuppressive effect on TNF-alpha production in CD patients, and whether this immunosuppressive action could be mediated by NF-kappaB down-regulation. TX 527 significantly decreased cell proliferation and TNF-alpha levels. On activation, NF-kappaB, rapidly released from its cytoplasmatic inhibitor (IKB-alpha), transmigrates into the nucleus and binds to DNA response elements in gene promoter regions. The activation of NF-kappaB, stimulated by TNF-alpha, and its nuclear translocation together with the degradation of IKB-alpha were blocked by TX 527. At the same time, NF-kappaB protein levels present in cytoplasmic extracts decreased in the presence of TNF-alpha and increased when PBMC were incubated with TX 527. The results of our studies indicate that TX 527 inhibits TNF-alpha mediated effects on PBMC and the activation of NF-kappaB and that its action is mediated by Vitamin D receptor (VDR), which is activated when the cells are stimulated with TX 527.     

High-fat diet–induced obesity and insulin resistance were ameliorated via enhanced fecal bile acid excretion in tumor necrosis factor-alpha receptor knockout mice

Tumor necrosis factor-α (TNF-α) is one of the main mediators of inflammatory response activated by fatty acids in obesity, and this signaling through TNF-α receptor (TNFR) is responsible for obesity-associated insulin resistance. Recently, TNF-α has shown to affect lipid metabolism including the regulation of lipase activity and bile acid synthesis. However, there is scanty in vivo evidence for the involvement of TNF-α in this process, and the mechanistic role of TNFR remains unclear. In this study, TNFR2 knockout mice (R2KO) and wild-type (WT) mice were fed commercial normal diet (ND) or high-fat diet (HFD) for 8 weeks. In R2KO/HFD mice, the increase in body weight and the accumulation of fat were significantly ameliorated compared with WT/HFD mice in association with the decrease in plasma total cholesterol (137.7±3.1 vs. 98.6±3.1 mg/dL, P<0.005), glucose (221.9±14.7 vs. 167.3±8.1 mg/dL, P<0.01), and insulin (5.1±0.3 vs. 3.4±0.3 ng/mL, P<0.05). Fecal excretion of lipid contents was significantly increased in R2KO mice. In R2KO/HFD mice, the decrease in hepatic cholesterol-7a-hydroxylase activity, the rate-limiting enzyme in bile acid synthesis, was inhibited (1.7±0.2 vs. 8.1±1.0 pmol/min/mg protein, P<0.01). These results suggested that HFD-induced obesity with metabolic derangements could be ameliorated in mice lacking TNF-α receptor 2 via increasing fecal bile acid and lipid content excretion. Therefore, TNF-α signaling through TNFR2 is essentially involved in the bile acid synthesis and excretion of lipids, resulting in its beneficial effects.     

Study of lipid changes in freeze-dried fish during storage. I. The interaction of relative humidity and tissue lipids.

Several studies on lipid stability of freeze-dried fish pointed out the relationship between relative humidity (RH) and autoxidation of highly unsaturated fatty acids. The present study shows the changes of tissue lipids in two different fish, freeze-dried and stored under various RH conditions. Fillets of Scomber scomber L and Sardina pilchardus sardina were freeze-dried and stored for 1, 3, 6 and 9 months. periodically, the sample of both fish were analyzed to evaluate the autoxidation of lipids. The results show that the autoxidation of lipids is quicker during 1st-3rd month of storage, while in a high humidity environment the tissue lipids change slower. According to these results both time of storage and relative humidity must be controlled to maintain the nutritive value of freeze-dried fish.     

CD4+ natural regulatory T cells prevent experimental cerebral malaria via CTLA-4 when expanded in vivo

Studies in malaria patients indicate that higher frequencies of peripheral blood CD4(+) Foxp3(+) CD25(+) regulatory T (Treg) cells correlate with increased blood parasitemia. This observation implies that Treg cells impair pathogen clearance and thus may be detrimental to the host during infection. In C57BL/6 mice infected with Plasmodium berghei ANKA, depletion of Foxp3(+) cells did not improve parasite control or disease outcome. In contrast, elevating frequencies of natural Treg cells in vivo using IL-2/anti-IL-2 complexes resulted in complete protection against severe disease. This protection was entirely dependent upon Foxp3(+) cells and resulted in lower parasite biomass, impaired antigen-specific CD4(+) T and CD8(+) T cell responses that would normally promote parasite tissue sequestration in this model, and reduced recruitment of conventional T cells to the brain. Furthermore, Foxp3(+) cell-mediated protection was dependent upon CTLA-4 but not IL-10. These data show that T cell-mediated parasite tissue sequestration can be reduced by regulatory T cells in a mouse model of malaria, thereby limiting malaria-induced immune pathology.     

A Novel Null Homozygous Mutation Confirms CACNA2D2 as a Gene Mutated in Epileptic Encephalopathy

Contribution to epileptic encephalopathy (EE) of mutations in CACNA2D2, encoding α2δ-2 subunit of Voltage Dependent Calcium Channels, is unclear. To date only one CACNA2D2 mutation altering channel functionality has been identified in a single family. In the same family, a rare CELSR3 polymorphism also segregated with disease. Involvement of CACNA2D2 in EE is therefore not confirmed, while that of CELSR3 is questionable. In a patient with epilepsy, dyskinesia, cerebellar atrophy, psychomotor delay and dysmorphic features, offspring to consanguineous parents, we performed whole exome sequencing (WES) for homozygosity mapping and mutation detection. WES identified extended autozygosity on chromosome 3, containing two novel homozygous candidate mutations: c.1295delA (p.Asn432fs) in CACNA2D2 and c.G6407A (p.Gly2136Asp) in CELSR3. Gene prioritization pointed to CACNA2D2 as the most prominent candidate gene. The WES finding in CACNA2D2 resulted to be statistically significant (p = 0.032), unlike that in CELSR3. CACNA2D2 homozygous c.1295delA essentially abolished α2δ-2 expression. In summary, we identified a novel null CACNA2D2 mutation associated to a clinical phenotype strikingly similar to the Cacna2d2 null mouse model. Molecular and statistical analyses together argued in favor of a causal contribution of CACNA2D2 mutations to EE, while suggested that finding in CELSR3, although potentially damaging, is likely incidental.     

EXCAVATOR: detecting copy number variants from whole-exome sequencing data

We developed a novel software tool, EXCAVATOR, for the detection of copy number variants (CNVs) from whole-exome sequencing data. EXCAVATOR combines a three-step normalization procedure with a novel heterogeneous hidden Markov model algorithm and a calling method that classifies genomic regions into five copy number states. We validate EXCAVATOR on three datasets and compare the results with three other methods. These analyses show that EXCAVATOR outperforms the other methods and is therefore a valuable tool for the investigation of CNVs in largescale projects, as well as in clinical research and diagnostics. EXCAVATOR is freely available at http://sourceforge.net/projects/excavatortool/.     

Elucidating Poor Decision-Making in a Rat Gambling Task

Although poor decision-making is a hallmark of psychiatric conditions such as attention deficit/hyperactivity disorder, pathological gambling or substance abuse, a fraction of healthy individuals exhibit similar poor decision-making performances in everyday life and specific laboratory tasks such as the Iowa Gambling Task. These particular individuals may provide information on risk factors or common endophenotypes of these mental disorders. In a rodent version of the Iowa gambling task – the Rat Gambling Task (RGT), we identified a population of poor decision makers, and assessed how these rats scored for several behavioral traits relevant to executive disorders: risk taking, reward seeking, behavioral inflexibility, and several aspects of impulsivity. First, we found that poor decision-making could not be well predicted by single behavioral and cognitive characteristics when considered separately. By contrast, a combination of independent traits in the same individual, namely risk taking, reward seeking, behavioral inflexibility, as well as motor impulsivity, was highly predictive of poor decision-making. Second, using a reinforcement-learning model of the RGT, we confirmed that only the combination of extreme scores on these traits could induce maladaptive decision-making. Third, the model suggested that a combination of these behavioral traits results in an inaccurate representation of rewards and penalties and inefficient learning of the environment. Poor decision-making appears as a consequence of the over-valuation of high-reward-high-risk options in the task. Such a specific psychological profile could greatly impair clinically healthy individuals in decision-making tasks and may predispose to mental disorders with similar symptoms.