A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Tablets as an Option for Telemedicine-Evaluation of Diagnostic Performance and Efficiency in Intracranial Arterial Aneurysm Detection.

Purpose: To evaluate a commercially available mobile device for the highly specialized task of detection of intracranial arterial aneurysm in telemedicine. Methods: Six radiologists with three different levels of experience retrospectively interpreted 60 computed tomography (CT) angiographies for the presence of intracranial arterial aneurysm, among them 30 cases with confirmed positive findings. Each radiologist reviewed the angiography datasets twice: once on a dedicated medical-grade workstation and on a commercially available mobile consumer-grade tablet with an interval of 3 months. Diagnostic performance, reading efficiency and subjective scorings including diagnostic confidence were analyzed and compared. Results: Diagnostic performance was comparable on both devices regardless of readers' experience, and no significant differences in sensitivity (66-87.5%) and specificity (79.4-87%) were found. Results obtained with tablets and medical workstations were also comparable in terms of subjective assessment across all reader groups. Conclusions: There was no significant difference between tablet and workstation readings of angiography datasets for the presence of intracranial arterial aneurysm. Sensitivity, specificity, efficiency and subjective scorings were similar with the two devices for all three reader groups. While medical workstations are 10 times more expensive, tablets allow higher mobility especially for radiologists on call.

Various forms of HIF-1α protein characterize the clear cell renal cell carcinoma cell lines.

Renal cell carcinoma (RCC) represents around 2-3% of all malignancies diagnosed in adult patients. Most frequent (around 70-80% cases) and the most aggressive subtype is clear cell RCC (ccRCC). Mutations in VHL (von Hippel Lindau) gene, characteristic for this cancer type, lead to altered activity of the trimeric VBC (pVHL-elongin B-C) complex and consequently to HIF-1α stabilization. In this study, we present results of exhaustive investigation of HIF-1α alternative transcript variants abundance in A498, CAKI-1, and 786-O ccRCC cell lines. We proved the existence of truncated HIF-1α protein form (HIF1A∆-6) in A498 and HIF1A gene rearrangements in 786-O cell lines. Subsequently, we found that HIF1A∆2-6 was more stable than the full-length HIF-1α. Moreover, the shorter HIF-1α was insensitive for hypoxia and was overaccumulated after proteasome inhibitor treatment indicative of potential diversified roles of full-length and truncated HIF-1α forms in the cell. We also showed that A498, CAKI-1, and 786-O exhibit differential expression of various regulatory genes involved in the control of metabolic processes, that is, glucose and lipid metabolism, and encoding subunits of such machineries like SWI/SNF chromatin remodeling complex. Furthermore, these cell lines exhibited differential responses to axitinib, everolimus, and sunitinib-anticancer drugs-in normoxia and hypoxia as well as various alterations in metabolism-related regulatory processes. Finally, we have shown that overexpression of truncated HIF1A∆2-6 form may affect the protein level of endogenous full-length HIF-1α protein. Thus, our study proves an important role of HIF-1α in the ccRCC development.

The SWI/SNF ATP-Dependent Chromatin Remodeling Complex in Arabidopsis Responds to Environmental Changes in Temperature-Dependent Manner.

SWI/SNF ATP-dependent chromatin remodeling complexes (CRCs) play important roles in the regulation of transcription, cell cycle, DNA replication, repair, and hormone signaling in eukaryotes. The core of SWI/SNF CRCs composed of a SWI2/SNF2 type ATPase, a SNF5 and two of SWI3 subunits is sufficient for execution of nucleosome remodeling in vitro. The Arabidopsis genome encodes four SWI2/SNF2 ATPases, four SWI3, a single SNF5 and two SWP73 subunits. Genes of the core SWI/SNF components have critical but not fully overlapping roles during plant growth, embryogenesis, and sporophyte development. Here we show that the Arabidopsis swi3c mutant exhibits a phenotypic reversion when grown at lower temperature resulting in partial restoration of its embryo, root development and fertility defects. Our data indicates that the swi3c mutation alters the expression of several genes engaged in low temperature responses. The location of SWI3C-containing SWI/SNF CRCs on the ICE1, MYB15 and CBF1 target genes depends on the temperature conditions, and the swi3c mutation thus also influences the transcription of several cold-responsive (COR) genes. These findings, together with genetic analysis of swi3c/ice1 double mutant and enhanced freezing tolerance of swi3c plants illustrate that SWI/SNF CRCs contribute to fine-tuning of plant growth responses to different temperature regimes.

Evaluation of the role of downregulation of SNF5/INI1 core subunit of SWI/SNF complex in clear cell renal cell carcinoma development.

Clear cell renal cell carcinoma (ccRCC) is characterized by stabilization of hypoxia-inducible factor (HIF1), and mutations in von Hippel-Lindau (VHL) gene. Additionally, in about 40% of ccRCC cases the mutation in PBRM1 (POLYBROMO1) gene coding for a non-core subunit of SWI/SNF chromatin remodeling complex was found suggesting potential impairment of this complex function in ccRCC. In this study we assessed the extent to which the core SWI/SNF complex subunit - INI1 (hSNF5/SMARCB1) is affected in ccRCC and whether it has any consequences on the development of this type of cancer. The evaluation of INI1 protein level in samples from 50 patients with diagnosed ccRCC, including three displaying rhabdoid features, showed the INI1 positive staining in rhabdoid cells while the conventional ccRCC cells exhibited reduced INI1 level. This indicated the rhabdoid component of ccRCC as distinct from other known rhabdoid tumors. The reduced INI1 protein level observed in all conventional ccRCC cases used in this study correlated with decreased SMARCB1 gene expression at the transcript level. Consistently, the overexpression of INI1 protein in A498 ccRCC cell line resulted in the elevation of endogenous SMARCB1 transcript level indicating that the INI1-dependent regulatory feedback loop controlling expression of this gene is affected in ccRCC Moreover, the set of INI1 target genes including i.e. CXCL12/CXCR7/CXCR4 chemokine axis was identified to be affected in ccRCC. In summary, we demonstrated that the inactivation of INI1 may be of high importance for ccRCC development and aggressiveness.

Validation and proposals for a refinement of the WHO 2008 classification of myelodysplastic syndromes without excess of blasts.

In 2008, the WHO proposed changes in the classification of MDS regarding RCUD and MDS unclassifiable. We validated these proposals by using 2032 patients of the Düsseldorf MDS Registry. 10% of the patients had RCUD and 6% MDS-U. Among patients with RCUD, only 9% had RN and 6% had RT. There was no correlation between dysplastic cell line and type of cytopenia. There was no difference in prognosis between RCMD and MDS-U and between RA, RT, and RN. The separation of RA, RN, and RT is not justified suggesting a consolidation as RCUD. MDS-U should be integrated into RCMD.

Effect of Medicago sativa Mhb1gene expression on defense response of Arabidopsis thaliana plants.

Besides the previously described nitric oxide-detoxification activity we identified new features of class-1 non-symbiotic hemoglobin from Medicago sativa (Mhb1). Under in vitro conditions, using peroxidase in-gel activity assay, the Mhb1 protein was shown to possess also peroxidase-like activity. Due to this activity, in the presence of nitrite and hydrogen peroxide, the protein can mediate autonitration and nitration of other proteins at tyrosine residues, as revealed by tandem mass spectrometry and immune assay approaches. Mhb1 through its multifunctional activities can affect different components of signal transduction cascades operating during plant response to infections. This influence is manifested by Mhb1-mediated selective up-regulation of expression of certain pathogen inducible genes in Pseudomonas syringae infected Arabidopsis thaliana plants which overproduce Mhb1, as revealed by reverse transcription-quantitative real-time PCR analysis. Changes in expression level of these genes can influence such processes as synthesis of secondary metabolites, protein degradation and biosynthesis of ethylene. They can also result in alteration of pathogen-induced defense response of Mhb1 transgenic plants.

NO-degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR-1a gene expression in Mhb1-overproducing tobacco plants.

Tobacco plants overproducing alfalfa class 1 hemoglobin (HOT plants) have been shown to have reduced necrotic symptom development. Here, we show that this altered pathogenic response is linked to a significant increase in the nitric oxide (NO)-affected pathogenesis-related (PR-1a) transcript accumulation in the transgenic plants. Homogenates of HOT transgenic seedlings were also found to have higher NO-scavenging activity than non-transformed ones. The NO-scavenging properties of recombinant alfalfa class1 hemoglobin have been examined. Recombinant Mhb1 (rMhb1) was produced in bacteria and purified using polyethylene glycol (10-25%) fractionation, chromatography on DEAE-Sephacel, and Phenyl Superose columns. After the final purification step, the obtained preparations were near homogeneous and had a molecular weight of 44 kDa determined by size-exclusion chromatography and 23 kDa by SDS-PAGE, indicating that rMhb1 is a dimer. The protein participated in NO-degradation activity with NAD(P)H as a cofactor. After ion-exchange columns, addition of FAD was necessary for exhibiting maximal NO-degradation activity. The NAD(P)H-dependent NO-scavenging activity of rMhb1, which is similar to that of barley hemoglobin, supports a conclusion that both monocot and dicot class 1 hemoglobins can affect cellular NO levels by scavenging NO formed during hypoxia, pathogen attack and other stresses.