Domain Of Alpha Research Articles

KRAB-containing zinc finger protein ZNF496 inhibits breast cancer cell proliferation by selectively repressing ERα activity

KRAB-containing zinc finger proteins (KZNF) constitute the largest family of transcriptional regulators in humans and play critical roles in normal development and tumorigenesis. However, the function and mechanism of most KZNFs remain unclear. Here, we report that ZNF496, a KZNF family member, interacts with the DNA binding domain (DBD) of estrogen receptor alpha (ERα) via its C2H2 domain. This interaction decreases ERα binding to chromatin DNA and results in the repression of ERα transactivation, the selective suppression of ERα target genes, and ultimately in a reduction of ERα-positive cell growth in the presence of E2. An analysis of clinical data revealed that the downregulation of ZNF496 expression is observed only in ERα-positive and not in ERα-negative breast cancer tissues when compared with that in matched adjacent tissues. Lastly, we also observed that the downregulation of ZNF496 is associated with poor recurrence-free survival among patients with breast cancer. Collectively, our findings demonstrate that ZNF496 is a novel ERα-binding protein that acts as a target gene-specific ERα corepressor and inhibits the growth of ERα-positive breast cancer cells.

Cadmium-induced conformational changes in type 2 metallothionein of medicinal plant Coptis japonica: insights from molecular dynamics studies of apo, partially and fully metalated forms

Plants play an important role in the removal of excess heavy metals from soil and water. Medicinal plants can also have non-traditional use in phytoremediation technologies. Among the heavy metals, Cadmium (Cd) is the most abundant and readily taken up by the crop plants. Plant metallothioneins (MTs) are small proteins having cysteine-rich residues and appear to play key roles in metal homoeostasis. Plant metallothionein 2 (MT 2) from Coptis japonica (Gold-thread; CjMT 2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by 42 amino acids long linker region. In-silico analysis of MT 2 protein sequences of C. japonica was performed. In this study, ab initio methods were utilised for the prediction of three-dimensional structure of CjMT 2. After structure validation, heavy metal-binding sites were predicted for the selected modelled structures of CjMT 2. To obtain Cdi-CjMT 2 (i = 1–7), metalated complex individual docking experiments were performed. The stability of the metalated docked structures was assessed by molecular dynamics (MD) simulation studies. Our study showed that CjMT 2 binds up to 4 Cd2+ ions in two distinct domains: a N-terminal β-domain that binds to 2 Cd2+ ions and a C-terminal α-domain that binds with 2 Cd2+ ions. Our analysis revealed that Cys residues of alpha and beta domain and some residues of spacer region of CjMT 2 protein might be important for the cadmium interaction. MD simulation studies provided insight into metal-induced conformational changes and mechanism of metalation of CjMT 2, an intrinsically disordered protein. This study provides useful insights into mechanism of cadmium-type 2 metallothionein interaction.

Mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines

The detection of recurrent mutations affecting the hormone binding domain (HBD) of estrogen receptor alpha (ERα/ESR1) in endocrine therapy-resistant and metastatic breast cancers has prompted interest in functional characterization of these genetic alterations. Here, we explored the role of HBD-ESR1 mutations in influencing the behavior of breast cancer stem cells (BCSCs), using various BC cell lines stably expressing wild-type or mutant (Y537 N, Y537S, D538G) ERα. Compared to WT-ERα clones, mutant cells showed increased CD44+/CD24- ratio, mRNA levels of stemness genes, Mammosphere Forming Efficiency (MFE), Self-Renewal and migratory capabilities. Mutant clones exhibited high expression of NOTCH receptors/ligands/target genes and blockade of NOTCH signaling reduced MFE and migratory potential. Mutant BCSC activity was dependent on ERα phosphorylation at serine 118, since its inhibition decreased MFE and NOTCH4 activation only in mutant cells. Collectively, we demonstrate that the expression of HBD-ESR1 mutations may drive BC cells to acquire stem cell traits through ER/NOTCH4 interplay. We propose the early detection of HBD-ESR1 mutations as a challenge in precision medicine strategy, suggesting the development of tailored-approaches (i.e. NOTCH inhibitors) to prevent disease development and metastatic spread in BC mutant-positive patients.

Genetic variation and selection of MHC class I loci differ in two congeneric frogs.

Major histocompatibility complex (MHC) genes encode proteins in the acquired immune response pathway that often show distinctive selection-driven patterns in wild vertebrate populations. We examined genetic variation and signatures of selection in the MHC class I alpha 1 (A1)- and alpha 2 (A2)-domain encoding exons of two frog congeners [Agalychnis callidryas (n = 20) and A. lemur (n = 20)] from a single locality in Panama. We also investigated how historical demographic processes may have impacted MHC genetic diversity by analyzing a neutral mitochondrial marker. We found that both MHC domains were highly variable in both species, with both species likely expressing three loci. Our analyses revealed different signatures of selection between the two species, most notably that the A. callidryas A2 domain had experienced positive selection while the A2 domain of A. lemur had not. Diversifying selection acted on the same number of A1 and A2 allelic lineages, but on a higher percentage of A1 sites compared to A2 sites. Neutrality tests of mitochondrial haplotypes predominately indicated that the two species were at genetic equilibrium when the samples were collected. In addition, two historical tests of demography indicated both species have had relatively stable population sizes over the past 100,000 years; thus large population size changes are unlikely to have greatly influenced MHC diversity in either species during this time period. In conclusion, our results suggest that the impact of selection on MHC diversity varied between these two closely related species, likely due to a combination of distinct ecological conditions and past pathogenic pressures.

Statistically Reliable Molecular Dynamics Simulations of Transient Conformational Changes in the Estrogen Receptor

Typical molecular dynamics (MD) simulations do not reach equilibrium and hence their trajectory data do not correspond to a well-defined statistical distribution. On the other hand, multiple trajectories initiated from the same configuration can be averaged to yield well-defined, time-dependent properties. This strategy can be implemented using conventional MD or the weighted ensemble (WE) approach. We apply both methods to study the conformational dynamics of the ligand binding domain of estrogen receptor alpha in several settings - the wild-type and two mutants important in metastatic breast cancer, as well as apo and ligand-bound conditions. Overall, the simulations enable us to make quantitative determinations of when the conformational behavior is statistically distinguishable under the various conditions.

Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. Samd7-null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the Samd7-deficient retina, showing that Samd7 deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity.

P069 Completion of HLA proased nearest-neighbor extrapolation of HLA database sequences

Aim The IMGT/HLA database contains every publicly available HLA sequence. However, most of these HLA protein sequences are restricted to the alpha-1/alpha-2 domain for HLA class I and alpha-1/beta-1 domain for HLA class II. Nevertheless, also polymorphism outside these domains may play a role in alloreactivity after transplantation. Several computational algorithms that aim for predicting alloreactivity after transplantation, HLAMatchmaker and the PIRCHE algorithm, require a major or the whole part of the HLA protein sequence as input for their prediction. Methods In this study we describe an automated homology-based nearest-neighbor method to extrapolate incomplete HLA protein sequences. To get insight in the reliability of our extrapolation method, we performed a 10-fold cross-validation. Results The majority of the amino acid positions of the individual HLA class I and II proteins were correctly predicted. Eplets as defined by HLAMatchmaker were located both at correctly predicted as well as at lower reliably predicted amino acid positions. Moreover, six out of seven completely sequenced HLA alleles with previously unknown exons sequences were in agreement with the extrapolated amino acid sequences. Conclusions Incomplete HLA sequences can be completed by a homology-based nearest-neighbor principle. Since this method is automated, future submitted incomplete HLA sequences can easily be extrapolated. M. Niemann: 5. Employee; Company/Organization; PIRCHE AG. E. Spierings: 7. Other (Identify); Company/Organization; The University Medical Center Utrecht has filed a patent application for the prediction of an alloimmune response against mismatched HLA. E.S. is listed as inventor on this patent.

Live and let die: ZBP1 senses viral and cellular RNAs to trigger necroptosis.

Necroptosis is a programmed form of inflammatory cell death involved in various pathologies, such as viral infections. In two new papers published in The EMBO Journal and EMBO Reports , Z‐DNA binding protein 1 (ZBP1) is now shown to sense RNAs during viral infection or after caspase inhibition and activate necroptosis. This may suggest that Z‐RNAs are molecular patterns for activation of necroptosis.

Abstract B59: Enhancing T-cell therapy through TCR signaling-responsive nanogel drug delivery

Abstract Adoptive cell transfer (ACT) with autologous tumor-reactive T-cells has recently shown great promise in the treatment of leukemia in clinical trials. Effective control of solid tumors, remain challenging. Glioblatoma multiforme (GM) is a lethal disease in need of clinically effective therapies. Ninety-seven percent of tumors in the ‘classical’ subtype carry extra copies of the epidermal growth factor receptor (EGFR) gene, and most have higher than normal expression of EGFR. In the present study we utilize a novel carrier-free delivery strategy to specifically support a EGFR directed chimeric antigen receptor (CAR) T-cell with recombinant Interleukin-15 super agonist (IL15-Sa), in a heterotopic mouse model of GM. CAR-T cell design: The CAR-T cell was designed based on the heavy and light chains of cetuximab to form a single-chain variable fragment, which was fused to a portion of the extracellular and transmembrane domains of human CD8 alpha, followed by the intracellular domains of 4-1BB and CD3 zeta. The bicistronic vector also encoded truncated human CD19 as a selectable marker. The plasmid coding hu EGFRscFv-BBz-CAR was synthesized and lentivirus packaging was made. Isolated T cells were derived from leukapheresis products and stimulated with Dynabeads Human T Activator CD3/CD28. T cell were cultured and were transduced and or left untransduced one day following bead stimulation, and then T cells were expanded for 10 days and cryopreserved. Surface expression of the CAR was confirmed and quantified with biotinylated human EGFR protein and by staining for CD19. CAR-T cells phenotype was identified. Coupling of NGs to T cells: anti CD45 antibody (aCD45) against highly expressed cell surface phosphatase CD45 was used as an anchor to stably link human IL-15Sa as a drug cargo/nanogel (NG) to the T cell surface. The aCD45/IL-15-Sa-NG was added to CAR T cells. In vivo therapy study using human Chimeric Antigen Receptor (CAR)-T cells: Luciferase-expressing U-87 MG human glioblastoma cells (1.0 × 106) were injected s.c. into NSG mice on Day 0. Animals received i.v. adoptive transfer of activated CAR-T cells (2.6×106 total T-cells -38% transduction equating to 106 CAR-T-cells)) alone or with surface coupled NGs on Day 7. In other groups, free IL-15Sa was injected i.v. after ACT at equivalent dose. Tumor area and body weight were measured every two days. Mice were also imaged for bioluminescence to monitor the tumor growth and were euthanized when body weight loss was beyond 20%, or tumor area reached 150 mm2 or the animal had become moribund. Findings: CAR T-cells backpacked with IL-15Sa-NGs were compared to CAR T-cells alone or T-cells supplemented with an equivalent systemic dose of free IL-15Sa. NG-backpacked CAR T-cells eradicated tumors in 80% of animals. By contrast, transfer of CAR-T cells alone had a small impact on tumor growth and survival, which did not reach statistical significance; responses were marginally improved by the addition of free IL-15Sa. We have demonstrated NG delivery of cytokines has the potential to enhance CAR T-cell therapy in a mice model of GM. This strategy should be broadly applicable in ACT strategies employing natural, TCR-engineered, or CAR T-cell therapies, and illustrates a general approach to couple the action of a synthetic drug delivery carrier to the function of therapeutic cells. Citation Format: Ana Castano, Li Tang, Sagar Kudchodkar, Irvine Darrell, Marcela Maus. Enhancing T-cell therapy through TCR signaling-responsive nanogel drug delivery. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B59.

ADME/Tox Properties and Biochemical Interactions of Silybin Congeners: In silico Study

Silymarin, the active constituent of Silybum marianum (milk thistle), and its main component, silybin, are products with well-known hepatoprotective, cytoprotective, antioxidant, and chemopreventative properties. Despite substantial in vitro and in vivo investigations of these flavonolignans, their mechanisms of action and potential toxic effects are not fully defined. In this study we explored important ADME/Tox properties and biochemical interactions of selected flavonolignans using in silico methods. A quantitative structure-activity relationship (QSAR) model based on data from a parallel artificial membrane permeability assay (PAMPA) was used to estimate bioavailability after oral administration. Toxic effects and metabolic transformations were predicted using the knowledge-based expert systems Derek Nexus and Meteor Nexus (Lhasa Ltd). Potential estrogenic activity of the studied silybin congeners was outlined. To address further the stereospecificity of this effect the stereoisomeric forms of silybin were docked into the ligand-binding domain of the human estrogen receptor alpha (ERa) (MOE software, CCG). According to our results both stereoisomers can be accommodated into the ERa active site, but different poses and interactions were observed for silybin A and silybin B.

Comparsion of Real EEG References with and Without Zero Potential According Resulting Topograthy Differencies

The problem to find an optimal EEG reference is the actual topic for discussion over 60 years. We have studied topographical differences in averaged EEG amplitudes of alpha domain recorded in 10–20 system during “eyes closed” test. These differences appeared due to the use of 13 reference schemes: top and bottom of the chin (Ch1, Ch2); nose (N); top and bottom of the neck (Nc1, Nc2); upper back (Bc); united electrodes at the base of the neck anteriorly and posteriorly (2Nc); united, ipsilateral, and individual ear electrodes (A12, Sym, A1, A2); vertex (Cz); and averaged reference (AR). Six experiments for each of the ten subjects were carried out with grounded and ungrounded states of three distant basic references Ch2, Bc, 2Nc. Pairwise comparisons of topographic consistency of 13 reference schemes were carried out on the proposed complex of three independent indicators with the evaluative criterion, followed by centroid-based clustering of the reference schemes and its discriminant verification. As a result, we have established: (1) that most coordinated topography is provided by the following reference electrodes —A12, Ch1, Ch2, Sym; (2) reference electrodes A1, Nc2, A2, Sh1, AR, Cz are characterized by individually varying topography, which may lead to contradictory conclusions obtained when they are used; (3) no significant reasons have been found for preferring the grounded (neutral) states of reference electrodes, that makes less important the search for or mathematical construct of an infinitely remote neutral reference electrode; (4) numerous distortions of EEG topography by reference electrode standardization technique (REST) raise serious doubts about its proclaimed advantages in EEG studies.

Universality and Domain Specificity of Emotion-A Quantitative Non Linear EEG Based Approach

Music has been present in human culture since time immemorial, some say music came even before speech. The effort to understand the wide variety of emotions evoked by music has started not long back. With the advent and rapid growth of various neurological bio-sensors we can now attempt to quantify various dimensions of emotional experience induced by music especially instrumental music – since it is free from any language barriers. In this study, we took eight (8) cross cultural instrumental clips originating mainly from Indian and Western music. A listening test comprising of 100 participants across the globe was conducted to associate each clip with its corresponding emotional valence. The participants were asked to mark each clip according to their perception of four basic emotions (joy/sorrow and anxiety/serenity) invoked by each instrumental clip. EEG study was then conducted on 10 participants to measure the response evoked by the same instrumental clips in the alpha and theta frequency regions. We took the help of latest non-linear multi-fractal analysis technique – MFDFA to estimate the change in multi-fractal spectral width (corresponding to alpha as well as theta waves) associated with each of the clips in frontal, temporal and occipital lobes. The response in the alpha domain reveals a hint in the direction of universality of music, while in theta domain we have culture specific response. Moreover, we tried to develop alpha as well as theta multi-fractal spectral width as a single parameter with which we can quantify the valence and arousal based effects corresponding to a particular musical clip. The results and implications are discussed in detail.

A Novel Functional Domain of Tab2 Involved in the Interaction with Estrogen Receptor Alpha in Breast Cancer Cells.

Tab2, originally described as a component of the inflammatory pathway, has been implicated in phenomena of gene de-repression in several contexts, due to its ability to interact with the NCoR corepressor. Tab2 interacts also with steroid receptors and dismisses NCoR from antagonist-bound Estrogen and Androgen Receptors on gene regulatory regions, thus modifying their transcriptional activity and leading to pharmacological resistance in breast and prostate cancer cells. We demonstrated previously that either Tab2 knock-down, or a peptide mimicking the Estrogen Receptor alpha domain interacting with Tab2, restore the antiproliferative response to Tamoxifen in Tamoxifen-resistant breast cancer cells. In this work, we map the domain of Tab2 responsible of Estrogen Receptor alpha interaction. First, using both co-immunoprecipitation and pull-down with recombinant proteins, we found that the central part of Tab2 is primarily responsible for this interaction, and that this region also interacts with Androgen Receptor. Then, we narrowed down the essential interaction region by means of competition assays using recombinant protein pull-down. The interaction motif was finally identified as a small region adjacent to, but not overlapping, the Tab2 MEKK1 phosphorylation sites. A synthetic peptide mimicking this motif efficiently displaced Tab2 from interacting with recombinant Estrogen Receptor alpha in vitro, prompting us to test its efficacy using derivatives of the MCF7 breast carcinoma cell lines that are spontaneously resistant to Tamoxifen. Indeed, we observed that this mimic peptide, made cell-permeable by addition of the TAT minimal carrier domain, reduced the growth of Tamoxifen-resistant MCF7 cells in the presence of Tamoxifen. These data indicate a novel functional domain of the Tab2 protein with potential application in drug design.

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia.

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death.

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

Genetic basis for high population diversity in Protea-associated Knoxdaviesia

Sexual reproduction is necessary to generate genetic diversity and, in ascomycete fungi, this process is controlled by a mating type (MAT) locus with two complementary idiomorphs. Knoxdaviesia capensis and K. proteae (Sordariomycetes; Microascales; Gondwanamycetaceae) are host-specific saprophytic fungi that show high population diversity within their Protea plant hosts in the Cape Floristic Region of South Africa. We hypothesise that this diversity is the result of outcrossing driven by a heterothallic mating system and sought to describe the MAT1 loci of both species. The available genome assembly of each isolate contained only one of the MAT1 idiomorphs necessary for sexual reproduction, implying that both species are heterothallic. Idiomorph segregation during meiosis, a 1:1 ratio of idiomorphs in natural populations and mating experiments also supported heterothallism as a sexual strategy. Long-range PCR and shot-gun sequencing to identify the opposite idiomorph in each species revealed no sequence similarity between MAT1-1 and MAT1-2 idiomorphs, but the homologous idiomorphs between the species were almost identical. The MAT1-1 idiomorph contained the characteristic MAT1-1-1 and MAT1-1-2 genes, whereas the MAT1-2 idiomorph consisted of the genes MAT1-2-7 and MAT1-2-1. This gene content was similar to that of the three species in the Ceratocystidaceae (Microascales) with characterized MAT loci. The Knoxdaviesia MAT1-2-7 protein contained and alpha domain and predicted intron, which suggests that this gene arose from MAT1-1-1 during a recombination event. In contrast to the Ceratocystidaceae species, Knoxdaviesia conformed to the ancestral Sordariomycete arrangement of flanking genes and is, therefore, a closer reflection of the structure of this locus in the Microascalean ancestor.

Estrogenic Receptor-Functionalized Magnetite Nanoparticles for Rapid Separation of Phytoestrogens in Plant Extracts.

With growing interests of phytoestrogens, many natural phytochemicals extracted from diverse plant species have been explored for their estrogenic-like activities and potential applications. In this work, a simple and rapid separation of phytoestrogenic compounds from crude plant extracts was purposed using magnetic nanoparticles (MNPs) of Fe3O4 immobilized with the ligand-binding domain (LBD) of estrogen receptor alpha (ERα). The recombinant LBD-ERα peptide of 40kDa was produced and subsequently covalently linked to MNPs. One milligram of the LBD-ERα-immobilized MNPs demonstrated a specific binding to the standard 17β-estradiol (E2) of 3.37nmol and 91.3-100% of the bound E2 were subsequently recovered. LBD-ERα-immobilized MNPs could separate phytoestrogens of 4.6nmol E2-equivalent activity from a 1-mg crude extract of Asparagus racemosus. The produced MNPs showed no separation yield when were applied to the negative controls, the crude extract of radish (Raphanus sativus), and the standard progesterone (P4). Thin-layer chromatography demonstrated a single phytochemical band of the separated phytoestrogens, which exhibited the activity to promote MCF-7 cell proliferation at 4.7 folds greater than the crude A. racemosus extract. The results of this work demonstrated a simple method to specifically separate phytoestrogens from crude plant extracts via the LBD-ERα-immobilized MNPs.

Differences in conformational stability of the two alpha domains of the disease-associated and non-disease-associated subtypes of HLA-B27

The MHC Class I molecule, HLA-B27, is strongly linked with development of the inflammatory arthritic disease, ankylosing spondylitis (AS); whereas the B*2705 subtype shows strong association, B*2709 is not associated with disease, even though the two subtypes differ in only a single residue at position 116. Currently, attention is focused on the misfolding propensities of these two subtypes, including studies of disulfide-linked dimers and non-covalently formed high molecular weight (HMW) aggregates. Using mutants retaining only a single cysteine at positions C67 or C164, and using a cysteine-reactive, environment-sensitive, fluorescence probe (acrylodan), we find that within the same overall population of identical single-cysteine HLA-B27 molecules, there exist sub-populations which (a) possess free cysteines which react with acrylodan, (b) form disulfide-linked dimers, and (c) form HMW aggregates. Further, using acrylodan fluorescence, we find (d) that the α1 and α2 domains unfold independently of each other in HMW aggregates, (e) that these two domains of B*2709 are less stable to chemical and thermal denaturation than the corresponding domains of B*2705, suggesting easier clearance of misfolded molecules in the former, and (f) C67 is much more exposed in B*2705 than in B*2709, which could potentially explain how B*2705 more easily forms C67-mediated disulfide-bonded dimers.

Characterizing regularity of domains via the Riesz transforms on their boundaries

Given a domain D in R^d with mild geometric measure theoretic assumptions on its boundary, we show that boundedness of the principal value Riesz tranforms (witn kernel of homogeneity -(d-1)) on H\"older spaces of order alpha on the boundary of D is equivalent to D being a Lyapunov domain of order alpha (i.e., the boundary of D is an hypersurface of class 1+alpha). Another equivalent condition involving Riesz transforms on D is discussed. We also prove that on Lyapunov domains of order alpha the higher order Riesz transforms associated with an odd polynomial are bounded on the H\"older space of order alpha on the boundary of D. Finally, a limiting case of the above results dealing with VMO and Semmes-Kenig-Toro domains is considered.

Optimal choice of a reference electrode for EEG recording

We have studied topographical differences in averaged EEG amplitudes in alpha domain recorded in 10–20 system during “eyes closed” test. These differences appeared due to the use of 13 reference schemes: top and bottom of the chin (P1, P2); nose (N); top and bottom of the back of the neck (Sh1, Sh2); upper back (C); united electrodes at the base of the neck anteriorly and posteriorly (2Sh); united, ipsilateral, and individual ear electrodes (A12, Sym, A1, A2); vertex (Cz); and averaged reference (AR). Six experiments for each of the ten subjects were carried out with grounded and ungrounded states of three distant basic references P2, C, and 2Sh. Pairwise comparisons of topographic consistency of 13 reference schemes were carried out on the proposed complex of three independent indicators with evaluative criterion, followed by centroid-based clustering of the reference schemes and its discriminant verification. As a result, we have established (1) that most coordinated topography is provided by the following reference electrodes—A12, P1, P2, Sym; (2) reference electrodes A1, Sh2, A2, Sh1, AR, Cz are characterized by individually varying topography, which may lead to contradictory conclusions obtained when they are used; (3) no significant reasons have been found for preferring a grounded (neutral) states of reference electrodes, that makes the search for or mathematical construct of an infinitely remote neutral reference electrode less important.