Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

Dissection of a Down syndrome-associated trisomy to separate the gene dosage-dependent and -independent effects of an extra chromosome.

As an aneuploidy, trisomy is associated with mammalian embryonic and postnatal abnormalities. Understanding the underlying mechanisms involved in mutant phenotypes is broadly important and may lead to new strategies to treat clinical manifestations in individuals with trisomies, such as trisomy 21 [Down syndrome (DS)]. Although increased gene dosage effects because of a trisomy may account for the mutant phenotypes, there is also the possibility that phenotypic consequences of a trisomy can arise because of the presence of a freely segregating extra chromosome with its own centromere, i.e. a 'free trisomy' independent of gene dosage effects. Presently, there are no reports of attempts to functionally separate these two types of effects in mammals. To fill this gap, here we describe a strategy that employed two new mouse models of DS, Ts65Dn;Df(17)2Yey/+ and Dp(16)1Yey/Df(16)8Yey. Both models carry triplications of the same 103 human chromosome 21 gene orthologs; however, only Ts65Dn;Df(17)2Yey/+ mice carry a free trisomy. Comparison of these models revealed the gene dosage-independent impacts of an extra chromosome at the phenotypic and molecular levels for the first time. They are reflected by impairments of Ts65Dn;Df(17)2Yey/+ males in T-maze tests when compared with Dp(16)1Yey/Df(16)8Yey males. Results from the transcriptomic analysis suggest the extra chromosome plays a major role in trisomy-associated expression alterations of disomic genes beyond gene dosage effects. This model system can now be used to deepen our mechanistic understanding of this common human aneuploidy and obtain new insights into the effects of free trisomies in other human diseases such as cancers.

An evolving view of complex II-noncanonical complexes, megacomplexes, respiration, signaling, and beyond.

Mitochondrial complex II is traditionally studied for its participation in two key respiratory processes: the electron transport chain and the Krebs cycle. There is now a rich body of literature explaining how complex II contributes to respiration. However, more recent research shows that not all of the pathologies associated with altered complex II activity clearly correlate with this respiratory role. Complex II activity has now been shown to be necessary for a range of biological processes peripherally related to respiration, including metabolic control, inflammation, and cell fate. Integration of findings from multiple types of studies suggests that complex II both participates in respiration and controls multiple succinate-dependent signal transduction pathways. Thus, the emerging view is that the true biological function of complex II is well beyond respiration. This review uses a semichronological approach to highlight major paradigm shifts that occurred over time. Special emphasis is given to the more recently identified functions of complex II and its subunits because these findings have infused new directions into an established field.

Does the urinary microbiome profile change after treatment of bladder cancer?

INTRODUCTION: We sought to investigate the change in the urinary microbiome profile after transurethral resection of bladder tumor (TURBT). METHODS: Urine specimens were collected from consecutive patients with bladder cancer. Patients were divided into those with bladder tumors ("Tumor group": de novo tumors or recurrent/progressed after TURBT ± intravesical therapy) versus those without evidence of recurrence after treatment "No Recurrent Tumor group". Samples were analyzed using 16S rRNA sequencing. Alteration in the urinary microbiome was described in terms of alpha (diversity within a sample measured by Observed, Chao, Shannon, and Simpson indices), beta diversities (diversity among different samples measured by Brady Curtis Diversity index), and differential abundance of bacteria at the genus level. Analyses were adjusted for gender, method of preservation (frozen vs preservative), and method of collection (mid-stream vs. catheter). RESULTS: Sixty-eight samples were analyzed (42 in "Tumor" vs 26 in "No Recurrent Tumor" groups). The median age was 70 years (IQR 64-74) and 85% were males. All patients in the "No Recurrent Tumor" group had non-muscle invasive bladder cancer and 85% received BCG compared to 69% and 43% for the "Tumor" group, respectively. There was no significant difference in alpha diversity (p > 0.05). Beta diversity was significantly different (p = 0.04). Veillonella and Bifidobacterium were more abundant in the "Tumor" group (> 2FC, p = 0.0002), while Escherichia-Shigella (> 2FC, p = 0.0002) and Helococcus (> 2FC, p = 0.0008) were more abundant in the "No Recurrent Tumor" group. CONCLUSION: Bladder cancer patients with no recurrence and/or progression exhibited a different urinary microbiome profile compared to those with tumors.

Comparable transforming growth factor beta-mediated immune suppression in ex vivo-expanded natural killer cells from cord blood and peripheral blood: implications for adoptive immunotherapy.

T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an "off-the-shelf" setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-ß) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors' group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells' functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-ß.  However, a direct comparison of the effects of TGF-ß-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-ß results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-ß-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-ß-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer.

Potassium-induced partial inhibition of lactoperoxidase: structure of the complex of lactoperoxidase with potassium ion at 2.20 Å resolution.

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.

Genes Relevant to Tissue Response to Cancer Therapy Display Diurnal Variation in mRNA Expression in Human Oral Mucosa.

BACKGROUND: To address a critical gap for application of cancer chronotherapy of when would be the best time(s) for treating an individual cancer patient, we conducted a pilot study to characterize diurnal variations of gene expression in oral mucosal tissue, which is vulnerable to damage from cancer therapies. METHODS: We conducted RNA-seq assay on individual oral mucosal samples collected from 11 healthy volunteers every 4 hours (6 time points). Using a cosine-based method, we estimated the individual and average values of peak-time and amplitude for each gene. Correlations between gene expression peak-times and age was examined, adjusting for individual's sleep timing. RESULTS: Among candidate gene pathways that are relevant to treatment response, 7 of 16 genes (PER3, CIART, TEF, PER1, PER2, CRY2, ARNTL) involved in circadian regulation and 1 of 118 genes (WEE1) involved in cell cycle regulation achieved p-value ≤ 0.1 and relative amplitude>0.1. The average peak times were approximately 10:15 for PER3, CIART and TEF, 10:45 for PER1, 13:00 for WEE1, PER2 and CRY2, and 19:30 for ARNTL. Ranges in peak times across individuals differed by gene (e.g., 8 hours for PER1; 16.7 hours for WEE1). Older people had later peak times for PER1 (r = 0.77, p = 0.03) and PER3 (r = 0.69, p-value = 0.06). CONCLUSION: In oral mucosa, expression of some genes relevant to treatment response displayed diurnal variation. These genes may be candidates for development of biomarkers for optimizing individual timing of cancer therapy using non-invasively collected oral mucosa.

An alternative N-terminal fold of the intestine-specific annexin A13a induces dimerization and regulates membrane-binding.

Annexin proteins function as Ca2+-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by in vitro binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with uracil and uridine.

Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (KD ) were 1.2 × 10-6 M and 1.4 × 10-7 M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10-6 M and 1.1 × 10-7 M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.

Accurate Quantification of Residual Cancer Cells in Pelvic Washing Reveals Association with Cancer Recurrence Following Robot-Assisted Radical Cystectomy.

PURPOSE: Bladder cancer recurrence following cystectomy remains a significant cause of bladder cancer specific mortality. Residual cancer cells contribute to cancer recurrence due to tumor spillage or undetectable preexisting micrometastatic tumor clones. We detected and quantified residual cancer cells in pelvic washing using ultradeep targeted sequencing. We compared the levels of residual cancer cells with clinical variables and cancer recurrence. MATERIALS AND METHODS: The primary tumor specimen was available in 17 patients who underwent robot-assisted radical cystectomy. All tumors had negative surgical margins. Pelvic washes and blood were collected intraoperatively before and after robot-assisted radical cystectomy, after pelvic lymph node dissection and in the suction fluid collected during the procedure. Two-step sequencing, including whole exome sequencing followed by ultradeep targeted sequencing (× greater than 50,000), was done to quantify residual cancer cells in each sample. Eight patients were excluded from study due to sample quality issues. The final analysis cohort comprised 9 patients. The residual cancer cell level was quantified for each sample as the relative cancer cell fraction and compared between time points. The peak relative cancer cell fraction of each patient was correlated with clinical and pathological variables. RESULTS: Residual cancer cells were detected in approximately half of the pelvic washing specimens during or after but not before robot-assisted radical cystectomy. Higher residual cancer cell levels were associated with aggressive variant histology and cancer recurrence. Verifying the feasibility of using residual cancer cells as a novel biomarker for recurrence requires larger cohorts. CONCLUSIONS: Detection of residual cancer cells in intraoperative peritoneal washes of patients with bladder cancer who undergo radical cystectomy may represent a robust biomarker of tumor aggressiveness and metastatic potential.

Search of multiple hot spots on the surface of peptidyl-tRNA hydrolase: structural, binding and antibacterial studies.

Peptidyl-tRNA hydrolase (Pth) catalyzes the breakdown of peptidyl-tRNA into peptide and tRNA components. Pth from Acinetobacter baumannii (AbPth) was cloned, expressed, purified and crystallized in a native unbound (AbPth-N) state and in a bound state with the phosphate ion and cytosine arabinoside (cytarabine) (AbPth-C). Structures of AbPth-N and AbPth-C were determined at 1.36 and 1.10 Šresolutions, respectively. The structure of AbPth-N showed that the active site is filled with water molecules. In the structure of AbPth-C, a phosphate ion is present in the active site, while cytarabine is bound in a cleft which is located away from the catalytic site. The cytarabine-binding site is formed with residues: Gln19, Trp27, Glu30, Gln31, Lys152, Gln158 and Asp162. In the structure of AbPth-N, the side chains of two active-site residues, Asn70 and Asn116, were observed in two conformations. Upon binding of the phosphate ion in the active site, the side chains of both residues were ordered to single conformations. Since Trp27 is present at the cytarabine-binding site, the fluorescence studies were carried out which gave a dissociation constant (KD) of 3.3 ± 0.8 × 10-7 M for cytarabine. The binding studies using surface plasmon resonance gave a KD value of 3.7 ± 0.7 × 10-7 M. The bacterial inhibition studies using the agar diffusion method and the biofilm inhibition assay established the strong antimicrobial potential of cytarabine. It also indicated that cytarabine inhibited Gram-negative bacteria more profoundly when compared with Gram-positive bacteria in a dose-dependent manner. Cytarabine was also effective against the drug-resistant bacteria both alone as well as in combination with other antibiotics.

New crystal forms of the integral membrane Escherichia coli quinol:fumarate reductase suggest that ligands control domain movement.

Quinol:fumarate reductase (QFR) is an integral membrane protein and a member of the respiratory Complex II superfamily. Although the structure of Escherichia coli QFR was first reported almost twenty years ago, many open questions of catalysis remain. Here we report two new crystal forms of QFR, one grown from the lipidic cubic phase and one grown from dodecyl maltoside micelles. QFR crystals grown from the lipid cubic phase processed as P1, merged to 7.5 Šresolution, and exhibited crystal packing similar to previous crystal forms. Crystals grown from dodecyl maltoside micelles processed as P21, merged to 3.35 Šresolution, and displayed a unique crystal packing. This latter crystal form provides the first view of the E. coli QFR active site without a dicarboxylate ligand. Instead, an unidentified anion binds at a shifted position. In one of the molecules in the asymmetric unit, this is accompanied by rotation of the capping domain of the catalytic subunit. In the other molecule, this is associated with loss of interpretable electron density for this same capping domain. Analysis of the structure suggests that the ligand adjusts the position of the capping domain.

Mapping the Damping Dynamics of Mega-Ampere Electron Pulses Inside a Solid.

We report the lifetime of intense-laser (2×10^{19} W/cm^{2}) generated relativistic electron pulses in solids by measuring the time evolution of their Cherenkov emission. Using a picosecond resolution optical Kerr gating technique, we demonstrate that the electrons remain relativistic as long as 50 picoseconds-more than 1000 times longer than the incident light pulse. Numerical simulations of the propagation of relativistic electrons and the emitted Cherenkov radiation with Monte Carlo geant4 package reproduce the striking experimental findings.

A glycoprotein from mammary gland secreted during involution promotes apoptosis: Structural and biological studies.

Secretory signalling glycoprotein (SPX-40) from mammary gland is highly expressed during involution. This protein is involved in a programmed cell death during tissue remodelling which occurs at the end of lactation. SPX-40 was isolated and purified from buffalo (SPB-40) from the samples obtained during involution. One solution of SPB-40 was made by dissolving it in buffer containing 25 mM Tris-HCl and 50 mM NaCl at pH 8.0. Another solution was made by adding 25% ethanol to the above solution. The biological effects of SPB-40 dissolved in above two solutions were evaluated on MCF-7 breast cancer cell lines. Free SPB-40 indicated significant pro-apoptotic effects while ethanol exposed SPB-40 showed considerably reduced effects on the apoptosis. SPB-40 was crystallized in the native state. The crystals of SPB-40 were soaked in four separate solutions containing 25% acetone, 25% ethanol, 25% butanol and 25% MPD. Four separate data sets were collected and their structures were determined at high resolutions. In all the four structures, the molecules of acetone, ethanol, butanol and MPD respectively were observed in the hydrophobic binding pocket of SPB-40. As a result of which, the conformation of Trp78 was altered thus blocking the binding site in SPB-40 leading to the loss of activity.

Design of anti-thyroid drugs: Binding studies and structure determination of the complex of lactoperoxidase with 2-mercaptoimidazole at 2.30 Å resolution.

Lactoperoxidase (LPO) belongs to mammalian heme peroxidase superfamily, which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO). LPO catalyzes the oxidation of a number of substrates including thiocyanate while TPO catalyzes the biosynthesis of thyroid hormones. LPO is also been shown to catalyze the biosynthesis of thyroid hormones indicating similar functional and structural properties. The binding studies showed that 2-mercaptoimidazole (MZY) bound to LPO with a dissociation constant of 0.63 µM. The inhibition studies showed that the value of IC50 was 17 µM. The crystal structure of the complex of LPO with MZY showed that MZY bound to LPO in the substrate-binding site on the distal heme side. MZY was oriented in the substrate-binding site in such a way that the sulfur atom is at a distance of 2.58 Å from the heme iron. Previously, a similar compound, 3-amino-1,2,4-triazole (amitrole) was also shown to bind to LPO in the substrate-binding site on the distal heme side. The amino nitrogen atom of amitrole occupied the same position as that of sulfur atom in the present structure indicating a similar mode of binding. Recently, the structure of the complex of LPO with a potent antithyroid drug, 1-methylimidazole-2-thiol (methimazole, MMZ) was also determined. It showed that MMZ bound to LPO in the substrate-binding site on the distal heme side with 2 orientations. The position of methyl group was same in the 2 orientations while the positions of sulfur atom differed indicating a higher preference for a methyl group.

Integration of VDR genome wide binding and GWAS genetic variation data reveals co-occurrence of VDR and NF-κB binding that is linked to immune phenotypes.

BACKGROUND: The nuclear hormone receptor superfamily acts as a genomic sensor of diverse signals. Their actions are often intertwined with other transcription factors. Nuclear hormone receptors are targets for many therapeutic drugs, and include the vitamin D receptor (VDR). VDR signaling is pleotropic, being implicated in calcaemic function, antibacterial actions, growth control, immunomodulation and anti-cancer actions. Specifically, we hypothesized that the biologically significant relationships between the VDR transcriptome and phenotype-associated biology could be discovered by integrating the known VDR transcription factor binding sites and all published trait- and disease-associated SNPs. By integrating VDR genome-wide binding data (ChIP-seq) with the National Human Genome Research Institute (NHGRI) GWAS catalog of SNPs we would see where and which target gene interactions and pathways are impacted by inherited genetic variation in VDR binding sites, indicating which of VDR's multiple functions are most biologically significant. RESULTS: To examine how genetic variation impacts VDR function we overlapped 23,409 VDR genomic binding peaks from six VDR ChIP-seq datasets with 191,482 SNPs, derived from GWAS-significant SNPs (Lead SNPs) and their correlated variants (r 2 > 0.8) from HapMap3 and the 1000 genomes project. In total, 574 SNPs (71 Lead and 503 SNPs in linkage disequilibrium with Lead SNPs) were present at VDR binding loci and associated with 211 phenotypes. For each phenotype a hypergeometric test was used to determine if SNPs were enriched at VDR binding sites. Bonferroni correction for multiple testing across the 211 phenotypes yielded 42 SNPs that were either disease- or phenotype-associated with seven predominately immune related including self-reported allergy; esophageal cancer was the only cancer phenotype. Motif analyses revealed that only two of these 42 SNPs reside within a canonical VDR binding site (DR3 motif), and that 1/3 of the 42 SNPs significantly impacted binding and gene regulation by other transcription factors, including NF-κB. This suggests a plausible link for the potential cross-talk between VDR and NF-κB. CONCLUSIONS: These analyses showed that VDR peaks are enriched for SNPs associated with immune phenotypes suggesting that VDR immunomodulatory functions are amongst its most important actions. The enrichment of genetic variation in non-DR3 motifs suggests a significant role for the VDR to bind in multimeric complexes containing other transcription factors that are the primary DNA binding component. Our work provides a framework for the combination of ChIP-seq and GWAS findings to provide insight into the underlying phenotype-associated biology of a given transcription factor.

Structure of bovine lactoperoxidase with a partially linked heme moiety at 1.98Å resolution.

Lactoperoxidase (LPO) is a member of mammalian heme peroxidase superfamily whose other members are myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). In these enzymes, the heme moiety is linked to protein through two or three covalent bonds. In the mature LPO, the heme moiety is linked to protein through two ester bonds with highly conserved glutamate and aspartate residues. The previously reported structures of LPO have confirmed the formation of two covalent linkages involving Glu258 and Asp108 with 1-methyl and 5-methyl groups of pyrrole rings A and C respectively. We report here a new form of structure of LPO where the covalent bond between Glu258 and 1-methyl group of pyrrole ring A is present only in a fraction of protein molecules. In this case, the side chain of Glu258 occupies two distinct positions, each of which has a 0.5 occupancy. In one position, it forms a normal ester covalent linkage while in the second position, the side chain of Glu258 is located in the middle of the substrate binding site on the distal heme side. In this position, the atom of the side chain of Glu258 forms several contacts with atoms of other residues and heme moiety. Out of the two observed positions of the side chain of Glu258, the former contributes to the stabilization of heme position and improved catalytic action of LPO while the latter is responsible for the reduced stability of the heme position as well as it blocks the substrate binding site.

Integrative genomic analysis in K562 chronic myelogenous leukemia cells reveals that proximal NCOR1 binding positively regulates genes that govern erythroid differentiation and Imatinib sensitivity.

To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib.

Structure of iron saturated C-lobe of bovine lactoferrin at pH 6.8 indicates a weakening of iron coordination.

The bilobal lactoferrin is an approximately 76 kDa glycoprotein. It sequesters two Fe(3+) ions together with two CO(3)(2-) ions. The C-terminal half (residues, Tyr342-Arg689, C-lobe) of bovine lactoferrin (BLF) (residues Ala1-Arg689) was prepared by limited proteolysis using trypsin. Both C-lobe and intact BLF were saturated to 100%. Both of them retained up to nearly 85% of iron at pH 6.5. At pH 5.0, C-lobe retained 75% of iron whereas intact protein could retain only slightly more than 60%. At pH 4.0 both contained 25% iron and at pH 2.0 they were left with iron concentration of only 10%. The structure of iron saturated C-lobe was determined at 2.79 Å resolution and refined to R(cryst) and R(free) factors of 0.205 and 0.273, respectively. The structure contains two crystallographically independent molecules, A and B. They were found to have identical structures with an r.m.s. shift of 0.5 Å for their C(α) atoms. A high solvent content of 66% was observed in the crystals. The average value of an overall B-factor was 68.0 Å(2). The distance of 2.9 Å observed for the coordination bond between Fe(3+) ion and N(e2) of His595 appeared to be considerably longer than the normally observed values of 1.9-2.2 Å. This indicated that the coordination bond involving His595 may be absent. Other coordination distances were observed in the range of 2.1-2.3 Å. Based on the present structure of iron saturated C-lobe, it may be stated that His595 is the first residue to dissociate from ferric ion when the pH is lowered.