Genomic Profiling of Childhood Tumor Patient-Derived Xenograft Models to Enable Rational Clinical Trial Design.

Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)-many of which are refractory to current standard-of-care treatments-from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector‐triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc‐finger‐like structure with a novel interleaved zinc‐binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P‐mediated recognition. The first zinc‐coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid‐binding and chromatin‐associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non‐conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.This research was supported by Australian Research Council (ARC) Discovery Projects DP120100685, DP130104098 and DP160102244. XZ was a recipient of an ANZ Trustees PhD Scholarship for Medical Research in Queensland. BK is a National Health and Medical Research Council (NHMRC) Principal Research Fellow (1003325 and 1110971). MB was a recipient of an ARC Discovery Early Career Research Award (DE130101292)

The yeast transcription elongation factor Spt4/5 is a sequence-specific RNA binding protein

The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co‐transcriptional pre‐mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence‐specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA‐binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5

A rapid method for assessing the RNA-binding potential of a protein

In recent years, evidence has emerged for the existence of many diverse types of RNA, which play roles in a wide range of biological processes in all kingdoms of life. These molecules generally do not, however, act in isolation, and identifying which proteins partner with RNA is a major challenge. Many methods, in vivo and in vitro , have been used to address this question, including combinatorial or high-throughput approaches, such as systematic evolution of ligands, cross-linking and immunoprecipitation and RNA immunoprecipitation combined with deep sequencing. However, most of these methods are not trivial to pursue and often require substantial optimization before results can be achieved. Here, we demonstrate a simple technique that allows one to screen proteins for RNA-binding properties in a gel-shift experiment and can be easily implemented in any laboratory. This assay should be a useful first-pass tool for assessing whether a protein has RNA- or DNA-binding properties, prior to committing resources to more complex procedures.ISSN:1362-4962ISSN:0301-561

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequencespecificity of four VapC proteins from two different organisms (VapCPAE0151 and VapCPAE2754 from Pyrobaculum aerophilum, and VapCRv0065 and VapCRv0617 from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms. Published by Cold Spring Harbor Laboratory Press

Exploring the suitability of RanBP2-type Zinc Fingers for RNA-binding protein design.

Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells. Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging

Spatial analysis of poverty and prosperity in the U.S. counties

Title from PDF of title page (University of Missouri--Columbia, viewed on October 22, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Kenneth E. Pigg.Vita.Ph. D. University of Missouri--Columbia 2010.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Prosperity is often associated with income. This study goes beyond the economic determinant of prosperity and develops a prosperity index by using indicators of education, employment, housing occupancy, and poverty status for all contiguous U.S. counties and non-metro counties. A spatial approach has been used to analyze the data as the data was spatially distributed. Using OLS, spatial lag, and spatial error methods, three models were developed and compared. Spatial error model explained higher percent of variation among three models. Labor markets variables were found to be important predictors of prosperity in all-counties and non-metro counties. The results showed that high prosperous counties had higher economic opportunities, higher urban influence, higher social capital, lower income inequality, lower percent of minority population, higher percent of employed female population, higher civic agricultural activities, lower percent of people working in low paying service sectors, and more jobs in manufacturing sectors. A major contribution of this study to sociology is that spatial effect should be taken into consideration while dealing with spatially correlated data.Includes bibliographical reference

PLK1 as a cooperating partner for BCL2-mediated antiapoptotic program in leukemia

Abstract The deregulation of BCL2 family proteins plays a crucial role in leukemia development. Therefore, pharmacological inhibition of this family of proteins is becoming a prevalent treatment method. However, due to the emergence of primary and acquired resistance, efficacy is compromised in clinical or preclinical settings. We developed a drug sensitivity prediction model utilizing a deep tabular learning algorithm for the assessment of venetoclax sensitivity in T-cell acute lymphoblastic leukemia (T-ALL) patient samples. Through analysis of predicted venetoclax-sensitive and resistant samples, PLK1 was identified as a cooperating partner for the BCL2-mediated antiapoptotic program. This finding was substantiated by additional data obtained through phosphoproteomics and high-throughput kinase screening. Concurrent treatment using venetoclax with PLK1-specific inhibitors and PLK1 knockdown demonstrated a greater therapeutic effect on T-ALL cell lines, patient-derived xenografts, and engrafted mice compared with using each treatment separately. Mechanistically, the attenuation of PLK1 enhanced BCL2 inhibitor sensitivity through upregulation of BCL2L13 and PMAIP1 expression. Collectively, these findings underscore the dependency of T-ALL on PLK1 and postulate a plausible regulatory mechanism

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin*

α-Hemoglobin (αHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with αHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting αHb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in αHb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro30, in loop 1 of AHSP. Mutation of Pro30 to a variety of residue types results in reduced ability to convert αHb. In complex with αHb, AHSP Pro30 adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in αHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the αHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of αHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops