Publications on this page begin in 2012. Some staff have a record of earlier publications on their personal pages.
A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor’s major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.
BACKGROUND:Meehl regarded schizotypy as a categorial liability for schizophrenia that is the product of genes, environment, and gene-environment interactions. We sought to test whether schizophrenia-related genotypes and environmental risk factors predict membership in classes defined by taxometric analyses of positive (cognitive-perceptual), negative (interpersonal), and disorganized schizotypy.
METHODS:Participants (n = 500) completed the Schizotypal Personality Questionnaire (SPQ) and provided information on the following risk factors: cannabis use, pregnancy and obstetric complications, social adjustment, and family history of psychosis. Saliva samples were obtained so that the frequency of single-nucleotide polymorphism (SNP) alleles associated with risk for developing schizophrenia could be determined. Genotyped SNPs were rs1625579 (MIR137), rs7004633 (MMP16), rs7914558 (CNNM2), and rs12966547 (CCDC68). Sets of SPQ items were subject to multiple coherent cut kinetic (CCK) analyses, including mean-above-minus-below-a-cut, maximum covariance, maximum eigenvalue, and latent modes analyses.
RESULTS:CCK analyses indicated latent taxonicity of schizotypy across the 3 item sets. The cognitive-perceptual class had a base rate of 25%, and membership was predicted by the rs7004633 SNP (odds ratio = 2.33, 95% confidence interval = 1.15-4.72 in adjusted analyses). Poor social adjustment predicted memberships in the interpersonal (16%) and disorganized (21%) classes. Classes were found not to be mutually exclusive.
CONCLUSIONS:Schizotypy is taxonic and schizotypy class membership is predicted by genetic and environmental factors that predict schizophrenia. The findings hold the promise that a more complete understanding of schizotypy as a schizophrenia liability state will come from investigation of other genes and environmental factors associated with schizophrenia.
Enzymes that utilize the cofactor pyridoxal 5'-phosphate play essential roles in amino acid metabolism in all organisms. The cofactor is used by proteins that adopt at least five different folds, which raises questions about the evolutionary processes that might explain the observed distribution of functions among folds. In this study, we show that a representative of fold type III, the Escherichia coli alanine racemase (ALR), is a promiscuous cystathionine β-lyase (CBL). Furthermore, E. coli CBL (fold type I) is a promiscuous alanine racemase. A single round of error-prone PCR and selection yielded variant ALR(Y274F), which catalyzes cystathionine β-elimination with a near-native Michaelis constant (Km = 3.3 mm) but a poor turnover number (kcat ≈10 h(-1)). In contrast, directed evolution also yielded CBL(P113S), which catalyzes l-alanine racemization with a poor Km (58 mm) but a high kcat (22 s(-1)). The structures of both variants were solved in the presence and absence of the l-alanine analogue, (R)-1-aminoethylphosphonic acid. As expected, the ALR active site was enlarged by the Y274F substitution, allowing better access for cystathionine. More surprisingly, the favorable kinetic parameters of CBL(P113S) appear to result from optimizing the pKa of Tyr-111, which acts as the catalytic acid during l-alanine racemization. Our data emphasize the short mutational routes between the functions of pyridoxal 5'-phosphate-dependent enzymes, regardless of whether or not they share the same fold. Thus, they confound the prevailing model of enzyme evolution, which predicts that overlapping patterns of promiscuity result from sharing a common multifunctional ancestor.
The mechanisms by which the fetus induces maternal physiological adaptations to pregnancy are unclear. Cellular debris, shed from the placental syncytiotrophoblast into the maternal blood and phagocytosed by maternal endothelial and immune cells, may be one of these mechanisms. Here we show that trophoblastic debris from normal first trimester placentae induces changes in the transcriptome and proteome of endothelial cells in vitro, which might contribute to the adaptation of the maternal cardiovascular system to pregnancy. Trophoblastic debris also induced endothelial cells to transcribe placenta-specific genes, including the vasodilator hormone CSH1, thereby expanding the effective functional size of the placenta. Our data suggest that the deportation of trophoblastic debris is an important part of the complex network of feto-maternal communication.
Biogenesis of the photosystems in oxygenic phototrophs requires co-translational insertion of chlorophyll a. The first committed step of chlorophyll a biosynthesis is the insertion of a Mg2+ ion into the tetrapyrrole intermediate protoporphyrin IX, catalyzed by Mg-chelatase. We have identified a Synechocystis sp. PCC 6803 strain with a spontaneous mutation in chlH that results in a Gly195 to Glu substitution in a conserved region of the catalytic subunit of Mg-chelatase. Mutant strains containing the ChlH Gly195 to Glu mutation were generated using a two-step protocol that introduced the chlH gene into a putative neutral site in the chromosome prior to deletion of the native gene. The Gly195 to Glu mutation resulted in strains with decreased chlorophyll a. Deletion of the PS II assembly factor Ycf48 in a strain carrying the ChlH Gly195 to Glu mutation did not grow photoautotrophically. In addition, the ChlH-G195E:ΔYcf48 strain showed impaired PS II activity and decreased assembly of PS II centers in comparison to a ΔYcf48 strain. We suggest decreased chlorophyll in the ChlH-G195E mutant provides a background to screen for the role of assembly factors that are not essential under optimal growth conditions.
This study reports a procedure for the simultaneous purification of glyco(caseino)macropeptide, immunoglobulin, lactoperoxidase, lactoferrin, α-lactalbumin and β-lactoglobulin from sheep cheese sweet whey, an under-utilized by-product of cheese manufacture generated by an emerging sheep dairy industry in New Zealand. These proteins have recognized value in the nutrition, biomedical and health-promoting supplements industries. A sequential fractionation procedure using economical anion and cation exchange chromatography on HiTrap resins was evaluated. The whey protein fractionation is performed under mild conditions, requires only the adjustment of pH between ion exchange chromatography steps, does not require buffer exchange and uses minimal amounts of chemicals. The purity of the whey protein fractions generated were analyzed by reversed phase-high performance liquid chromatography and the identity of the proteins was confirmed by mass spectrometry. This scalable procedure demonstrates that several proteins of recognized value can be fractionated in reasonable yield and purity from sheep cheese whey in one streamlined process.
Hemp protein isolates (HPIs) were hydrolysed by proteases (AFP, HT, ProG, actinidin and zingibain). The enzymatic hydrolysis of HPIs was evaluated through the degree of hydrolysis and SDS-PAGE profiles. The bioactive properties of the resultant hydrolysates (HPHs) were accessed through ORAC, DPPḢ scavenging and ACE-inhibitory activities. The physical properties of the resultant HPHs were evaluated for their particle sizes, zeta potential and surface hydrophobicity. HT had the highest rate of caseinolytic activity at the lowest concentration (0.1mgmL(-1)) compared to other proteases that required concentration of 100mgmL(-1) to achieve their maximum rate of caseinolytic activity. This led to the highest degree of hydrolysis of HPIs by HT in the SDS-PAGE profiles. Among all proteases and substrates, HT resulted in the highest bioactivities (ORAC, DPPḢ scavenging and ACE-inhibitory activities) generated from alkali extracted HPI in the shortest time (2h) compared to the other protease preparations.
Establishing causality of risk factors is important to determine the pathogenetic mechanisms underlying rheumatic diseases, and can facilitate the design of interventions to improve care for affected patients. The presence of unmeasured confounders, as well as reverse causation, is a challenge to the assignment of causality in observational studies. Alleles for genetic variants are randomly inherited at meiosis. Mendelian randomization analysis uses these genetic variants to test whether a particular risk factor is causal for a disease outcome. In this Review of the Mendelian randomization technique, we discuss published results and potential applications in rheumatology, as well as the general clinical utility and limitations of the approach.
Protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources were used to hydrolyze the red blood cell fractions (RBCFs) separated from deer, sheep, pig, and cattle abattoir-sourced blood. After 1, 2, 4 and 24 h of hydrolysis, the antioxidant and antibacterial activities of the peptide hydrolysates obtained were investigated. The increase in trichloroacetic acid-soluble peptides over the hydrolysis period was examined using the o-phthaldialdehyde (OPA) assay and the hydrolysis profiles were illustrated using SDS-PAGE. Papain generated RBCF hydrolysates exhibited higher ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) compared to those generated with bromelain, FP400 and FPII. At certain concentrations, 24 h hydrolysates of RBCF using FP400 and FPII were able to inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The results indicated that the use of proteases from plant or fungal sources can produce animal blood hydrolysates with antioxidant and antimicrobial activities.
Senescent cancer-associated fibroblasts (CAF) develop a senescence-associated secretory phenotype (SASP) that is believed to contribute to cancer progression. The mechanisms underlying SASP development are, however, poorly understood. Here we examined the functional role of microRNA in the development of the SASP in normal fibroblasts and CAF. We identified a microRNA, miR-335, up-regulated in the senescent normal fibroblasts and CAF and able to modulate the secretion of SASP factors and induce cancer cell motility in co-cultures, at least in part by suppressing the expression of phosphatase and tensin homologue (PTEN). Additionally, elevated levels of cyclo-oxygenase 2 (PTGS2; COX-2) and prostaglandin E2 (PGE2) secretion were observed in senescent fibroblasts, and inhibition of COX-2 by celecoxib reduced the expression of miR-335, restored PTEN expression and decreased the pro-tumourigenic effects of the SASP. Collectively these data demonstrate the existence of a novel miRNA/PTEN-regulated pathway modulating the inflammasome in senescent fibroblasts.