Defective monocyte plasticity and altered cAMP pathway characterize USB1-mutated poikiloderma with neutropenia Clericuzio type.

Poikiloderma with neutropenia (PN) Clericuzio type (OMIM #604173) is a rare disease with areas of skin hyper- and hypopigmentation caused by biallelic USB1 variants. The current study was spurred by poor healing of a perianal tear wound in one affected child homozygous for c.266-1G>A (p.E90Sfster8) mutation, from a family reported previously. Treatment with G-CSF/CSF3 or GM-CSF/CSF2 transiently increased neutrophil/monocytes count with no effect on wound healing. Analysis of peripheral blood revealed a lack of non-classical (CD14+/- CD16+ ) monocytes, associated with a systemic inflammatory cytokine profile, in the two affected brothers. Importantly, despite normal expression of cognate receptors, monocytes from PN patients did not respond to M-CSF or IL-34 in vitro, as determined by cytokine secretion or CD16 expression. RNAseq of monocytes showed 293 differentially expressed genes, including significant downregulation of GATA2, AKAP6 and PDE4DIP that are associated with leucocyte differentiation and cyclic adenosine monophosphate (cAMP) signalling. Notably, the plasma cAMP was significantly low in the PN patients. Our study revealed a novel association of PN with a lack of non-classical monocyte population. The defects in monocyte plasticity may contribute to disease manifestations in PN and a defective cAMP signalling may be the primary effect of the splicing errors caused by USB1 mutation.

Electrophoretic mobility of individual molecules of alkaline phosphatase.

The electrophoretic mobilities and catalytic rates of individual molecules of bovine intestinal alkaline phosphatase were determined in CHES and borate buffers of identical pH using a capillary electrophoresis based method. Both properties were found to be heterogeneous. In the presence of CHES, the mobility and rate were found to be -1.9 ± 0.2 × 10-9 m2 V-1 s-1 and 9.8 ± 7.4 × 104 min-1 (N = 38), respectively. In the presence of borate, the mobility and rate were found to be -6.9 ± 0.5 × 10-9 m2 V-1 s-1 and 2.0 ± 1.3 × 104 min-1 (N = 41), respectively. The means and variances for both properties were found to differ significantly between the two buffers. The difference in average mobility was attributed to an increase in negative charge caused by borate complexing with the carbohydrate moieties attached to the enzyme. The difference in variance was attributed to heterogeneous complexation with borate due to heterogeneity in the glycosylation. The differences in mean values for the catalytic rate were attributed to the inhibitory effect of borate and the difference in variance may suggest that the KI of this binding may also be heterogeneous.

Citrate analysis using capillary electrophoresis and complexation with Eu3+-tetracycline.

A sensitive assay for citrate was developed. Citrate was incubated with 50 µM Eu3+-tetracycline and the complex separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. Signal was linear with citrate concentration from 10 µM to 200 nM. Injection volumes were 320 pL. For the 200 nM sample, this corresponds to the injection of 64 amoles of citrate. Separation time was < 90 s with a total run time of 5 min. As an application the method was used to analyze citrate in agricultural and medicinal products. The method was also used to develop an assay for the enzyme citrate synthase.

Analysis of complexes of metabolites with europium tetracycline using capillary electrophoresis coupled with laser-induced luminescence detection.

Glycolysis and Krebs cycle intermediates were incubated with Eu3+-tetracycline and separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. 3-phopshoglycerate, phosphoenolpyruvate, adenosine diphosphate, phosphate, citrate and oxaloacetate were detected at a concentration of 100 µM or lower. When all these detected metabolites were contained within the same sample it was found that they interfered with one another. Of all the metabolites, oxaloacetate showed the highest detectability. The system was found to yield a linear response for oxaloacetate from 50 nM to 10 µM. The injected volume of sample was 400 pL. This corresponds to 2 × 10-17 mol of injected oxaloacetate from the 50 nM sample. As an application, the system was used to assay the enzyme aspartate aminotransferase, for whom oxaloacetate is a product. After a 1 h incubation period, 1.2 × 10-13 M (3.3 µU/mL) enzyme was sufficient to form a detectable product signal. Extension of this incubation to 18 h permitted the detection of the activity of 1.2 × 10-14 M (330 nU/mL) enzyme. This is the equivalent of 4.8 ymoles (2.9 molecules) of enzyme in the 400 pL injection volume. The enzyme's catalytic rate was determined to be 240 s-1 under the conditions used. In a second application, homogenates of Drosophila melanogaster were analyzed for metabolites, providing several peaks, including one which had the same retention time as citrate.

Determination of the inhibitor dissociation constant of an individual unmodified enzyme molecule in free solution.

Single enzyme molecule assays on E. coli ß-galactosidase were performed using a capillary electrophoresis-based method. Three types of assays were performed. The catalytic rate of 20 individual molecules was assayed in duplicate in the presence of 50 µM substrate. The ratio of rates for the second incubation relative to the first was 0.96 ± 0.03, showing the reproducibility of the method. In the second assay, the rates were determined in the absence and presence of 210 µM L-ribose, a competitive inhibitor. The ratio of the rate in the presence of inhibitor to that in its absence for 19 individual molecules was 0.44 ± 0.23. This large relative standard deviation suggests that each individual enzyme molecule was affected to a different extent by the presence of the inhibitor, which is consistent with KI being heterogeneous. To estimate KI for individual molecules, a third assay was performed. Each molecule was incubated in the presence of 30 and 50 µM substrate and then in the presence of 50 µM substrate plus 210 µM inhibitor. Comparison of the rates in the two substrate concentrations allowed for the determination of the individual Km of each molecule. From this value and the difference in rates in the presence and absence of inhibitor, the individual molecule KI values were determined. This value was found to differ between individual molecules and was found to increase with an increase in Km . Modeling showed that a heterogeneity in KI results in an alteration in the Michaelis-Menten curve for a population of enzymes in the presence of a competitive inhibitor.

Conformational change in individual enzyme molecules.

Single ß-galactosidase molecule assays were performed using a capillary electrophoresis based protocol, employing post-column laser-induced fluorescence detection. In a first set of experiments, the distribution of single ß-galactosidase molecule catalytic rates and electrophoretic mobilities were determined from lysates of Escherichia coli strains containing deletions for different heat shock proteins and grown under normal and heat shock conditions. There was no clear observed pattern of effect of heat shock protein expression on these distributions. In a second set of experiments, individual enzyme molecule catalytic rates were determined at 21 °C before and after 2 sequential brief periods of incubation at 50, 28, and 10 °C. The brief incubations at 50 °C caused a change in the enzyme molecules resulting in a different catalytic rate. Any given molecule was just as likely to show an increase in rate as a decrease, resulting in no significant difference in the average rate of the population. The average change in individual molecule rate was dependent upon the temperature of the brief incubation period, with a lesser average change occurring at 28 °C and negligible change at 10 °C. A third set of experiments was similar to that of the second with the exception that it was electrophoretic mobility that was considered. This provided a similar result. Incubation at higher temperature resulted in a change in electrophoretic mobility. The probability of an individual molecules switching to a higher mobility was approximately equal to that of switching to a lower mobility, resulting in no net average change in the population. The magnitude of the changes in electrophoretic mobilities suggest that the associated conformational changes are subtle.

Comparison of the single molecule activity distributions of recombinant and non-recombinant bovine intestinal alkaline phosphatase.

Single molecule assays were performed on bovine intestinal alkaline phosphatase and the recombinant enzyme expressed in Pichia pastoris using a capillary electrophoresis-based method. The catalytic rates for the bovine and recombinant enzymes were found to be 11,000±7000min(-1) (N=161) and 12,000±7000min(-1) (N=173), respectively. Mean catalytic rates and variances did not differ significantly between the enzyme from both sources. Furthermore, the distribution of catalytic rates were indistinguishable.

12-Channel Peltier array temperature control unit for single molecule enzymology studies using capillary electrophoresis.

Capillary electrophoresis has been used to demonstrate that individual molecules of a given enzyme support different catalytic rates. In order to determine how rate varies with temperature, and determine activation energies for individual ß-galactosidase molecules, a 12-channel Peltier array temperature control device was constructed where the temperature of each cell was separately controlled. This array was used to control the temperature of the central 30 cm of a 50 cm long capillary, producing a temperature gradient along its length. Continuous flow single ß-galactosidase molecule assays were performed allowing measurement of the catalytic rates at different temperatures. Arrhenius plots were produced and the distribution of activation energies for individual ß-galactosidase molecules was found to be 56 ± 10 kJ/mol with a range of 34-72 kJ/mol.

Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins.

BACKGROUND: Disulfide engineering is an important biotechnological tool that has advanced a wide range of research. The introduction of novel disulfide bonds into proteins has been used extensively to improve protein stability, modify functional characteristics, and to assist in the study of protein dynamics. Successful use of this technology is greatly enhanced by software that can predict pairs of residues that will likely form a disulfide bond if mutated to cysteines. RESULTS: We had previously developed and distributed software for this purpose: Disulfide by Design (DbD). The original DbD program has been widely used; however, it has a number of limitations including a Windows platform dependency. Here, we introduce Disulfide by Design 2.0 (DbD2), a web-based, platform-independent application that significantly extends functionality, visualization, and analysis capabilities beyond the original program. Among the enhancements to the software is the ability to analyze the B-factor of protein regions involved in predicted disulfide bonds. Importantly, this feature facilitates the identification of potential disulfides that are not only likely to form but are also expected to provide improved thermal stability to the protein. CONCLUSIONS: DbD2 provides platform-independent access and significantly extends the original functionality of DbD. A web server hosting DbD2 is provided at http://cptweb.cpt.wayne.edu/DbD2/.

A novel approach for predicting upstream regulators (PURE) that affect gene expression.

External factors such as exposure to a chemical, drug, or toxicant (CDT), or conversely, the lack of certain chemicals can cause many diseases. The ability to identify such causal CDTs based on changes in the gene expression profile is extremely important in many studies. Furthermore, the ability to correctly infer CDTs that can revert the gene expression changes induced by a given disease phenotype is a crucial step in drug repurposing. We present an approach for Predicting Upstream REgulators (PURE) designed to tackle this challenge. PURE can correctly infer a CDT from the measured expression changes in a given phenotype, as well as correctly identify drugs that could revert disease-induced gene expression changes. We compared the proposed approach with four classical approaches as well as with the causal analysis used in Ingenuity Pathway Analysis (IPA) on 16 data sets (1 rat, 5 mouse, and 10 human data sets), involving 8 chemicals or drugs. We assessed the results based on the ability to correctly identify the CDT as indicated by its rank. We also considered the number of false positives, i.e. CDTs other than the correct CDT that were reported to be significant by each method. The proposed approach performed best in 11 out of the 16 experiments, reporting the correct CDT at the very top 7 times. IPA was the second best, reporting the correct CDT at the top 5 times, but was unable to identify the correct CDT at all in 5 out of the 16 experiments. The validation results showed that our approach, PURE, outperformed some of the most popular methods in the field. PURE could effectively infer the true CDTs responsible for the observed gene expression changes and could also be useful in drug repurposing applications.

Immunological modifications following chemotherapy are associated with delayed recurrence of ovarian cancer.

Introduction: Ovarian cancer recurs in most High Grade Serous Ovarian Cancer (HGSOC) patients, including initial responders, after standard of care. To improve patient survival, we need to identify and understand the factors contributing to early or late recurrence and therapeutically target these mechanisms. We hypothesized that in HGSOC, the response to chemotherapy is associated with a specific gene expression signature determined by the tumor microenvironment. In this study, we sought to determine the differences in gene expression and the tumor immune microenvironment between patients who show early recurrence (within 6 months) compared to those who show late recurrence following chemotherapy. Methods: Paired tumor samples were obtained before and after Carboplatin and Taxol chemotherapy from 24 patients with HGSOC. Bioinformatic transcriptomic analysis was performed on the tumor samples to determine the gene expression signature associated with differences in recurrence pattern. Gene Ontology and Pathway analysis was performed using AdvaitaBio's iPathwayGuide software. Tumor immune cell fractions were imputed using CIBERSORTx. Results were compared between late recurrence and early recurrence patients, and between paired pre-chemotherapy and post-chemotherapy samples. Results: There was no statistically significant difference between early recurrence or late recurrence ovarian tumors pre-chemotherapy. However, chemotherapy induced significant immunological changes in tumors from late recurrence patients but had no impact on tumors from early recurrence patients. The key immunological change induced by chemotherapy in late recurrence patients was the reversal of pro-tumor immune signature. Discussion: We report for the first time, the association between immunological modifications in response to chemotherapy and the time of recurrence. Our findings provide novel opportunities to ultimately improve ovarian cancer patient survival.

Augmented annotation and orthologue analysis for Oryctolagus cuniculus: Better Bunny.

BACKGROUND: The rabbit is an important model organism used in a wide range of biomedical research. However, the rabbit genome is still sparsely annotated, thus prohibiting extensive functional analysis of gene sets derived from whole-genome experiments. We developed a web-based application that provides augmented annotation and orthologue analysis for rabbit genes. Importantly, the application allows comprehensive functional analysis through the use of orthologous relationships. RESULTS: Using data extracted from several public bioinformatics repositories we created Better Bunny, a database and query tool that extensively augments the available functional annotation for rabbit genes. Using the complete set of target genes from a commercial rabbit gene expression microarray as our benchmark, we are able to obtain functional information for 88 % of the genes on the microarray. Previously, functional information was available for fewer than 10 % of the rabbit genes. CONCLUSIONS: We have developed a freely available, web-accessible bioinformatics tool that enables investigators to quickly and easily perform extensive functional analysis of rabbit genes (http://cptweb.cpt.wayne.edu). The software application fills a critical void for a wide range of biomedical research that relies on the rabbit model and requires characterization of biological function for large sets of genes.

Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of ß-galactosidase.

Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional ß-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized ß-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.

Arrhenius plot for a reaction catalyzed by a single molecule of ß-galactosidase.

The activity of a single enzyme molecule of Escherichia coli ß-galactosidase was measured using a capillary electrophoresis continuous flow assay. As the enzyme molecule traversed the capillary the incubation temperature was increased from 27 to 37 °C, providing a continuous record of the change in rate with temperature. This data was used to develop a single enzyme molecule Arrhenius plot, from which the activation energy of the reaction was determined to be 31 kJ mol(-1).

Measurement of the activity of individual subunits of single molecules of the tetrameric enzyme ß-galactosidase.

Escherichia coli ß-galactosidase was incubated in the presence of the slow-release inhibitor D-galactal for 30 min at a concentration of 70 times its K(i). The sample was then diluted 20000-fold into buffer containing the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-on-7-yl) ß-D-galactoside, reducing the inhibitor concentration to K(i)/280. The sample was subjected to a capillary electrophoresis continuous flow single enzyme molecule assay. As the inhibitor dissociated while the enzyme traveled the length of the capillary, a fraction of molecules showed stepwise increases in activity. This was due to the activation of individual subunits within single molecules. The changes in activity can be largely explained in terms of each molecule containing subunits of indistinguishable activity.

Analysis of cyanide using fluorogenic derivatization and capillary electrophoresis.

Samples containing cyanide were incubated at 85 °C in the presence of the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and glutamic acid, and analyzed by capillary electrophoresis utilizing post-separation laser-induced fluorescence detection in a sheath flow cuvette. The separation time on a 25 cm long capillary at 800 Vcm-1 was 3 min with the fluorescent product eluting at 107 s. Flushing of the capillary was not required between runs. Signal was proportional with cyanide concentration from 50 nM to 1.5 µM. LOD and LOQ were determined to be 26 and 87 nM respectively. As an application, free cyanide in five individual apple seeds was measured and found to range from 12 to 86 ng/mg, with a mean of 55 ± 32 ng/mg. As a means for the detection of amygdalin, cyanide was enzymatically produced from amygdalin using the enzymes ß-glucosidase and mandelonitrile lyase. The cyanide was then reacted with FQ and injected onto the capillary. Amygdalin was detected at a concentration of 1 µM.

FOXQ1 recruits the MLL complex to activate transcription of EMT and promote breast cancer metastasis.

Aberrant expression of the Forkhead box transcription factor, FOXQ1, is a prevalent mechanism of epithelial-mesenchymal transition (EMT) and metastasis in multiple carcinoma types. However, it remains unknown how FOXQ1 regulates gene expression. Here, we report that FOXQ1 initiates EMT by recruiting the MLL/KMT2 histone methyltransferase complex as a transcriptional coactivator. We first establish that FOXQ1 promoter recognition precedes MLL complex assembly and histone-3 lysine-4 trimethylation within the promoter regions of critical genes in the EMT program. Mechanistically, we identify that the Forkhead box in FOXQ1 functions as a transactivation domain directly binding the MLL core complex subunit RbBP5 without interrupting FOXQ1 DNA binding activity. Moreover, genetic disruption of the FOXQ1-RbBP5 interaction or pharmacologic targeting of KMT2/MLL recruitment inhibits FOXQ1-dependent gene expression, EMT, and in vivo tumor progression. Our study suggests that targeting the FOXQ1-MLL epigenetic axis could be a promising strategy to combat triple-negative breast cancer metastatic progression.

Discovery of primary prostate cancer biomarkers using cross cancer learning.

Prostate cancer (PCa), the second leading cause of cancer death in American men, is a relatively slow-growing malignancy with multiple early treatment options. Yet, a significant number of low-risk PCa patients are over-diagnosed and over-treated with significant and long-term quality of life effects. Further, there is ever increasing evidence of metastasis and higher mortality when hormone-sensitive or castration-resistant PCa tumors are treated indistinctively. Hence, the critical need is to discover clinically-relevant and actionable PCa biomarkers by better understanding the biology of PCa. In this paper, we have discovered novel biomarkers of PCa tumors through cross-cancer learning by leveraging the pathological and molecular similarities in the DNA repair pathways of ovarian, prostate, and breast cancer tumors. Cross-cancer disease learning enriches the study population and identifies genetic/phenotypic commonalities that are important across diseases with pathological and molecular similarities. Our results show that ADIRF, SLC2A5, C3orf86, HSPA1B are among the most significant PCa biomarkers, while MTRNR2L1, EEPD1, TEPP and VN1R2 are jointly important biomarkers across prostate, breast and ovarian cancers. Our validation results have further shown that the discovered biomarkers can predict the disease state better than any randomly selected subset of differentially expressed prostate cancer genes.

Kinetic studies of unmodified individual Escherichia coli beta-galactosidase molecules in free solution.

Assays were performed on individual Escherichia coli beta-galactosidase molecules at 2 different concentrations of the substrate DDAO-beta-D-galactoside using a free zone capillary electrophoresis-based protocol with an in-laboratory-constructed instrument utilizing laser-induced fluorescence detection. In a typical run, 2 enzyme molecules were injected into the capillary. They were separated from each other by a brief period of electrophoresis and incubated on the capillary in the presence of the substrate. They were then mobilized on the capillary into a zone of substrate at a different concentration, re-incubated, and the product peaks mobilized past the detector . The relative change in activity as the concentration was increased differed between molecules, suggesting differences in Km. In a different experiment, the capillary was filled with on average 13 enzyme molecules per run, incubated, and the activities of the individual molecules determined. The shapes of the distribution curves of single molecule activities obtained at different concentrations of the substrate resorufin-beta-D-galactoside were indistinguishable, suggesting a homogeneous Km. To explain why individual enzyme molecules behaved as if they were heterogeneous with respect to Km but the population behaved as if it were homogeneous, theoretical Michaelis-Menten curves were constructed. The curves for populations with heterogeneous Km values were found to be indistinguishable from that of a homogeneous population.

Heritable Susceptibility to Breast Cancer among African-American Women in the Detroit Research on Cancer Survivors Study.

BACKGROUND: African-American women have high rates of breast cancer associated with hereditary features. However, no studies have reported the prevalence of inherited variation across all genes known to be breast cancer risk factors among African-American patients with breast cancer not selected for high-risk characteristics. METHODS: We evaluated 182 African-American women diagnosed with invasive breast cancer in metropolitan Detroit via targeted capture and multiplex sequencing of 13 well-established breast cancer risk genes and five suggested breast cancer risk genes. RESULTS: We identified 24 pathogenic variants in 23 women [12.6%; 95% confidence interval (CI), 8.2%-18.4%] and five genes (BRCA2, BRCA1, ATM, RAD50, CDH1). BRCA1 and BRCA2 accounted for 58.3% of all pathogenic variants. An additional six pathogenic variants were found in suggested breast cancer risk genes (MSH6, MUTYH, NF1, BRIP1). CONCLUSIONS: The prevalence of germline pathogenic variants is relatively high among African-American patients with breast cancer unselected for high-risk characteristics across a broad spectrum of genes. IMPACT: This study helps to define the genomic landscape of breast cancer susceptibility in African-American women who could benefit from enhanced surveillance and screening.