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.

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.

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.

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.

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.

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.

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.

The extrema of circulating miR-17 are identified as biomarkers for aggressive prostate cancer.

MicroRNAs (miRNAs) constitute short non-coding RNAs that can post-transcriptionally modulate the expression of many oncogenes and tumor suppressor genes engaged in key cellular processes. Deregulated serum miRNA signatures have been detected in various solid cancers including prostate cancer, suggesting that circulating miRNAs could function as non-invasive biomarkers of tumor emergence and progression. To determine whether serum miRNA expression levels are different between patients with aggressive and non-aggressive prostate cancer, we analyzed a panel of miRNAs from the blood of African American (AA) prostate cancer patients using a new recursive partitioning method that allows hypothesis testing of each split. We observed that both extrema of circulating miR-17, i.e. upregulation and downregulation, are associated with aggressive prostate cancer. A similar effect was observed in tumor samples from a separate dataset representing a different population of prostate cancer patients and in AA prostate cancer samples from the TCGA. The dual effect is consistent with the contradictory findings on the role of miR-17 in prostate cancer progression, whereby it controls important oncogenic and tumor-suppressive genes.

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.

Cooperative oncogenic effect and cell signaling crosstalk of co­occurring HER2 and mutant PIK3CA in mammary epithelial cells.

Though incidence of PI3K oncogenic mutation is prominent in breast cancer (20-30%), pharmacological targeting of this signaling pathway alone has failed to provide meaningful clinical benefit. To better understand and address this problem, we conducted genome-wide analysis to study the association of mutant PI3K with other gene amplification events. One of the most significant copy number gain events associated with PIK3CA mutation was the region within chromosome 17 containing HER2. To investigate the oncogenic effect and cell signaling regulation of co-occurring PIK3CA-H1047R and or HER2 gene, we generated cell models ectopically expressing mutant PIK3CA, HER2 or both genetic alterations. We observed that cells with both genetic alterations demonstrate increased aggressiveness and invasive capabilities than cells with either genetic change alone. Furthermore, we found that the combination of the HER2 inhibitor (CP-724714) and pan PI3K inhibitor (LY294002) is more potent than either inhibitor alone in terms of inhibition of cell proliferation and colony formation. Significantly, four cell signaling pathways were found in common for cells with HER2, mutant PIK3CA and cells with both genetic alterations through an Affymetric microarray analysis. Moreover, the cells with both genetic alterations acquired more significant replication stress as shown by enriched signaling pathways of cell cycle checkpoint control and DNA damage response signaling. Our study suggests co-occurrence of oncogenic HER2 and mutant PIK3CA cooperatively drives breast cancer progression. The cells with both genetic alterations obtain additional features of replication stress which could open new opportunity for cancer diagnostics and treatment.

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.

Electrophoretic mobility, catalytic rate, and activation energy of catalysis of single molecules of the enzyme ß-glucuronidase from Escherichia coli.

Single molecule assays were performed on the enzyme E. coli ß-glucuronidase using a capillary electrophoresis-based protocol. Electrophoretic mobility, catalytic rate and activation energy of catalysis were all found to be heterogeneous. The average mobility at 22°C was -1.1×10-8±0.1m2V-1s-1 (N=49) with a total range of -0.6 to -1.3×10-8m2V-1s-1. The range in electrophoretic mobility suggests that the differences in shape or charge of the individual molecules underlying the heterogeneity are likely minimal. The average catalytic rate at 22°C was 37,000±19,000min-1 (N=49) with a total range of 14,000 to 130,000min-1. Both of these properties were measured simultaneously for each of the molecules. There was a weak correlation (r2=0.43) between mobility and rate with the molecules with a less negative mobility having a tendency to have a higher rate. The average activation energy of catalysis, as determined by comparing rates at 22 and 35°C, was found to be 48±18kJmol-1 (N=7) with a total range of 18-66kJmol-1.

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.

Protein disulfide engineering.

Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

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/.

Electrophoretic heterogeneity limits the utility of streptavidin-ß-galactosidase as a probe in free zone capillary electrophoresis separations.

Single molecule assays were performed on streptavidin-ß-galactosidase using a capillary electrophoresis-based protocol in order to assess the suitability of single molecule ß-galactosidase assays for adaptation to the detection of single copies of target DNA. The conjugate was found to have a heterogeneous catalytic rate, showing an average rate of 44,000 ± 24,000 min(-1), which is similar to that of the unmodified enzyme. Electrophoretic mobility was also measured on individual molecules and determined to be -1.32 × 10(-4) ± 0.19 × 10(-4) cm(2)V(-1)s(-1). The variance in mobility was several times that reported for the unmodified enzyme. The electrophoretic heterogeneity was found to result in the formation of a broad window of peaks in the resultant electropherograms of free zone separations of small plugs of streptavidin-ß-galactosidase. This range of mobilities largely overlapped with that of the conjugate bound to primer and plasmid containing a target DNA sequence. This overlap suggests that the separation of free conjugate from that bound to target DNA, which is a requirement for application of the single enzyme molecule assay to the detection of target DNA sequences, is not plausible using free zone capillary electrophoresis.