The Mechanistic Basis of Myxococcus xanthus Rippling Behavior and Its Physiological Role during Predation

Myxococcus xanthus cells self-organize into periodic bands of traveling waves, termed ripples, during multicellular fruiting body development and predation on other bacteria. To investigate the mechanistic basis of rippling behavior and its physiological role during predation by this Gram-negative soil bacterium, we have used an approach that combines mathematical modeling with experimental observations. Specifically, we developed an agent-based model (ABM) to simulate rippling behavior that employs a new signaling mechanism to trigger cellular reversals. The ABM has demonstrated that three ingredients are sufficient to generate rippling behavior: (i) side-to-side signaling between two cells that causes one of the cells to reverse, (ii) a minimal refractory time period after each reversal during which cells cannot reverse again, and (iii) physical interactions that cause the cells to locally align. To explain why rippling behavior appears as a consequence of the presence of prey, we postulate that prey-associated macromolecules indirectly induce ripples by stimulating side-toside contact-mediated signaling. In parallel to the simulations, M. xanthus predatory rippling behavior was experimentally observed and analyzed using time-lapse microscopy. A formalized relationship between the wavelength, reversal time, and cell velocity has been predicted by the simulations and confirmed by the experimental data. Furthermore, the results suggest that the physiological role of rippling behavior during M. xanthus predation is to increase the rate of spreading over prey cells due to increased side-to-side contact-mediated signaling and to allow predatory cells to remain on the prey longer as a result of more periodic cell motility

Pathways of Resistance to Thymineless Death in Escherichia coli and the Function of UvrD

Thymineless death (TLD) is the rapid loss of viability in bacterial, yeast, and human cells starved of thymine. TLD is the mode of action of common anticancer drugs and some antibiotics. TLD in Escherichia coli is accompanied by blocked replication and chromosomal DNA loss and recent work identified activities of recombination protein RecA and the SOS DNA-damage response as causes of TLD. Here, we examine the basis of hypersensitivity to thymine deprivation (hyper-TLD) in mutants that lack the UvrD helicase, which opposes RecA action and participates in some DNA repair mechanisms, RecBCD exonuclease, which degrades double-stranded linear DNA and works with RecA in double-strand-break repair and SOS induction, and RuvABC Holliday-junction resolvase. We report that hyper-TLD in ∆uvrD cells is partly RecA dependent and cannot be attributed to accumulation of intermediates in mismatch repair or nucleotide-excision repair. These data imply that both its known role in opposing RecA and an additional as-yet-unknown function of UvrD promote TLD resistance. The hyper-TLD of ∆ruvABC cells requires RecA but not RecQ or RecJ. The hyper-TLD of recB cells requires neither RecA nor RecQ, implying that neither recombination nor SOS induction causes hyper-TLD in recB cells, and RecQ is not the sole source of double-strand ends (DSEs) during TLD, as previously proposed; models are suggested. These results define pathways by which cells resist TLD and suggest strategies for combating TLD resistance during chemotherapies

A family-based study of gene variants and maternal folate and choline in neuroblastoma: a report from the Children’s Oncology Group

Neuroblastoma is a childhood cancer of the sympathetic nervous system with embryonic origins. Previous epidemiologic studies suggest maternal vitamin supplementation during pregnancy reduces the risk of neuroblastoma. We hypothesized offspring and maternal genetic variants in folate-related and choline-related genes are associated with neuroblastoma and modify the effects of maternal intake of folate, choline and folic acid

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

A G316A polymorphism in the ornithine decarboxylase gene promoter modulates MYCN-driven childhood neuroblastoma

Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein

A Modified LC/MS/MS Method with Enhanced Sensitivity for the Determination of Scopolamine in Human Plasma

Intranasal scopolamine is a choice drug for the treatment of motion sickness during space flight because of its quick onset of action, short half-life and favorable sideeffects profile. The dose administered usually ranges between 0.1 and 0.4 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids using existing sensitive LC/MS/MS method, especially when the biological sample volumes are limited. To measure scopolamine in human plasma to facilitate pharmacokinetic evaluation of the drug, we developed a sensitive LC/MS/MS method using 96 well micro elution plates for solid phase extraction (SPE) of scopolamine in human plasma. Human plasma (100-250 micro L) were loaded onto Waters Oasis HLB 96 well micro elution plate and eluted with 50 L of organic solvent without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 3 minutes. The mobile phase for separation was 80:20 (v/v) methanol: ammonium acetate (30 mM) in water. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 right arrow 138.1 and internal standard hyoscyamine m/z = 290.2 right arrow 124.1. The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at about 1.1 and 1.7 min respectively. The linear range is 25-10000 pg/mL for scopolamine in human plasma with correlation coefficients greater than 0.99 and CV less than 0.5%. The intra-day and inter-day CVs are less than 15% for quality control samples with concentrations of 75,300, and 750 pg/mL of scopolamine in human plasma. SPE using 96 well micro elution plates allows rapid sample preparation and enhanced sensitivity for the LC/MS/MS determination of scopolamine in a small volume of biological samples. The new method is also cost effective since it uses a small volume of organic solvents compared to the methods using SPE cartridges or regular 96 well SPE plates. This method can be successfully used for bioavailability and pharmacokinetic evaluations of scopolamine, especially when volumes of biological samples are limited. Further investigation to use automated SPE system with 96 well micro elution plates is planned

Determination of Scopolamine in Human Saliva Using Solid Phase Extraction and LC/MS/MS

Purpose: Scopolamine is the preferred treatment for motion sickness during space flight because of its quick onset of action, short half-life and favorable side-effect profile. The dose administered depends on the mode of administration and usually ranges between 0.1 and 0.8 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids by using conventional HPLC methods. To measure scopolamine in saliva and thereby to evaluate the pharmacokinetics of scopolamine, we developed an LC/MS/MS method using off-line solid phase extraction. Method: Samples (0.5mL) were loaded onto Waters Oasis HLB co-polymer cartridges (10 mg, 1 mL) and eluted with 0.5 mL methanol without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 4 minutes. The mobile phase for separation was 90:10 (v/v) methanol: ammonium acetate (2 mM) in water, pH 5.0 +/- 0.1. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 yields 138.1 and internal standard (IS) hyoscyamine m/z = 290.2 yields 124.1. Results: The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at 1.7 and 3.2 min respectively. The linear range is 50-5000 pg/mL for scopolamine in saliva with correlation coefficients > 0.99 with a CV < 0.5 %. The intra-day and inter-day CVs are < 15 % for quality control samples with concentrations of 75, 300, 750 and 3000 pg/mL of scopolamine in human saliva. Conclusion: Solid phase extraction allows more rapid sample preparation and greater precision than liquid extraction. Furthermore, we increased the sensitivity and specificity by adjusting the LC mobile phase and using an MS/MS detector

Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies

<div><p>Microsatellites (MST), tandem repeats of 1–6 nucleotide motifs, are mutational hot-spots with a bias for insertions and deletions (INDELs) rather than single nucleotide polymorphisms (SNPs). The majority of MST instability studies are limited to a small number of loci, the Bethesda markers, which are only informative for a subset of colorectal cancers. In this paper we evaluate non-haplotype alleles present within next-gen sequencing data to evaluate somatic MST variation (SMV) within DNA repair proficient and DNA repair defective cell lines. We confirm that alleles present within next-gen data that do not contribute to the haplotype can be reliably quantified and utilized to evaluate the SMV without requiring comparisons of matched samples. We observed that SMV patterns found in DNA repair proficient cell lines without DNA repair defects, MCF10A, HEK293 and PD20 RV:D2, had consistent patterns among samples. Further, we were able to confirm that changes in SMV patterns in cell lines lacking functional BRCA2, FANCD2 and mismatch repair were consistent with the different pathways perturbed. Using this new exome sequencing analysis approach we show that DNA instability can be identified in a sample and that patterns of instability vary depending on the impaired DNA repair mechanism, and that genes harboring minor alleles are strongly associated with cancer pathways. The MST Minor Allele Caller used for this study is available at <a href="https://github.com/zalmanv/MST_minor_allele_caller" target="_blank">https://github.com/zalmanv/MST_minor_allele_caller</a>.</p></div

Relative Bioavailability of Scopolamine Dosage Forms and Interaction with Dextroamphetamine

The NASA Reduced Gravity Office (RGO) uses scopolamine (SCOP) and in combination with dextoamphetamine (DEX) to manage motion sickness symptoms during parabolic flights. The medications are dispensed as custom dosage forms as gelatin capsules. Anecdotal evidence of efficacy suggests that these formulations are unreliable and less efficacious for the treatment of motion sickness. We estimated bioavailability of four different oral formulations used by NASA for the treatment of motion sickness. Twelve healthy, non-smoking subjects between 21and 48 years of age received four treatments on separate days in a randomized fashion; the treatments were 0.8 mg SCOP alone as tablet, 0.8 mg SCOP alone in gel cap, 0.8 mg SCOP and 10 mg DEX as tablets, and 0.8 mg SCOP and 10 mg DEX in gel cap. After each treatment, blood, saliva, and urine samples were collected at scheduled time intervals for 24 h after dosing. Bioavailability and pharmacokinetic parameters were calculated and compared using ANOVA. After administration of SCOP tablets alone, maximum concentration (C(sub max)) and time for maximum concentration (t(sub max)) were 0.26 plus or minus 0.04 ng/mL and 0.71 plus or minus 0.02 h, respectively; volume of distribution, and clearance were 47.6 plus or minus 4.72 L/kg and 23.0 plus or minus 4.58 L/h/kg, respectively. SCOP t(sub max) after administration as gelcaps was significantly longer than that with tablets (1.04 h, p less than 0.05), but no significant differences in other pharmacokinetic parameters of SCOP were observed between the two dosage forms. When coadministered with DEX, the area underneath the concentration versus time curve (AUC) of SCOP was significantly reduced to 0.61 plus or minus 0.09 and 0.64 plus or minus 0.11 ng (raised dot) h/mL after administration as a tablet or gelcap formulation, respectively; SCOP C(sub max) was lower after coadministration with DEX, this difference, however, was not statistically significant. Delayed absorption with gelcaps coupled with reduced bioavailability with DEX coadministration may have resulted in the observed treatment failure with some of these formulations. This could result from failure to reach minimum effective concentrations after treatment with gelcap formulations before parabolic flights. A new nomogram for dosing is proposed for treatment with SCOP gelcaps or SCOP-DEX combination

Effects of sequencing error and the minimum number of reads required to call an allele on the number of alleles called in sequencing data.

<p>Modeling data with different error frequencies (0.5%–5%) showed an increase in loci with multiple alleles as error increased when both 2 (A) and 3 (B) reads were minimally required to call an allele. In contrast, standard exome sequencing data from DNA repair proficient cells (PD20 RV:D2 cells) and exome sequencing after whole genome amplification from a single cell were insensitive to the cut-off used.</p