A highly sensitive and specific system for large-scale gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Rapid progress in the field of gene expression-based molecular network integration has generated strong demand on enhancing the sensitivity and data accuracy of experimental systems. To meet the need, a high-throughput gene profiling system of high specificity and sensitivity has been developed.</p> <p>Results</p> <p>By using specially designed primers, the new system amplifies sequences in neighboring exons separated by big introns so that mRNA sequences may be effectively discriminated from other highly related sequences including their genes, unprocessed transcripts, pseudogenes and pseudogene transcripts. Probes used for microarray detection consist of sequences in the two neighboring exons amplified by the primers. In conjunction with a newly developed high-throughput multiplex amplification system and highly simplified experimental procedures, the system can be used to analyze >1,000 mRNA species in a single assay. It may also be used for gene expression profiling of very few (<it>n </it>= 100) or single cells. Highly reproducible results were obtained from duplicate samples with the same number of cells, and from those with a small number (100) and a large number (10,000) of cells. The specificity of the system was demonstrated by comparing results from a breast cancer cell line, MCF-7, and an ovarian cancer cell line, NCI/ADR-RES, and by using genomic DNA as starting material.</p> <p>Conclusion</p> <p>Our approach may greatly facilitate the analysis of combinatorial expression of known genes in many important applications, especially when the amount of RNA is limited.</p

Pharmacokinetic (Pk) Activity of Cabazitaxel (Cbz) in Patients (Pts) with Renal Impairment (Ri)

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Lipopolysaccharide (LPS) potentiates hydrogen peroxide toxicity in T98G astrocytoma cells by suppression of anti-oxidative and growth factor gene expression

<p>Abstract</p> <p>Background</p> <p>Lipopolysaccharide (LPS) is a cell wall component of Gram-negative bacteria with proved role in pathogenesis of sepsis. Brain injury was observed with both patients dead from sepsis and animal septic models. However, <it>in vitro </it>administration of LPS has not shown obvious cell damage to astrocytes and other relative cell lines while it does cause endothelial cell death <it>in vitro</it>. These observations make it difficult to understand the role of LPS in brain parenchymal injury.</p> <p>Results</p> <p>To test the hypothesis that LPS may cause biological changes in astrocytes and make the cells to become vulnerable to reactive oxygen species, a recently developed highly sensitive and highly specific system for large-scale gene expression profiling was used to examine the gene expression profile of a group of 1,135 selected genes in a cell line, T98G, a derivative of human glioblastoma of astrocytic origin. By pre-treating T98G cells with different dose of LPS, it was found that LPS treatment caused a broad alteration in gene expression profile, but did not cause obvious cell death. However, after short exposure to H₂O₂, cell death was dramatically increased in the LPS pretreated samples. Interestingly, cell death was highly correlated with down-regulated expression of antioxidant genes such as cytochrome b561, glutathione s-transferase a4 and protein kinase C-epsilon. On the other hand, expression of genes encoding growth factors was significantly suppressed. These changes indicate that LPS treatment may suppress the anti-oxidative machinery, decrease the viability of the T98G cells and make the cells more sensitive to H₂O₂stress.</p> <p>Conclusion</p> <p>These results provide very meaningful clue for further exploring and understanding the mechanism underlying astrocyte injury in sepsis <it>in vivo</it>, and insight for why LPS could cause astrocyte injury <it>in vivo</it>, but not <it>in vitro</it>. It will also shed light on the therapeutic strategy of sepsis.</p

A phase I pharmacokinetic and safety study of cabazitaxel in adult cancer patients with normal and impaired renal function

$\textbf{PURPOSE}$ Limited data are available on cabazitaxel pharmacokinetics in patients with renal impairment. This open-label, multicenter study assessed cabazitaxel in patients with advanced solid tumors and normal or impaired renal function. $\textbf{METHODS}$ Cohorts A (normal renal function: creatinine clearance [CrCL] >80 mL/min/1.73 m$^{2}$), B (moderate renal impairment: CrCL 30 to <50 mL/min/1.73 m$^{2}$) and C (severe impairment: CrCL <30 mL/min/1.73 m(2)) received cabazitaxel 25 mg/m$^{2}$ (A, B) or 20 mg/m(2) (C, could be escalated to 25 mg/m$^{2}$), once every 3 weeks. Pharmacokinetic parameters and cabazitaxel unbound fraction (F$_{U}$) were assessed using linear regression and mixed models. Geometric mean (GM) and GM ratios (GMRs) were determined using mean CrCL intervals (moderate and severe renal impairment: 40 and 15 mL/min/1.73 m$^{2}$) versus a control (90 mL/min/1.73 m$^{2}$). $\textbf{RESULTS}$ Overall, 25 patients received cabazitaxel (median cycles: 3 [range 1-20]; Cohort A: 5 [2-13]; Cohort B: 3 [1-15]; and Cohort C: 5 [1-20]), of which 24 were eligible for pharmacokinetic analysis (eight in each cohort). For moderate and severe renal impairment versus normal renal function, GMR estimates were: clearance normalized to body surface area (CL/BSA) 0.95 (90% CI 0.80-1.13) and 0.89 (0.61-1.32); area under the curve normalized to dose (AUC/dose) 1.06 (0.88-1.27) and 1.14 (0.76-1.71); and F U 0.99 (0.94-1.04) and 0.97 (0.87-1.09), respectively. Estimated slopes of linear regression of log parameters versus log CrCL (renal impairment) were: CL/BSA 0.06 (-0.15 to 0.28); AUC/dose -0.07 (-0.30 to 0.16); and F U 0.02 (-0.05 to 0.08). Cabazitaxel safety profile was consistent with previous reports. $\textbf{CONCLUSIONS}$ Renal impairment had no clinically meaningful effect on cabazitaxel pharmacokinetics.This study was supported by Sanofi. Javier Garcia-Corbacho acknowledges clinical fellowship support from SEOM. Experimental Cancer Medicine Centre (ECMC) and NIHR Biomedical Research Centre (BRC) funding is also acknowledged for the Cambridge Cancer Centre

Identification and characterization of the dif Site from Bacillus subtilis

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in Escherichia coli is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as dif (Ecdif). In Bacillus subtilis the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bsdif), lying 6° counterclockwise from the B. subtilis terminus of replication (172°), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bsdif in vivo recombination did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the B. subtilis SPβ-bacteriophage, and in particular its yopP gene product, appears to strongly modulate the extent of the partitioning defects seen in codV strains and, to a lesser extent, those seen in ripX and dif strains

Genetic Structures of Copy Number Variants Revealed by Genotyping Single Sperm

Copy number variants (CNVs) occupy a significant portion of the human genome and may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases may be underlain by CNVs. However, because of the presence of their multiple copies, variability in copy numbers and the diploidy of the human genome, detailed genetic structure of CNVs cannot be readily studied by available techniques.Single sperm samples were used as the primary subjects for the study so that CNV haplotypes in the sperm donors could be studied individually. Forty-eight CNVs characterized in a previous study were analyzed using a microarray-based high-throughput genotyping method after multiplex amplification. Seventeen single nucleotide polymorphisms (SNPs) were also included as controls. Two single-base variants, either allelic or paralogous, could be discriminated for all markers. Microarray data were used to resolve SNP alleles and CNV haplotypes, to quantitatively assess the numbers and compositions of the paralogous segments in each CNV haplotype.This is the first study of the genetic structure of CNVs on a large scale. Resulting information may help understand evolution of the human genome, gain insight into many genetic processes, and discriminate between CNVs and SNPs. The highly sensitive high-throughput experimental system with haploid sperm samples as subjects may be used to facilitate detailed large-scale CNV analysis

Molecular correlates of response to capmatinib in advanced non-small-cell lung cancer: clinical and biomarker results from a phase I trial

Background: Dysregulation of receptor tyrosine kinase MET by various mechanisms occurs in 3%–4% of non-small-cell lung cancer (NSCLC) and is associated with unfavorable prognosis. While MET is a validated drug target in lung cancer, the best biomarker strategy for the enrichment of a susceptible patient population still remains to be defined. Towards this end we analyze here primary data from a phase I dose expansion study of the MET inhibitor capmatinib in patients with advanced MET-dysregulated NSCLC. Patients and methods: Eligible patients [≥18 years; Eastern Cooperative Oncology Group (ECOG) performance status ≤2] with MET-dysregulated advanced NSCLC, defined as either (i) MET status by immunohistochemistry (MET IHC) 2+ or 3+ or H-score ≥150, or MET/centromere ratio ≥2.0 or gene copy number (GCN) ≥5, or (ii) epidermal growth factor receptor wild-type (EGFRwt) and centrally assessed MET IHC 3+, received capmatinib at the recommended dose of 400 mg (tablets) or 600 mg (capsules) b.i.d. The primary objective was to determine safety and tolerability; the key secondary objective was to explore antitumor activity. The exploratory end point was the correlation of clinical activity with different biomarker formats. Results: Of 55 patients with advanced MET-dysregulated NSCLC, 40/55 (73%) had received two or more prior systemic therapies. All patients discontinued treatment, primarily due to disease progression (69.1%). The median treatment dur

A Phase Ib, open-label, dose-finding study of alpelisib in combination with paclitaxel in patients with advanced solid tumors

Phosphatidylinositol 3-kinase (PI3K) pathway activation is associated with resistance to paclitaxel in solid tumors. We assessed the safety and activity of alpelisib, an oral, selective PI3K p110\u3b1 inhibitor, plus paclitaxel in patients with advanced solid tumors. This Phase Ib, multicenter, open-label, dose-finding study, with a planned dose-expansion phase of alpelisib once daily (QD) plus fixed-dose paclitaxel, recruited patients with advanced solid tumors. For the dose-finding phase, the primary objective was determination of maximum tolerated and/or recommended Phase II dose of alpelisib plus paclitaxel, and the secondary objectives included the assessment of safety for this combination. From March 2014 to August 2016, 19 patients with advanced solid tumors were treated with alpelisib QD (300 mg, n=6; 250 mg, n=4; 150 mg, n=9) plus paclitaxel (80 mg/m2, per standard of care). During dose finding, five of 12 (41.7%) evaluable patients for MTD determination experienced dose-limiting toxicities: alpelisib 300 mg, Grade 2 hyperglycemia (n=1); alpelisib 250 mg, Grade 2 hyperglycemia (n=1), Grade 4 hyperglycemia and Grade 3 acute kidney injury (n=1); and alpelisib 150 mg, Grade 2 hyperglycemia (n=1) and Grade 4 leukopenia (n=1). The MTD of alpelisib when administered with paclitaxel was 150 mg QD. Most frequent all-grade AEs were diarrhea (73.7%; Grade 3/4 10.5%) and hyperglycemia (57.9%; Grade 3/4 31.6%). The planned dose-expansion phase was not initiated. Alpelisib plus paclitaxel has a challenging safety profile in patients with advanced solid tumors. This study was closed following the completion of the dosefinding phase. Clinical trial registration: ClinicalTrials.gov NCT02051751