Olaparib for childhood tumors harboring defects in DNA damage repair genes: arm H of the NCI-COG Pediatric MATCH trial.

BACKGROUND: The National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice (MATCH) precision oncology platform trial enrolled children aged 1-21 years with treatment-refractory solid tumors and predefined actionable genetic alterations. Patients with tumors harboring alterations in DNA damage repair (DDR) genes were assigned to receive olaparib. METHODS: Tumor and blood samples were submitted for centralized molecular testing. Tumor and germline sequencing were conducted in parallel. Olaparib was given twice daily for 28-day cycles starting at a dose 30% lower than the adult recommended phase 2 dose (RP2D). The primary endpoint was the objective response. RESULTS: Eighteen patients matched (1.5% of those screened) based on the presence of a deleterious gene alteration in BRCA1/2, RAD51C/D, or ATM detected by tumor sequencing without germline subtraction or analysis of loss of heterozygosity (LOH). Eleven (61%) harbored a germline mutation, with only one exhibiting LOH. Six patients enrolled and received the olaparib starting dose of 135 mg/m2/dose. Two participants were fully evaluable; 4 were inevaluable because <85% of the prescribed dose was administered during cycle 1. There were no dose-limiting toxicities or responses. Minimal hematologic toxicity was observed. CONCLUSION: Most DDR gene alterations detected in Pediatric MATCH were germline, monoallelic, and unlikely to confer homologous recombination deficiency predicting sensitivity to olaparib monotherapy. The study closed due to poor accrual. CLINICALTRIALS.GOV IDENTIFIER: NCT03233204. IRB approved: initial July 24, 2017.

The Exceptional Responders Initiative: Feasibility of a National Cancer Institute Pilot Study.

BACKGROUND: Tumor molecular profiling from patients experiencing exceptional responses to systemic therapy may provide insights into cancer biology and improve treatment tailoring. This pilot study evaluates the feasibility of identifying exceptional responders retrospectively, obtaining pre-exceptional response treatment tumor tissues, and analyzing them with state-of-the-art molecular analysis tools to identify potential molecular explanations for responses. METHODS: Exceptional response was defined as partial (PR) or complete (CR) response to a systemic treatment with population PR or CR rate less than 10% or an unusually long response (eg, duration >3 times published median). Cases proposed by patients' clinicians were reviewed by clinical and translational experts. Tumor and normal tissue (if possible) were profiled with whole exome sequencing and, if possible, targeted deep sequencing, RNA sequencing, methylation arrays, and immunohistochemistry. Potential germline mutations were tracked for relevance to disease. RESULTS: Cases reflected a variety of tumors and standard and investigational treatments. Of 520 cases, 476 (91.5%) were accepted for further review, and 222 of 476 (46.6%) proposed cases met requirements as exceptional responders. Clinical data were obtained from 168 of 222 cases (75.7%). Tumor was provided from 130 of 168 cases (77.4%). Of 117 of the 130 (90.0%) cases with sufficient nucleic acids, 109 (93.2%) were successfully analyzed; 6 patients had potentially actionable germline mutations. CONCLUSION: Exceptional responses occur with standard and investigational treatment. Retrospective identification of exceptional responders, accessioning, and sequencing of pretreatment archived tissue is feasible. Data from molecular analyses of tumors, particularly when combining results from patients who received similar treatments, may elucidate molecular bases for exceptional responses.

Effect of Capivasertib in Patients With an AKT1 E17K-Mutated Tumor: NCI-MATCH Subprotocol EAY131-Y Nonrandomized Trial.

Importance: In the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial, agents targeting genetic tumor abnormalities are administered to patients. In the NCI-MATCH subprotocol EAY131-Y trial, patients with an AKT1 E17K-mutated metastatic tumor received the pan-AKT inhibitor capivasertib. Objective: To assess the objective response rate (ORR) of capivasertib in patients with an AKT1 E17K-mutated tumor. Design, Setting, and Participants: Between July 13, 2016, and August 10, 2017, patients in the NCI-MATCH trial were enrolled and assigned to the subprotocol EAY131-Y nonrandomized trial. Patients included adults with an AKT1 E17K-mutated metastatic tumor that had progressed with standard treatment, and these patients were assigned to receive capivasertib. Tumor assessments were repeated every 2 cycles. Data analysis of this evaluable population was performed from November 8, 2019, to March 12, 2020. Interventions: The study treatment was capivasertib, 480 mg, orally twice daily for 4 days on and 3 days off weekly in 28-day cycles until disease progression or unacceptable toxic effect. If patients continued hormone therapy for metastatic breast cancer, the capivasertib dose was 400 mg. Main Outcomes and Measures: The primary end point was the ORR (ie, complete response [CR] and partial response) according to the Response Evaluation Criteria in Solid Tumors criteria, version 1.1. Secondary end points included progression-free survival (PFS), 6-month PFS, overall survival, and safety. Results: In total, 35 evaluable and analyzable patients were included, of whom 30 were women (86%), and the median (range) age was 61 (32-73) years. The most prevalent cancers were breast (18 [51%]), including 15 patients with hormone receptor (HR)-positive/ERBB2-negative and 3 with triple-negative disease, and gynecologic (11 [31%]) cancers. The ORR rate was 28.6% (95% CI, 15%-46%). One patient with endometrioid endometrial adenocarcinoma achieved a CR and remained on therapy at 35.6 months. Patients with confirmed partial response had the following tumor types: 7 had HR-positive/ERBB2-negative breast cancer, 1 had uterine leiomyosarcoma, and 1 had oncocytic parotid gland carcinoma and continued receiving treatment at 28.8 months. Sixteen patients (46%) had stable disease as the best response, 2 (6%) had progressive disease, and 7 (20%) were not evaluable. With a median follow-up of 28.4 months, the overall 6-month PFS rate was 50% (95% CI, 35%-71%). Capivasertib was discontinued because of adverse events in 11 of 35 patients (31%). Grade 3 treatment-related adverse events included hyperglycemia (8 [23%]) and rash (4 [11%]). One grade 4 hyperglycemic adverse event was reported. Conclusions and Relevance: This nonrandomized trial found that, in patients with an AKT1 E17K-mutated tumor treated with capivasertib, a clinically significant ORR was achieved, including 1 CR. Clinically meaningful activity with single-agent capivasertib was demonstrated in refractory malignant neoplasms, including rare cancers. Trial Registration: ClinicalTrials.gov Identifier: NCT00700882.

Molecular Features of Cancers Exhibiting Exceptional Responses to Treatment.

A small fraction of cancer patients with advanced disease survive significantly longer than patients with clinically comparable tumors. Molecular mechanisms for exceptional responses to therapy have been identified by genomic analysis of tumor biopsies from individual patients. Here, we analyzed tumor biopsies from an unbiased cohort of 111 exceptional responder patients using multiple platforms to profile genetic and epigenetic aberrations as well as the tumor microenvironment. Integrative analysis uncovered plausible mechanisms for the therapeutic response in nearly a quarter of the patients. The mechanisms were assigned to four broad categories-DNA damage response, intracellular signaling, immune engagement, and genetic alterations characteristic of favorable prognosis-with many tumors falling into multiple categories. These analyses revealed synthetic lethal relationships that may be exploited therapeutically and rare genetic lesions that favor therapeutic success, while also providing a wealth of testable hypotheses regarding oncogenic mechanisms that may influence the response to cancer therapy.

The Molecular Analysis for Therapy Choice (NCI-MATCH) Trial: Lessons for Genomic Trial Design.

BACKGROUND: The proportion of tumors of various histologies that may respond to drugs targeted to molecular alterations is unknown. NCI-MATCH, a collaboration between ECOG-ACRIN Cancer Research Group and the National Cancer Institute, was initiated to find efficacy signals by matching patients with refractory malignancies to treatment targeted to potential tumor molecular drivers regardless of cancer histology. METHODS: Trial development required assumptions about molecular target prevalence, accrual rates, treatment eligibility, and enrollment rates as well as consideration of logistical requirements. Central tumor profiling was performed with an investigational next-generation DNA-targeted sequencing assay of alterations in 143 genes, and protein expression of protein expression of phosphatase and tensin homolog, mutL homolog 1, mutS homolog 2, and RB transcriptional corepressor 1. Treatments were allocated with a validated computational platform (MATCHBOX). A preplanned interim analysis evaluated assumptions and feasibility in this novel trial. RESULTS: At interim analysis, accrual was robust, tumor biopsies were safe (<1% severe events), and profiling success was 87.3%. Actionable molecular alteration frequency met expectations, but assignment and enrollment lagged due to histology exclusions and mismatch of resources to demand. To address this lag, we revised estimates of mutation frequencies, increased screening sample size, added treatments, and improved assay throughput and efficiency (93.9% completion and 14-day turnaround). CONCLUSIONS: The experiences in the design and implementation of the NCI-MATCH trial suggest that profiling from fresh tumor biopsies and assigning treatment can be performed efficiently in a large national network trial. The success of such trials necessitates a broad screening approach and many treatment options easily accessible to patients.

Current defence strategies in some contested drink-drive prosecutions: Is it now time for some additional statutory assumptions?

The prosecution of those socially irresponsible individuals who drive when they have had too much to drink - in that their breath, blood or urine alcohol level exceeds the prescribed legal limit - has been standard practice since October 1967. The forensic analysis of whichever type of specimen is taken from the suspect operates to a high degree of accuracy. Despite this a defence industry continues to operate in which specialist privately-funded legal practitioners [solicitors and counsel], in conjunction with several 'experts' whom they routinely instruct, seek to persuade the Courts that the alcohol reading on which the Crown's case depends cannot be correct as it does not accord with what their client subsequently states he had to drink. The defence team then seek copious disclosure of documents regarding the instrumentation that was used for the analysis, in the underlying hope that these are not produced: they can then make an application to the Court that their client is unable to have a fair trial as a result. Recent case law has improved the disclosure situation, but such cases still involve a quite disproportionate time input from the Police, the CPS and the Courts. Most drink-drive prosecutions today are based on evidential breath analysis: only instruments that have been Type Approved to a Government specification are used. Each analyser is periodically tested and then checked as a part of each subject's analysis procedure to ensure it is operating accurately at that time. There is no recorded case of such an instrument having given an inaccurate reading. It is therefore time that a Statutory Assumption should be enshrined in the legislation to the effect that an evidential breath alcohol reading should be assumed to be accurate unless there is some direct evidence [as opposed to inferential evidence] to the contrary. Likewise with blood and urine specimen analysis, before a subject can challenge the Crown's result he should have his own part of the specimen analysed. The result of this should be significantly different to that of the Crown's analysis, and it should also be consistent with his stated consumption. Failing these defence requirements the Crown's reading should be assumed to be accurate. In the continued absence of these suggested Statutory Assumptions we might as well dispense with breath, blood and urine analysis altogether and simply base all drink-drive prosecutions simply on the calculation result based on the suspect's stated consumption.

Analytical Validation and Application of a Targeted Next-Generation Sequencing Mutation-Detection Assay for Use in Treatment Assignment in the NCI-MPACT Trial.

Robust and analytically validated assays are essential for clinical studies. We outline an analytical validation study of a targeted next-generation sequencing mutation-detection assay used for patient selection in the National Cancer Institute Molecular Profiling-Based Assignment of Cancer Therapy (NCI-MPACT) trial (NCT01827384). Using DNA samples from normal or tumor cell lines and xenografts with known variants, we assessed the sensitivity, specificity, and reproducibility of the NCI-MPACT assay in five variant types: single-nucleotide variants (SNVs), SNVs at homopolymeric (HP) regions (≥3 identical bases), small insertions/deletions (indels), large indels (gap ≥4 bp), and indels at HP regions. The assay achieved sensitivities of 100% for 64 SNVs, nine SNVs at HP regions, and 11 large indels, 83.33% for six indels, and 93.33% for 15 indels at HP regions. Zero false positives (100% specificity) were found in 380 actionable mutation loci in 96 runs of haplotype map cells. Reproducibility analysis showed 96.3% to 100% intraoperator and 98.1% to 100% interoperator mean concordance in detected variants and 100% reproducibility in treatment selection. To date, 38 tumors have been screened, 34 passed preanalytical quality control, and 18 had actionable mutations for treatment assignment. The NCI-MPACT assay is well suited for its intended investigational use and can serve as a template for developing next-generation sequencing assays for other cancer clinical trial applications.

Expression array analysis of the hepatocyte growth factor invasive program.

Signaling by human hepatocyte growth factor (hHGF) via its cell surface receptor (MET) drives mitogenesis, motogenesis and morphogenesis in a wide spectrum of target cell types and embryologic, developmental and homeostatic contexts. Oncogenic pathway activation also contributes to tumorigenesis and cancer progression, including tumor angiogenesis and metastasis, in several prevalent malignancies. The HGF gene encodes full-length hHGF and two truncated isoforms known as NK1 and NK2. NK1 induces all three HGF activities at modestly reduced potency, whereas NK2 stimulates only motogenesis and enhances HGF-driven tumor metastasis in transgenic mice. Prior studies have shown that mouse HGF (mHGF) also binds with high affinity to human MET. Here we show that, like NK2, mHGF stimulates cell motility, invasion and spontaneous metastasis of PC3M human prostate adenocarcinoma cells in mice through human MET. To identify target genes and signaling pathways associated with motogenic and metastatic HGF signaling, i.e., the HGF invasive program, gene expression profiling was performed using PC3M cells treated with hHGF, NK2 or mHGF. Results obtained using Ingenuity Pathway Analysis software showed significant overlap with networks and pathways involved in cell movement and metastasis. Interrogating The Cancer Genome Atlas project also identified a subset of 23 gene expression changes in PC3M with a strong tendency for co-occurrence in prostate cancer patients that were associated with significantly decreased disease-free survival.

Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

Single-walled carbon nanotubes: differential genotoxic potential associated with physico-chemical properties.

Single-walled carbon nanotubes (SWCNTs) have recently attracted great attention because of their fibrous structure and high aspect ratio. Here the genotoxic potential of 400-800 nm, 1-3 µm and 5-30 µm SWCNT with respect to their geometry and surface characteristics was studied. Following thorough physico-chemical characterisation, human bronchial epithelial (BEAS-2B) and lymphoblastoid (MCL-5) cells were treated with SWCNT for 24 or 48 h. This showed significant increases in micronucleus frequency in a time- and dose-dependent manner in both cell types in the absence of cytotoxicity. Over the same dose range, only 1-3 µm SWCNT gave rise to significant increases in hprt point mutations at doses ≥25 µg/ml. Cellular 2,7-dichlorodihydrofluoresceindiacetate (DCFH-DA) fluorescence assay and RT-PCR for oxidative pathway gene profiling revealed a possible oxidative mechanism for the genotoxicity observed in the 1-3 µm SWCNT. Consequently, this study has demonstrated that SWCNT genotoxicity is dependent on its secondary structure under experimental conditions and oxidative stress alone cannot account for the observed damage.

Integration of health and social care: a case of learning and knowledge management.

This paper considers integration of health and social care as an exercise in learning and knowledge management (KM). Integration assembles diverse actors and organisations in a collective effort to design and deliver new service models underpinned by multidisciplinary working and generic practice. Learning and KM are integral to this process. A critical review of the literature is undertaken to identify theoretical insights and models in this field, albeit grounded mainly in a private sector context. The findings from a research study involving two integrated services are then used to explore the role of, and approach to, learning and KM. This case study research was qualitative in nature and involved an interrogation of relevant documentary material, together with 25 in-depth interviews with a cross-section of strategic managers and professionals undertaken between March and May 2011. The evidence emerging indicated no planned strategies for learning and KM, but rather, interventions and mechanisms at different levels to support integration processes. These included formal activities, particularly around training and appraisal, but also informal ones within communities of practice and networking. Although structural enablers such as a co-location of facilities and joint appointments were important, the value of trust and inter-personal relationships was highlighted especially for tacit knowledge exchange. The infrastructure for learning and KM was constructed around a collaborative culture characterised by a coherent strategic framework; clarity of purpose based on new models of service; a collaborative leadership approach that was facilitative and distributed; and, a focus on team working to exploit the potential of multidisciplinary practice, generic working and integrated management. The discussion and conclusion use Nonaka's knowledge conversation model to reflect on the research findings, to comment on the absence of an explicit approach to learning and KM, and to develop a template to assist policy-makers with the design of planned strategies.

Review of the dielectric properties of nanofiltration membranes and verification of the single oriented layer approximation.

The structuring of water at soft solid surfaces remains an area of great interest to colloid science as a whole and has many applications in relation to colloid stability, foams, and wetting films as well as being central to membrane separations. Quantitatively calculating the structural components of thin layers of water and the interaction forces of hydrated molecules with the surface of pores through a layer of water having modified structure is one of the most important challenges in the physics of surface phenomenon. In this paper these effects are reviewed and discussed in relation to the confines of a capillary pore. Membrane nanofiltration is extremely complex and is dependent on the micro-hydrodynamics and interfacial events occurring at the membrane surface and within the membrane nanopores. There is significant debate as to the exact nature of these complex phenomena and rejection is typically attributed to a combination of steric and electrical effects. The electrical effects are less well understood and in particular the contribution of dielectric exclusion. A review of the two competing descriptions of dielectric exclusion is presented along with the theories currently used in modelling this phenomena. A series of rejection experiments of 0.01 M salt solutions at the membrane isoelectric point has been performed for the NF270 and NF99HF membranes. The dielectric constants inside the nanopore are calculated and these values were consistent for three of the salts studied, indicating that a simplistic model based on Born theory is accurate enough for engineering calculations and that ion solvation is most likely to be the more appropriate dielectric exclusion mechanism for true nanofiltration membranes.

Characterisation and application of a novel positively charged nanofiltration membrane for the treatment of textile industry wastewaters.

The present study demonstrates the high potential for the application of a novel self assembled positively charged nanofiltration membrane, PA6DT-C, in processes such as the recovery of valuable cationic macromolecules in the bioprocess and pharmaceutical industries or removal of multi-valent cations such as dyes and heavy metals in the paper and pulp, textiles, nuclear, and automotive industries. The nanofiltration membrane, prepared in this laboratory, is further characterised and then tested for the removal and recovery of Methylene Blue from a synthetic dye house wastewater. The characterisation process involved the construction of a rejection profile for NaCl over a wide range of pH and concentration, which illustrates that the optimal process conditions for the removal of small cations using this membrane is in the region pH <8.0 and concentration less than 15 mol m(-3). The salt rejection data was used to calculate the magnitude of the effective membrane charge density and this was found to be significantly higher for the PA6DT-C membrane than two commercially available membranes (Desal-DK and Nanomax-50). The membrane flux for this new membrane is also superior to the commercial membranes with an approximate increase of 3-4 fold. The PA6DT-C membrane was successful in removal of Methylene Blue dye from synthetic dye house wastewaters achieving 98% rejection and a membrane flux of ≈ 17 LMH bar(-1). Thus, this new membrane both adds to and complements the existing short supply of positively charged NF membranes.

The role of iron redox state in the genotoxicity of ultrafine superparamagnetic iron oxide nanoparticles.

Ultrafine superparamagnetic iron oxide nanoparticles (USPION) hold great potential for revolutionising biomedical applications such as MRI, localised hyperthermia, and targeted drug delivery. Though evidence is increasing regarding the influence of nanoparticle physico-chemical features on toxicity, data however, is lacking that assesses a range of such characteristics in parallel. We show that iron redox state, a subtle though important physico-chemical feature of USPION, dramatically modifies the cellular uptake of these nanoparticles and influences their induction of DNA damage. Surface chemistry was also found to have an impact and evidence to support a potential mechanism of oxidative DNA damage behind the observed responses has been demonstrated. As human exposure to ferrofluids is predicted to increase through nanomedicine based therapeutics, these findings are important in guiding the fabrication of USPION to ensure they have characteristics that support biocompatibility.

Amino acid rejection behaviour as a function of concentration.

The solute rejection versus concentration behaviour of five different amino acids has been investigated using a Nitto Denko NTR7450 nanofiltration membrane. The experimental data for amino acid rejection was also compared against a combined steric and charge rejection model. At its isoelectric point, lysine was effectively neutral and its behaviour was well described by the model incorporating a steric function only. For phenylalanine, the combined model was found to fit the data well. In contrast there was poor agreement between the model and rejection data for glutamine, glutamic acid and glycine whose rejection values at first increased with concentration. This result implied that another governing process was in operation. Dimerisation as an explanation for the observed phenomena was also investigated. Size analysis of amino acid molecules as a function of the prevailing concentration using dynamic light scattering was limited but showed no evidence of dimerisation. This data was supported by osmotic pressure measurements which demonstrated no evidence of non-linearity in the relation between osmotic pressure and concentration.

Dextran coated ultrafine superparamagnetic iron oxide nanoparticles: compatibility with common fluorometric and colorimetric dyes.

Due to the unique physicochemical properties of nanomaterials (NM) and their unknown reactivity, the possibility of NM altering the optical properties of fluorometric/colorimetric probes that are used to measure their cyto- and genotoxicity may lead to inaccurate readings. This could have potential implications given that NM, such as ultrafine superparamagnetic iron oxide nanoparticles (USPION), are increasingly finding their use in nanomedicine and the absorbance/fluorescence based assays are used to assess their toxicity. This study looks at the potential of dextran-coated USPION (dUSPION) (maghemite and magnetite) to alter the background signal of common probes used for evaluating cytotoxicity (MTS, CyQUANT, Calcein, and EthD-1) and oxidative stress (DCFH-DA and APF). In the present study, both forms of dUSPION caused an increase in MTS signal but a decrease in background signal from calcein and 3'-(p-aminophenyl) fluorescein (APF) and no effect on CyQUANT and EthD-1 fluorescence responses. Magnetite caused a decrease in fluorescence signal of DCFH, but it did not decrease fluorescence signal in the presence of the reactive oxygen species-inducer tert-butyl hydroperoxide (TBHP). In contrast, maghemite caused an increase in fluorescence, which was substantially reduced in the presence of the antioxidant N-acetyl cysteine. This study emphasizes the importance of considering and controlling for possible interactions between NM and fluorometric/colorimetric dyes and, most importantly, the oxidation state of dUSPION that may confound their sensitivity and specificity.

Positively charged nanofiltration membranes: review of current fabrication methods and introduction of a novel approach.

A review of the fabrication processes currently available to produce positively charged nanofiltration membranes has been conducted. The review highlights that there are few membranes and studies currently available. The preparation of a novel positively charged nanofiltration membrane is also described. This membrane was fabricated by surface modification of a prepared base membrane using polyethyleneimine followed by cross linking with butanedioldiglycidylether. The fabrication process uses standard organic solvents and avoids the need for hazardous materials, such as concentrated sulphuric acid, which significantly benefits the scale up potential of any future commercial manufacturing process. The new membrane was characterised using a number of state-of-the-art techniques, including a novel use of atomic force microscopy to determine pore size. Streaming potential measurements confirmed that this new membrane is indeed positively charged in the pH range below pH 9, which covers the majority of normal operating conditions. The performance characteristics for the new membrane were very favourable, with a pure water flux determined to be 20 LMH bar(-1) and a rejection of MgCl of 96%. Thus, this new membrane both adds to and complements the existing short supply of positively charged NF membranes and is suitable for applications such as the recovery of valuable cationic macromolecules in the bioprocess and pharmaceutical industries or removal of multi-valent cations such as dyes and heavy metals in the paper and pulp, textiles, nuclear, and automotive industries.

Analysis of quantum dot fluorescence stability in primary blood mononuclear cells.

A quantitative assessment of fluorescence signal generation and persistence in blood cells, measured at multiple points over a time course, is presented. Quantum dots (QDs) are inorganic fluorophores that are photostable and nonmetabolized and so can provide quantitative measures of cell biology over multiple cell generations. However, if the potential of these nanoparticles for long-term reporting is to be realized, an understanding of the stability of their fluorescence in living cells is essential. CdTe/ZnS and CdSe/ZnS core/shell dots with peak emission wavelengths of 705 nm and 585 nm, respectively, were loaded, via endocytosis into mononuclear cells extracted from primary blood and flow cytometry used to measure the average fluorescence intensity per cell within populations >104. Time-based study showed a saturation-limited uptake of QDs with a characteristic time of 20 min and a maximum fluorescence signal that is linearly proportional to dot solution concentration. The fluorescence signal decreases after attachment and internalization within cells and is accurately described by a biexponential decay with a rapid initial decay followed by a much slower signal loss with characteristic times of 435 and 7,000 min respectively. Comparison with control samples indicates that interaction with the culture media is a major contributory factor to the initial signal decay. These results provide phenomenological descriptions of the evolving QD fluorescence within live cells with associated analytical equations that allow quantitative assessment of QD-based assays.

NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials.

With the rapid expansion in the nanotechnology industry, it is essential that the safety of engineered nanomaterials and the factors that influence their associated hazards are understood. A vital area governing regulatory health risk assessment is genotoxicology (the study of genetic aberrations following exposure to test agents), as DNA damage may initiate and promote carcinogenesis, or impact fertility. Of late, considerable attention has been given to the toxicity of engineered nanomaterials, but the importance of their genotoxic potential on human health has been largely overlooked. This comprehensive review focuses on the reported abilities of metal nanoparticles, metal-oxide nanoparticles, quantum dots, fullerenes, and fibrous nanomaterials, to damage or interact with DNA, and their ecogenotoxicity is also considered. Many of the engineered nanomaterials assessed were found to cause genotoxic responses, such as chromosomal fragmentation, DNA strand breakages, point mutations, oxidative DNA adducts and alterations in gene expression profiles. However, there are clear inconsistencies in the literature and it is difficult to draw conclusions on the physico-chemical features of nanomaterials that promote genotoxicity, largely due to study design. Hence, areas that require that further attention are highlighted and recommendations to improve our understanding of the genotoxic potential of engineered nanomaterials are addressed.

An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase.

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.