European flow cytometry quality assurance guidelines for the diagnosis of primary immune deficiencies and assessment of immune reconstitution following B cell depletion therapies and transplantation.

Over the last 15 years activity of diagnostic flow cytometry services have evolved from monitoring of CD4 T cell subsets in HIV-1 infection to screening for primary and secondary immune deficiencies syndromes and assessment of immune constitution following B cell depleting therapy and transplantation. Changes in laboratory activity in high income countries have been driven by initiation of anti-retroviral therapy (ART) in HIV-1 regardless of CD4 T cell counts, increasing recognition of primary immune deficiency syndromes and the wider application of B cell depleting therapy and transplantation in clinical practice. Laboratories should use their experience in standardization and quality assurance of CD4 T cell counting in HIV-1 infection to provide immune monitoring services to patients with primary and secondary immune deficiencies. Assessment of immune reconstitution post B cell depleting agents and transplantation can also draw on the expertise acquired by flow cytometry laboratories for detection of CD34 stem cell and assessment of MRD in hematological malignancies. This guideline provides recommendations for clinical laboratories on providing flow cytometry services in screening for immune deficiencies and its emerging role immune reconstitution after B cell targeting therapies and transplantation.

Prebiotic fibre mixtures counteract the manifestation of gut microbial dysbiosis induced by the chemotherapeutic 5-Fluorouracil (5-FU) in a validated in vitro model of the colon.

BACKGROUND: 5-Fluorouracil (5-FU) is used as an antineoplastic agent in distinct cancer types. Increasing evidence suggests that the gut microbiota might modulate 5-FU efficacy and toxicity, potentially affecting the patient's prognosis. The current experimental study investigated 5-FU-induced microbiota alterations, as well as the potential of prebiotic fibre mixtures (M1-M4) to counteract these shifts. METHODS: A pooled microbial consortium was derived from ten healthy donors, inoculated in an in vitro model of the colon, and treated with 5-FU, with or without prebiotic fibre mixtures for 72 h. Four different prebiotic fibre mixtures were tested: M1 containing short-chain galacto-oligosaccharides (sc GOS), long-chain fructo-oligosaccharides (lcFOS), and low viscosity pectin (lvPect), M2 consisting of arabinoxylan, beta-glucan, pectin, and resistant starch, M3 which was a mixture of scGOS and lcFOS, and M4 containing arabinoxylan, beta-glucan, pectin, resistant starch, and inulin. RESULTS: We identified 5-FU-induced changes in gut microbiota composition, but not in microbial diversity. Administration of prebiotic fibre mixtures during 5-FU influenced gut microbiota composition and taxa abundance. Amongst others, prebiotic fibre mixtures successfully stimulated potentially beneficial bacteria (Bifidobacterium, Lactobacillus, Anaerostipes, Weissella, Olsenella, Senegalimassilia) and suppressed the growth of potentially pathogenic bacteria (Klebsiella, Enterobacter) in the presence of 5-FU. The short-chain fatty acid (SCFA) acetate increased slightly during 5-FU, but even more during 5-FU with prebiotic fibre mixtures, while propionate was lower due to 5-FU with or without prebiotic fibre mixtures, compared to control. The SCFA butyrate and valerate did not show differences among all conditions. The branched-chain fatty acids (BCFA) iso-butyrate and iso-valerate were higher in 5-FU, but lower in 5-FU + prebiotics, compared to control. CONCLUSIONS: These data suggest that prebiotic fibre mixtures represent a promising strategy to modulate 5-FU-induced microbial dysbiosis towards a more favourable microbiota, thereby possibly improving 5-FU efficacy and reducing toxicity, which should be evaluated further in clinical studies.

Algerian Propolis from Distinct Geographical Locations: Chemical Profiles, Antioxidant Capacity, Cytotoxicity and Inhibition of 5-Lipoxygenase Product Biosynthesis.

Propolis was collected from honeybee hives in three geographically distinct Algerian climates and extracts were characterized for composition and bioactivity. Bees were identified as native subspecies using an in-silico DraI mtDNA COI-COII test. Over 20 compounds were identified in extracts by LC-MS. Extracts from the Medea region were more enriched in phenolic content (302±28 mg GAE/g of dry extract) than those from Annaba and Ghardaia regions. Annaba extracts had the highest flavonoid content (1870±385 mg QCE/g of dry extract). Medea extracts presented the highest free-radical scavenging activity (IC50=13.5 µg/mL) using the DPPH radical assay while Ghardaia extracts from the desert region were weak (IC50>100 µg/mL). Antioxidant activities measured using AAPH oxidation of linoleic acid were similar in all extracts with IC50 values ranging from 2.9 to 4.9 µg/mL. All extracts were cytotoxic (MTT assay) and proapoptotic (Annexin-V) against human leukemia cell lines in the low µg/mL range, although the Annaba extract was less active against the Reh cell line. Extracts inhibited cellular 5-lipoxygenase product biosynthesis with IC50 values ranging from 0.6 to 3.2 µg/mL. Overall, examined propolis extracts exhibited significant biological activity that warrant further characterization in cellular and in vivo models.

Caffeic acid phenethyl ester analogues as selective inhibitors of 12-lipoxygenase product biosynthesis in human platelets.

The inflammatory response is an essential process for the host defence against pathogens. Lipid mediators are important in coordinating the pro-inflammatory and pro-resolution phases of the inflammatory process. However, unregulated production of these mediators has been associated with chronic inflammatory diseases such as arthritis, asthma, cardiovascular diseases, and several types of cancer. Therefore, it is not surprising that enzymes implicated in the production of these lipid mediators have been targeted for potential therapeutic approaches. Amongst these inflammatory molecules, the 12-hydroxyeicosatetraenoic acid (12(S)-HETE) is abundantly produced in several diseases and is primarily biosynthesized via the platelet's 12-lipoxygenase (12-LO) pathway. To this day, very few compounds selectively inhibit the 12-LO pathway, and most importantly, none are currently used in the clinical settings. In this study, we investigated a series of polyphenol analogues of natural polyphenols that inhibit the 12-LO pathway in human platelets without affecting other normal functions of the cell. Using an ex vivo approach, we found one compound that selectively inhibited the 12-LO pathway, with IC50 values as low as 0.11 µM, with minimal inhibition of other lipoxygenase or cyclooxygenase pathways. More importantly, our data show that none of the compounds tested induced significant off-target effects on either the platelet's activation or its viability. In the continuous search for specific and better inhibitors targeting the regulation of inflammation, we characterized two novel inhibitors of the 12-LO pathway that could be promising for subsequent in vivo studies.

Decrease of Intracellular Glutamine by STF-62247 Results in the Accumulation of Lipid Droplets in von Hippel-Lindau Deficient Cells.

Kidney cancer is one of the top ten cancer diagnosed worldwide and its incidence has increased the last 20 years. Clear Cell Renal Cell Carcinoma (ccRCC) are characterized by mutations that inactivate the von Hippel-Lindau (VHL) tumor suppressor gene and evidence indicated alterations in metabolic pathways, particularly in glutamine metabolism. We previously identified a small molecule, STF-62247, which target VHL-deficient renal tumors by affecting late-stages of autophagy and lysosomal signaling. In this study, we investigated ccRCC metabolism in VHL-deficient and proficient cells exposed to the small molecule. Metabolomics profiling using 1H NMR demonstrated that STF-62247 increases levels of glucose, pyruvate, glycerol 3-phosphate while glutamate, asparagine, and glutathione significantly decreased. Diminution of glutamate and glutamine was further investigated using mass spectrometry, western blot analyses, enzymatic activities, and viability assays. We found that expression of SLC1A5 increases in VHL-deficient cells treated with STF-62247, possibly to stimulate glutamine uptake intracellularly to counteract the diminution of this amino acid. However, exogenous addition of glutamine was not able to rescue cell viability induced by the small molecule. Instead, our results showed that VHL-deficient cells utilize glutamine to produce fatty acid in response to STF-62247. Surprisingly, this occurs through oxidative phosphorylation in STF-treated cells while control cells use reductive carboxylation to sustain lipogenesis. We also demonstrated that STF-62247 stimulated expression of stearoyl-CoA desaturase (SCD1) and peripilin2 (PLIN2) to generate accumulation of lipid droplets in VHL-deficient cells. Moreover, the carnitine palmitoyltransferase 1A (CPT1A), which control the entry of fatty acid into mitochondria for ß-oxidation, also increased in response to STF-62247. CPT1A overexpression in ccRCC is known to limit tumor growth. Together, our results demonstrated that STF-62247 modulates cellular metabolism of glutamine, an amino acid involved in the autophagy-lysosome process, to support lipogenesis, which could be implicated in the signaling driving to cell death.

The Role of Intestinal Microbiota in Metastatic Colorectal Cancer Patients Treated With Capecitabine.

BACKGROUND: Previous pre-clinical research has indicated that the intestinal microbiota can potentiate anti-tumour efficacy of capecitabine and that capecitabine treatment impacts intestinal microbiota composition and diversity. Using a longitudinal design, this study explores the associations between the intestinal microbiota and treatment response in patients with metastatic colorectal cancer (mCRC) during capecitabine treatment. PATIENTS AND METHODS: Patients with mCRC treated with capecitabine were prospectively enrolled in a multicentre cohort study. Patients collected a faecal sample and completed a questionnaire before, during, and after three cycles of capecitabine. Several clinical characteristics, including tumour response, toxicity and antibiotic use were recorded. Intestinal microbiota were analysed by amplicon sequencing of the 16S rRNA V4 gene-region. RESULTS: Thirty-three patients were included. After three cycles of capecitabine, six patients (18%) achieved a partial response, 25 (76%) showed stable disease, and one (3%) experienced progressive disease. Of the 90 faecal samples were collected. Microbial diversity (α-diversity), community structure (ß-diversity), and bacterial abundance on phylum and genus level were not significantly different between responders and non-responders and were not significantly affected by three cycles of capecitabine. CONCLUSION: This is the first clinical study with longitudinal intestinal microbiota sampling in mCRC patients that explores the role of the intestinal microbiota during treatment with capecitabine. Intestinal microbiota composition and diversity before, during, and after three cycles of capecitabine were not associated with response in this study population. Capecitabine did not induce significant changes in the microbiota composition and diversity during the treatment period. Individual effects of antibiotics during capecitabine treatment were observed.

The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids.

Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy-based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently-available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non-toxic, and/or clinically-amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non-immunogenicity, biodegradability, and low toxicity. Chitosan-precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic-based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture-conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method.

A multiparametric extraction method for Vn96-isolated plasma extracellular vesicles and cell-free DNA that enables multi-omic profiling.

Extracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and 'omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.

Optimization of cysteine residue alkylation using an on-line LC-MS strategy: Benefits of using a cocktail of haloacetamide reagents.

Several common reagents for the alkylation of cysteine residues of model intact proteins were evaluated for reaction speed, yield of alkylated product and degree of over-alkylation using an online LC-MS platform. The efficiency of the alkylation reaction is found to be dependent on the (1) reagent, (2) peptide/protein, (3) reagent concentration and (4) reaction time. At high reagent concentrations, iodoacetic acid was found to produce significant levels of over-alkylation products wherein methionine residues become modified. For optimal performance of the alkylation reaction, we found the use of a cocktail of chloroacetamide, bromoacetamide and iodoacetamide worked best. The alkylating efficiency of each haloacetamide is a balance between the characteristics of the halogen leaving group and the steric hindrance of the alkylation site on the peptide or protein. A key aspect of using a cocktail of haloacetamides is that they all produce the same modification (+57.0209 Da) to the cysteine residues of the protein while the alkylation efficiency of each site may differ for each of the three reagents. Over-alkylation effects appear to be lower with the cocktail due to a lower concentration of each reagent. The haloacetamide cocktail could be useful when considering complex mixtures of proteins.

EBF1 drives hallmark B cell gene expression by enabling the interaction of PAX5 with the MLL H3K4 methyltransferase complex.

PAX5 and EBF1 work synergistically to regulate genes that are involved in B lymphocyte differentiation. We used the KIS-1 diffuse large B cell lymphoma cell line, which is reported to have elevated levels of PAX5 expression, to investigate the mechanism of EBF1- and PAX5-regulated gene expression. We demonstrate the lack of expression of hallmark B cell genes, including CD19, CD79b, and EBF1, in the KIS-1 cell line. Upon restoration of EBF1 expression we observed activation of CD19, CD79b and other genes with critical roles in B cell differentiation. Mass spectrometry analyses of proteins co-immunoprecipitated with PAX5 in KIS-1 identified components of the MLL H3K4 methylation complex, which drives histone modifications associated with transcription activation. Immunoblotting showed a stronger association of this complex with PAX5 in the presence of EBF1. Silencing of KMT2A, the catalytic component of MLL, repressed the ability of exogenous EBF1 to activate transcription of both CD19 and CD79b in KIS-1 cells. We also find association of PAX5 with the MLL complex and decreased CD19 expression following silencing of KMT2A in other human B cell lines. These data support an important role for the MLL complex in PAX5-mediated transcription regulation.

Improved intact peptide and protein quantitation by LC-MS: Battling the deleterious effects of analyte adsorption.

Peptide and protein quantitation by liquid chromatography-mass spectrometry relies on the assumption of linear signal response with concentration. At low concentrations, analyte adsorption to pipette tips, sample vials and equipment can have significant deleterious effects on signal response. Meanwhile at high concentrations, linearity breaks down due to competitive ionization, signal suppression, and the formation of peptide or protein multimers. These effects result in calibration curves that are more sigmoidal than linear. Linearity at low protein levels for identification and quantitation is of paramount importance in the discovery and validation of biomarker molecules. Herein, we demonstrate the benefits of using commercial low-bind microcentrifuge tubes and LC vials on the response of a 27-mer peptide, Vn96, and the intact proteins apomyoglobin and carbonic anhydrase. Linear curves were acquired for Vn96 while apomyoglobin required the addition of intact carbonic anhydrase as an adsorption competitor to achieve linearity. A linear calibration curve for carbonic anhydrase was also acquired by using the polypeptide ubiquitin as an adsorption competitor and internal standard. Linear response was recorded for approximately two orders of magnitude for apomyoglobin and carbonic anhydrase and three orders of magnitude for Vn96 with detection limits ranging from 0.33 to 19 fmol/µL. Finally, we used low-bind vials for the online enzymatic digestion of apomyoglobin where a high concentration of apomyoglobin acted as an adsorption blocker for the low level trypsin enzyme. Fortunately, the liberated tryptic peptides showed no affinity for the walls of the low-bind vials. In this study, we take a comprehensive approach to combat analyte adsorption by showing the significance of utilizing low-bind vials and adsorption competitors to greatly improve upon signal sensitivity at low concentrations of target molecules. The use of these methodologies should improve the low-level detection of molecules by mass spectrometry.

Assessment of plasma cell myeloma minimal residual disease testing by flow cytometry in an international inter-laboratory study: Is it ready for primetime use?

BACKGROUND: Minimal/measurable residual disease (MRD) testing by flow cytometry (FC) has been proposed as a potential surrogate clinical endpoint in plasma cell myeloma (PCM) clinical trials. As a result, effort has gone into standardizing this approach on PCM patients. AIMS: To assess inter-laboratory variation in FC MRD testing of PCM patients in an independent inter-laboratory study. METHODS: A dilution series of five stabilized bone marrow samples manufactured to contain 0%, 0.1%, 0.01%, 0.001%, and 0.0001% neoplastic plasma cells (PCs) were tested blind, using standardized FC PCM MRD assays by 10 international laboratories. RESULTS: Laboratories' assays broadly adhered to the consensus guidelines; however, some deviations were identified in panel design, fluorochrome conjugates, and lysis reagents. Despite this, all laboratories that returned results detected neoplastic PCs down to 0.001% of leucocytes. 6/8 laboratories detected neoplastic PCs at a level of 0.0001%. Quantitative data returned by laboratories showed good consensus and linearity with increasing variation at lower levels of MRD. However, examples of analytical and post analytical error were identified. SUMMARY/CONCLUSION: Broadly standardized PCM MRD FC assays can attain the lower limit of detection (LOD) required by current and future clinical trials, an important consideration in establishing PCM MRD testing as a surrogate clinical marker in PCM clinical trials. Laboratories' assays showed good linearity, encouraging the prediction of survival based on log reduction in neoplastic PC populations in future clinical trials. However, the deviations from consensus guidelines identified in this study would suggest that if PCM MRD assays are further standardized interlaboratory variation could be reduced. © 2018 International Clinical Cytometry Society.

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein-extraction methods.

Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of '-omics' research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol

CD24 induces changes to the surface receptors of B cell microvesicles with variable effects on their RNA and protein cargo.

The CD24 cell surface receptor promotes apoptosis in developing B cells, and we recently found that it induces B cells to release plasma membrane-derived, CD24-bearing microvesicles (MVs). Here we have performed a systematic characterization of B cell MVs released from WEHI-231 B lymphoma cells in response to CD24 stimulation. We found that B cells constitutively release MVs of approximately 120 nm, and that CD24 induces an increase in phosphatidylserine-positive MV release. RNA cargo is predominantly comprised of 5S rRNA, regardless of stimulation; however, CD24 causes a decrease in the incorporation of protein coding transcripts. The MV proteome is enriched with mitochondrial and metabolism-related proteins after CD24 stimulation; however, these changes were variable and could not be fully validated by Western blotting. CD24-bearing MVs carry Siglec-2, CD63, IgM, and, unexpectedly, Ter119, but not Siglec-G or MHC-II despite their presence on the cell surface. CD24 stimulation also induces changes in CD63 and IgM expression on MVs that is not mirrored by the changes in cell surface expression. Overall, the composition of these MVs suggests that they may be involved in releasing mitochondrial components in response to pro-apoptotic stress with changes to the surface receptors potentially altering the cell type(s) that interact with the MVs.

Built environmental correlates of older adults' total physical activity and walking: a systematic review and meta-analysis.

BACKGROUND: Identifying attributes of the built environment associated with health-enhancing levels of physical activity (PA) in older adults (≥65 years old) has the potential to inform interventions supporting healthy and active ageing. The aim of this study was to first systematically review and quantify findings on built environmental correlates of older adults' PA, and second, investigate differences by type of PA and environmental attribute measurement. METHODS: One hundred articles from peer-reviewed and grey literature examining built environmental attributes related to total PA met inclusion criteria and relevant information was extracted. Findings were meta-analysed and weighted by article quality and sample size and then stratified by PA and environmental measurement method. Associations (p < .05) were found in relation to 26 individual built environmental attributes across six categories (walkability, residential density/urbanisation, street connectivity, access to/availability of destinations and services, infrastructure and streetscape, and safety) and total PA and walking specifically. Reported individual- and environmental-level moderators were also examined. RESULTS: Positive environmental correlates of PA, ranked by strength of evidence, were: walkability (p < .001), safety from crime (p < .001), overall access to destinations and services (p < .001), recreational facilities (p < .001), parks/public open space (p = .002) and shops/commercial destinations (p = .006), greenery and aesthetically pleasing scenery (p = .004), walk-friendly infrastructure (p = .009), and access to public transport (p = .016). There were 26 individual differences in the number of significant associations when the type of PA and environmental measurement method was considered. No consistent moderating effects on the association between built environmental attributes and PA were found. CONCLUSIONS: Safe, walkable, and aesthetically pleasing neighbourhoods, with access to overall and specific destinations and services positively influenced older adults' PA participation. However, when considering the environmental attributes that were sufficiently studied (i.e., in ≥5 separate findings), the strength of evidence of associations of specific categories of environment attributes with PA differed across PA and environmental measurement types. Future research should be mindful of these differences in findings and identify the underlying mechanisms. Higher quality research is also needed.

Quantitative proteomics to study a small molecule targeting the loss of von Hippel-Lindau in renal cell carcinomas.

Inactivation of the tumor suppressor gene, von Hippel-Lindau (VHL), is known to play an important role in the development of sporadic clear cell renal cell carcinomas (ccRCCs). Even if available targeted therapies for metastatic RCCs (mRCCs) have helped to improve progression-free survival rates, they have no durable clinical response. We have previously shown the feasibility of specifically targeting the loss of VHL with the identification of a small molecule, STF-62247. Understanding its functionality is crucial for developing durable personalized therapeutic agents differing from those available targeting hypoxia inducible factor (HIF-) pathways. By using SILAC proteomics, we identified 755 deregulated proteins in response to STF-62247 that were further analyzed by ingenuity pathway analysis (IPA). Bioinformatics analyses predicted alterations in 37 signaling pathways in VHL-null cells in response to treatment. Validation of some altered pathways shows that STF-62247's selectivity is linked to an important inhibition of mTORC1 activation in VHL-null cells leading to protein synthesis arrest, a mechanism differing from two allosteric inhibitors Rapamycin and Everolimus. Altogether, our study identified signaling cascades driving STF-62247 response and brings further knowledge for this molecule that shows selectivity for the loss of VHL. The use of a global SILAC approach was successful in identifying novel affected signaling pathways that could be exploited for the development of new personalized therapeutic strategies to target VHL-inactivated RCCs.

Measurement of BCR-ABL1 by RT-qPCR in chronic myeloid leukaemia: findings from an International EQA Programme.

Sequential measurement of BCR-ABL1 mRNA levels by reverse transcription quantitative polymerase chain reaction (RT-qPCR) is embedded in the management of patients with chronic myeloid leukaemia (CML), and has played an important role in the remarkable improvement in patient outcomes seen in this disease. As a provider of external quality assessment (EQA) in this area, UK NEQAS for Leucocyte Immunophenotyping (UKNEQAS LI) has a unique perspective on the changing face of BCR-ABL1 testing in CML. To assess the impact of technical standardisation and the development of the International Scale (IS) upon the accuracy of BCR-ABL1 testing, we reviewed EQA trial data from 2007 to 2015. Comparison of participant results identified considerable variability at both high and low levels of disease, including therapeutically important decision points; however, results converted to the IS showed less variability compared to unconverted data sets. We also found that different methods of converting to the IS produce consistently different median results within UKNEQAS LI IS data sets. This data suggests that whilst the development of the IS has improved the comparability of results between centres, there is still the need for further improvement in the processes of converting raw results to the IS in order to fully realise the benefits of molecular monitoring of CML.

Measuring moderate-intensity walking in older adults using the ActiGraph accelerometer.

BACKGROUND: Accelerometry is the method of choice for objectively assessing physical activity in older adults. Many studies have used an accelerometer count cut point corresponding to 3 metabolic equivalents (METs) derived in young adults during treadmill walking and running with a resting metabolic rate (RMR) assumed at 3.5 mL · kg-1 · min-1 (corresponding to 1 MET). RMR is lower in older adults; therefore, their 3 MET level occurs at a lower absolute energy expenditure making the cut point derived from young adults inappropriate for this population. The few studies determining older adult specific moderate-to-vigorous intensity physical activity (MVPA) cut points had methodological limitations, such as not measuring RMR and using treadmill walking. METHODS: This study determined a MVPA hip-worn accelerometer cut point for older adults using measured RMR and overground walking. Following determination of RMR, 45 older adults (mean age 70.2 ± 7 years, range 60-87.6 years) undertook an outdoor, overground walking protocol with accelerometer count and energy expenditure determined at five walking speeds. RESULTS: Mean RMR was 2.8 ± 0.6 mL · kg-1 · min-1. The MVPA cut points (95% CI) determined using linear mixed models were: vertical axis 1013 (734, 1292) counts · min-1; vector magnitude 1924 (1657, 2192) counts · min-1; and walking speed 2.5 (2.2, 2.8) km · hr-1. High levels of inter-individual variability in cut points were found. CONCLUSIONS: These MVPA accelerometer and speed cut points for walking, the most popular physical activity in older adults, were lower than those for younger adults. Using cut points determined in younger adults for older adult population studies is likely to underestimate time spent engaged in MVPA. In addition, prescription of walking speed based on the adult cut point is likely to result in older adults working at a higher intensity than intended.

A spectroscopic study of substituted anthranilic acids as sensitive environmental probes for detecting cancer cells.

Small-molecule fluorescent reporters of disease states are highly sought after, yet they remain elusive. Anthranilic acids are extremely sensitive environmental probes, and hold promise as general but selective agents for cancer-cell detection if they can be equipped with the appropriate targeting groups. The optical properties of a small library of N-isopropyl invariant anthranilic acids were investigated in methanol and chloroform. Points of variation included: fluoro, trifluoromethyl, or cyano substitution on the aromatic ring, and derivitization of the parent carboxylic acid as esters or secondary carboxamides. Phenylboronic acid conjugation at the carboxylic acid alongside un-, mono-, and dimethylated 2-amino groups was also explored. The boron-containing anthranilic acids were also evaluated as sensitive fluorescent probes for cancer cells using laser scanning confocal microscopy. In general, the compounds produced blue fluorescence that was strongly influenced by substitution and environment. 4-Trifluoromethyl and 4-cyano esters proved to be the most sensitive environmental probes with quantum yields as large as 100% in chloroform, and enhancements of up to 30-fold on going from methanol to chloroform. Stokes shifts ranged from 63 to 120nm, generally increasing with ortho-substitution and environmental polarity. It was demonstrated that phenylboronic acid conjugation was an attractive method for cancer cell detection via boronate ester formation with overexpressed glycoproteins (with no interference from normal, healthy cells), presumably due to favorable boron-sialic acid interactions.