Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb^™/™, pRb^™/™ utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb^™/™ cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of pRb^™/™ cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb^™/™ cochlea and utricle is centered on e2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb^™/™ cochlea or utricle. In pRb^™/™ cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involved in proliferation and survival are enriched in pRb^™/™ utricle. Clustering analysis showed that the pRb^™/™ inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRb^flox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.Key words: hair cells, retinoblastoma, Rb1, proliferation, regeneration, apoptosis, inner ear     

A Significant Reduction in the Plasma Levels and Gene Expression of CCL2 in Patients with Osteoarthritis following Intervention with Krocina™

Background:inflammatory chemokines such as CCL2 and CCL5 are involved in the progress of osteoarthritis. Crocin with antioxidant and anti-inflammatory properties can reduce the symptoms of osteoarthritis (OA). This study was performed investigate the effect of Krocina™, on the gene expressions and plasma levels of CCL2 and CCL5 in OA patients.Methods:The study included 35 patients that were randomized in the Krocina™ and placebo groups. The intervention was Krocina™ 15mg daily for four months. Clinical and paraclinical parameters were measured. CCL2 and CCL5 genes expression and plasma levels were determined using the SYBR Green Real-Time RT-PCR and Enzyme-linked Immunosorbent Assay (ELISA) techniques.Results:The C-reactive protein (CRP) value in the Krocina™ group and the visual analogue scale (VAS) value in the Krocina™ and placebo groups decreased significantly after the intervention. The gene expression of CCL2 in the Krocina™ and placebo groups decreased significantly. On the contrary, the gene expression of CCL5 in the Krocina™ and placebo groups increased significantly. Moreover, the plasma levels of CCL2 in the Krocina™ and placebo groups decreased meaningfully. There was no difference regarding the plasma levels of CCL5 within the Krocina™ and placebo groups before and after the intervention in either of the groups.Conclusion:Administration of Krocina™ reduced the clinical signs of inflammation and CRP and VAS value. Also, Krocina™ significantly decreased the plasma levels and gene expression of CCL2 in osteoarthritis patients.Key Words: CCL2, CCL5, Krocina™, Osteoarthritis     

Treatment of Colorectal Cancer Using a Combination of Liposomal Irinotecan (Irinophore C?) and 5-Fluorouracil

Purpose

To investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer.

Experimental Design

The effect of irinotecan (IRI) and/or 5-FU exposure times on cytotoxicity was assessed in vitro against HT-29 or LS174T human colon carcinoma cells. The pharmacokinetics and biodistribution of Irinophore C™ (IrC™) and 5-FU, administered alone or in combination, were compared in vivo. A subcutaneous model of HT-29 human colorectal cancer in Rag2-M mice was utilized to assess the efficacy of IrC™ alone, and in combination with 5-FU.

Results

The cytotoxicity of IRI and 5-FU were strongly dependent on exposure time. Synergistic interactions were observed following prolonged exposure to IRI/5-FU combinations. Pharmacokinetics/biodistribution studies demonstrated that the 5-FU elimination rate was decreased significantly when 5-FU was co-administered intravenously with IrC™, versus alone. Significant decreases in 5-FU elimination were also observed in plasma, with an associated increase of 5-FU in some tissues when 5-FU was given by intraperitoneal injection and IrC™ was given intravenously. The elimination of IrC™ was not significantly different when administered alone or in combination with 5-FU. Therapeutic studies demonstrated that single agent IrC™ was significantly more effective than the combination of IRI/5-FU; surprisingly, IrC™/5-FU combinations were no more effective than IrC™ alone. The administration of combinations of 5-FU (16 mg/kg) and IrC™ (60 mg IRI/kg) showed increased toxicity when compared to IrC™ alone. Treatment with IrC™ alone (60 mg IRI/kg) delayed the time required for a 5-fold increase in initial tumor volume to day 49, compared to day 23 for controls. When IrC™ (40 mg IRI/kg) was used in combination with 5-FU (16 mg/kg), the time to increase tumor volume 5-fold was 43 days, which was comparable to that achieved when using IrC™ alone (40 mg IRI/kg).

Conclusions

Single agent IrC™ was well tolerated and has significant therapeutic potential. IrC™ may be a suitable replacement for IRI treatment, but its use with free 5-FU is complicated by IrC™-engendered changes in 5-FU pharmacokinetics/biodistribution which are associated with increased toxicity when using the combination.     

A Comparison of the Performance of the I-gel? vs. the LMA-S?during Anesthesia: A Meta-Analysis of Randomized Controlled Trials

Background and Objective

Conflicting results were found between the I-gel™ and the LMA-Supreme™ during anesthesia, so we conducted a meta-analysis of randomized controlled trials (RCTs) to compare the effectiveness and safety of the I-gel™ vs. the LMA-Supreme™during anesthesia.

Methods

A comprehensive search was conducted using Pubmed, EMbase, ISI Web of Knowledge, the Cochrane Library, China Journal Full-text Database, Chinese Biomedical Database, Chinese Scientific Journals Full-text Database, CMA Digital Periodicals, and Google scholar to find RCTs that compare the LMA-S™ with the i-gel™during anesthesia. Two reviewers independently selected trials, extracted data, and assessed the methodological qualities and evidence levels. Data were analyzed by RevMan 5.0 and comprehensive meta-analysis software.

Results

Ten RCTs were included. There were no significant differences in oropharyngeal leak pressures (mean difference [MD] 0.72, 95% confidence interval [CI] –1.10 2.53), device placement time (MD –1.3, 95%CI –4.07 1.44), first attempt insertion success (risk ratio [RR] 1.01, 95% CI 0.9 1.14), grade 3 and 4 fiberoptic view (RR 0.89, 95%CI 0.65 1.21), and blood on removal (RR 0.62, 95%CI 0.32 1.22) between the i-gel™ and the LMA-Supreme™, respectively. However, the LMA-Supreme™was associated with easier gastric tube insertion (RR 1.17, 95%CI 1.07 1.29), and more sore throat (RR 2.56, 95%CI 1.60 4.12) than the i-gel™ group.

Conclusions

The LMA-Supreme™ and i-gel™ were similarly successful and rapidly inserted. However, the LMA-Supreme™ was shown to be easier for gastric tube insertion and associated with more sore throat compared with the i-gel™.     

The spherulites(TM): a promising carrier for oligonucleotide delivery

Concentric multilamellar microvesicles, named spherulites™, were evaluated as an oligonucleotide carrier. Up to 80% oligonucleotide was encapsulated in these vesicles. The study was carried out on two different spherulite™ formulations. The spherulite™ size and stability characteristics are presented. Delivery of encapsulated oligonucleotide was performed on a rat hepatocarcinoma and on a lymphoblastoid T cell line, both expressing the luciferase gene. We showed that spherulites™ were able to transfect both adherent and suspension cell lines and deliver the oligonucleotide to the nucleus. Moreover, 48–62% luciferase inhibition was obtained in the rat hepatocarcinoma cell line when the antisense oligonucleotide targeted to the luciferase coding region was encapsulated at 500 nM concentration in spherulites™ of different compositions.     

Evaluation of PIMA?? Point of Care Technology for CD4 T Cell Enumeration in Kenya

CD4+ T cell enumeration is used to determine eligibility for antiretroviral therapy (ART) and to monitor the immune status of HIV-positive patients; however, many patients do not have access to this essential diagnostic test. Introducing point of care (POC) testing may improve access. We have evaluated Alere’s PIMA™, one such POC device, against conventional CD4+ testing platforms to determine its performance and validity for use in Kenya. In our hands, Alere PIMA™ had a coefficient of variability of 10.3% and of repeatability of 175.6 cells/µl. It differed from both the BD FACSCalibur™ (r² = 0.762, mean bias −64.8 cells/µl), and the BD FACSCount™ (r² = 0.874, mean bias 7.8 cells/µl). When compared to the FACSCalibur™ at a cutoff of 350 cells/µl, it had a sensitivity of 89.6% and a specificity of 86.7% in those aged 5 years and over (Kw = 0.7566). With the BD FACSCount™, it had a sensitivity of 79.4% and a specificity of 83.4% in those aged 5 years and over (Kw = 0.7790). The device also differed from PARTEC Cyflow™ (r² = 0.781, mean bias −24.2 cells/µl) and GUAVA™ (r² = 0.658, mean bias −0.3 cells/µl) platforms, which are used in some facilities in Kenya. We conclude that with refinement, Alere PIMA™ technology has potential benefits for HIV-positive patients. This study highlights the difficulty in selecting the most appropriate reference technology for technical evaluations.     

INVESTIGATION OF URINARY EXCRETION OF HYDROXYETHYL STARCH AND DEXTRAN BY UHPLC-HRMS IN DIFFERENT ACQUISITION MODES

Plasma volume expanders (PVEs) such as hydroxyethyl starch (HES) and dextran are misused in sports because they can prevent dehydration and reduce haematocrit values to mask erythropoietin abuse. Endogenous hydrolysis generates multiple HES and dextran oligosaccharides which are excreted in urine. Composition of the urinary metabolic profiles of PVEs varies depending on post-administration time and can have an impact on their detectability. In this work, different mass spectrometry data acquisition modes (full scan with and without in-source collision-induced dissociation) were used to study urinary excretion profiles of HES and dextran, particularly by investigating time-dependent detectability of HES and dextran urinary oligosaccharide metabolites in post-administration samples. In-source fragmentation yielded the best results in terms of limit of detection (LOD) and detection times, whereas detection of HES and dextran metabolites in full scan mode with no in-source fragmentation is related to recent administration (< 24 hours). Urinary excretion studies showed detection windows for HES and dextran respectively of 72 and 48 hours after administration. Dextran concentrations were above the previously proposed threshold of 500 µg · mL⁻¹ for 12 hours. A “dilute-and-shoot” method for the detection of HES and dextran in human urine by ultra-high-pressure liquid chromatography-electrospray ionization-high resolution Orbitrap™ mass spectrometry was developed for this study. Validation of the method showed an LOD in the range of 10-500 µg · mL⁻¹ for the most significant HES and dextran metabolites in the different modes. The method allows retrospective data analysis and can be implemented in existing high-resolution mass spectrometry-based doping control screening analysis.     

Two-Step Versus One-Step RNA-to-CT™ 2-Step and One-Step RNA-to-CT™ 1-Step: Validity, Sensitivity, and Efficiency

Quantitative RT-PCR can be carried out as a one- or a two-step reaction. However, the choice of method raises controversy from the perspective of the researcher and manufacturer, because of advantages and disadvantages with both systems. We therefore hypothesize that running the RNA-to-C_T™ 2-Step kit [(Applied Biosystems (AB), Foster City, CA] using a one-step protocol (as recommended) is not appropriate for quantitation of gene expression levels and should not be performed. Consequently, we ran comparative studies of the two suggested methods to evaluate their efficiency, sensitivity, and accuracy. To ensure precession, two different PCR machines were used: the StepOnePlus system and Chromo4. In addition, the RNA-to-C_T™ 1-Step kit (recently launched by AB) was also used to compare its efficiency with these methods. Efficiency, sensitivity, and linearity were determined by standard curves generated using RNA isolated from C2 myoblasts to amplify the housekeeping gene GAPDH. When the RNA-to-C_T™ 2-Step kit was run as a two-step reaction on the Chromo4 or StepOnePlus, respectively, not only did the efficiency increase (100±1.5% and 99.7±0.95%) but also the sensitivity (comparative threshold cycle for the lowest standard: 33.2±0.5 and 32.5±0.7) and linearity (0.997±0.001 and 0.993±0.006) compared with RNA-to-C_T™ 2-Step run as one-step and RNA-to-C_T™ 1-Step kit. This is the first study to demonstrate that the RNA-to-C_T™ 2-Step kit is not reliable to be performed as a one-step reaction but as a two-step reaction, is even more sensitive than the newly launched RNA-to-C_T™ 1-Step kit.     

Red Wine Polyphenols Prevent Metabolic and Cardiovascular Alterations Associated with Obesity in Zucker Fatty Rats (Fa/Fa)

Background

Obesity is associated with increased risks for development of cardiovascular diseases. Epidemiological studies report an inverse association between dietary flavonoid consumption and mortality from cardiovascular diseases. We studied the potential beneficial effects of dietary supplementation of red wine polyphenol extract, Provinols™, on obesity-associated alterations with respect to metabolic disturbances and cardiovascular functions in Zucker fatty (ZF) rats.

Methodology/Principal Findings

ZF rats or their lean littermates received normal diet or supplemented with Provinols™ for 8 weeks. Provinols™ improved glucose metabolism by reducing plasma glucose and fructosamine in ZF rats. Moreover, it reduced circulating triglycerides and total cholesterol as well as LDL-cholesterol in ZF rats. Echocardiography measurements demonstrated that Provinols™ improved cardiac performance as evidenced by an increase in left ventricular fractional shortening and cardiac output associated with decreased peripheral arterial resistances in ZF rats. Regarding vascular function, Provinols™ corrected endothelial dysfunction in aortas from ZF rats by improving endothelium-dependent relaxation in response to acetylcholine (Ach). Provinols™ enhanced NO bioavailability resulting from increased nitric oxide (NO) production through enhanced endothelial NO-synthase (eNOS) activity and reduced superoxide anion release via decreased expression of NADPH oxidase membrane sub-unit, Nox-1. In small mesenteric arteries, although Provinols™ did not affect the endothelium-dependent response to Ach; it enhanced the endothelial-derived hyperpolarizing factor component of the response.

Conclusions/Significance

Use of red wine polyphenols may be a potential mechanism for prevention of cardiovascular and metabolic alterations associated with obesity.     

Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10–15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na⁺, osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.     

Introducing a Mobile-Connected Umbilical Doppler Device (UmbiFlow?) into a Primary Care Maternity Setting: Does This Reduce Unnecessary Referrals to Specialised Care? Results of a Pilot Study in Kraaifontein,South Africa

Objectives

UmbiFlow™ is a mobile-connected Doppler device that utilises a continuous waveform to measure resistance in the umbilical artery. The main aim of this pilot study was to determine whether the use of UmbiFlow™ for umbilical artery Doppler in patients with a suspected decreased symphysis fundal (SF) growth could safely lead to a decreased number of patients requiring referral to a more specialised level of care. A secondary aim of the study was to evaluate the effectiveness of UmbiFlow™ Doppler as a screening tool for concealed placental insufficiency in late bookers by using a single screening cut-off value that will be abnormal for any gestation >28 weeks.

Methods

The cohort comprised two groups of patients: The first group included all follow-up patients with suspected intra-uterine growth restriction (a decreased symphysis-fundus measurement based on serial assessment) who underwent on-site UmbiFlow™Doppler testing performed by the midwife directly after the clinical examination. The second group included late bookers, where gestation was uncertain; but estimated >28 weeks based on clinical grounds. This group was comprised of unselected patients who report to antenatal care late for the first time and received an UmbiFlow™Doppler test for concealed placental insufficiency.

Results

UmbiFlow™Doppler could reduce the number of false referrals to hospital by 55%. A single UmbiFlow™Doppler test in late bookers appeared to identify a group of women at moderate risk of lower birth weight babies.     

Play to Become a Surgeon: Impact of Nintendo WII Training on Laparoscopic Skills

Background

Video-games have become an integral part of the new multimedia culture. Several studies assessed video-gaming enhancement of spatial attention and eye-hand coordination. Considering the technical difficulty of laparoscopic procedures, legal issues and time limitations, the validation of appropriate training even outside of the operating rooms is ongoing. We investigated the influence of a four-week structured Nintendo® Wii™ training on laparoscopic skills by analyzing performance metrics with a validated simulator (Lap Mentor™, Simbionix™).

Methodology/Principal Findings

We performed a prospective randomized study on 42 post-graduate I–II year residents in General, Vascular and Endoscopic Surgery. All participants were tested on a validated laparoscopic simulator and then randomized to group 1 (Controls, no training with the Nintendo® Wii™), and group 2 (training with the Nintendo® Wii™) with 21 subjects in each group, according to a computer-generated list. After four weeks, all residents underwent a testing session on the laparoscopic simulator of the same tasks as in the first session. All 42 subjects in both groups improved significantly from session 1 to session 2. Compared to controls, the Wii group showed a significant improvement in performance (p<0.05) for 13 of the 16 considered performance metrics.

Conclusions/Significance

The Nintendo® Wii™ might be helpful, inexpensive and entertaining part of the training of young laparoscopists, in addition to a standard surgical education based on simulators and the operating room.     

Monitoring the initial pulmonary absorption of two different beclomethasone dipropionate aerosols employing a human lung reperfusion model

Background

The pulmonary residence time of inhaled glucocorticoids as well as their rate and extend of absorption into systemic circulation are important facets of their efficacy-safety profile. We evaluated a novel approach to elucidate the pulmonary absorption of an inhaled glucocorticoid. Our objective was to monitor and compare the combined process of drug particle dissolution, pro-drug activation and time course of initial distribution from human lung tissue into plasma for two different glucocorticoid formulations.

Methods

We chose beclomethasone dipropionate (BDP) delivered by two different commercially available HFA-propelled metered dose inhalers (Sanasthmax^®/Becloforte™ and Ventolair^®/Qvar™). Initially we developed a simple dialysis model to assess the transfer of BDP and its active metabolite from human lung homogenate into human plasma. In a novel experimental setting we then administered the aerosols into the bronchus of an extracorporally ventilated and reperfused human lung lobe and monitored the concentrations of BDP and its metabolites in the reperfusion fluid.

Results

Unexpectedly, we observed differences between the two aerosol formulations Sanasthmax^®/Becloforte™ and Ventolair^®/Qvar™ in both the dialysis as well as in the human reperfusion model. The HFA-BDP formulated as Ventolair^®/Qvar™ displayed a more rapid release from lung tissue compared to Sanasthmax^®/Becloforte™. We succeeded to explain and illustrate the observed differences between the two aerosols with their unique particle topology and divergent dissolution behaviour in human bronchial fluid.

Conclusion

We conclude that though the ultrafine particles of Ventolair^®/Qvar™ are beneficial for high lung deposition, they also yield a less desired more rapid systemic drug delivery. While the differences between Sanasthmax^®/Becloforte™ and Ventolair^®/Qvar™ were obvious in both the dialysis and lung perfusion experiments, the latter allowed to record time courses of pro-drug activation and distribution that were more consistent with results of comparable clinical trials. Thus, the extracorporally reperfused and ventilated human lung is a highly valuable physiological model to explore the lung pharmacokinetics of inhaled drugs.     

Rapid Screening of Antibody Aggregates and Protein Impurities Using Short Gel Filtration Columns

Gel filtration (GF) is an excellent tool to acquire information about sizing (identity), purity, and the multimeric state of a protein of interest. Superdex™ 200 and Superdex 75 are outstanding GF media for such analysis. To speed up analysis and keep sample and buffer consumption at minimum, two prepacked short GF columns have been developed, Superdex™ 200 5/150 GL and Superdex 75 5/150 GL. With lengths of only 15 cm and volume of 3 ml, these columns allow rapid analysis (6–12 min/run) with minimal sample (4–50μl) and buffer consumption.Purification of antibodies often generates dimers and higher aggregated forms, and during optimization of purification protocols, many samples may need to be analyzed for aggregate content. Gel filtration (GF) provides reliable information about size and the purity of a protein of interest, especially when it is in a multimeric state. GF, however, is often time-consuming, and the analyses become a bottleneck. A new column format—3 ml bed vol and 15 cm long—has been developed, enabling rapid and reliable GF with low sample and buffer consumption, using Superdex™ 75 and Superdex™ 200 media for determination of size and purity status.     

Oral Vaccination with Lipid-Formulated BCG Induces a Long-lived,Multifunctional CD4+ T Cell Memory Immune Response

Oral delivery of BCG in a lipid formulation (Liporale™-BCG) targets delivery of viable bacilli to the mesenteric lymph nodes and confers protection against an aerosol Mycobacterium tuberculosis challenge. The magnitude, quality and duration of the effector and memory immune response induced by Liporale™-BCG vaccination is unknown. Therefore, we compared the effector and memory CD4⁺ T cell response in the spleen and lungs of mice vaccinated with Liporale™-BCG to the response induced by subcutaneous BCG vaccination. Liporale™-BCG vaccination induced a long-lived CD4⁺ T cell response, evident by the detection of effector CD4⁺ T cells in the lungs and a significant increase in the number of Ag85B tetramer-specific CD4⁺ T cells in the spleen up to 30 weeks post vaccination. Moreover, following polyclonal stimulation, Liporale™-BCG vaccination, but not s.c. BCG vaccination, induced a significant increase in both the percentage of CD4⁺ T cells in the lungs capable of producing IFNγ and the number of multifunctional CD4⁺ T cells in the lungs at 30 weeks post vaccination. These results demonstrate that orally delivered Liporale™-BCG vaccine induces a long-lived multifunctional immune response, and could therefore represent a practical and effective means of delivering novel BCG-based TB vaccines.     

Preparation,Biodistribution and Neurotoxicity of Liposomal Cisplatin following Convection Enhanced Delivery in Normal and F98 Glioma Bearing Rats

The purpose of this study was to evaluate two novel liposomal formulations of cisplatin as potential therapeutic agents for treatment of the F98 rat glioma. The first was a commercially produced agent, Lipoplatin™, which currently is in a Phase III clinical trial for treatment of non-small cell lung cancer (NSCLC). The second, produced in our laboratory, was based on the ability of cisplatin to form coordination complexes with lipid cholesteryl hemisuccinate (CHEMS). The in vitro tumoricidal activity of the former previously has been described in detail by other investigators. The CHEMS liposomal formulation had a Pt loading efficiency of 25% and showed more potent in vitro cytotoxicity against F98 glioma cells than free cisplatin at 24 h. In vivo CHEMS liposomes showed high retention at 24 h after intracerebral (i.c.) convection enhanced delivery (CED) to F98 glioma bearing rats. Neurotoxicologic studies were carried out in non-tumor bearing Fischer rats following i.c. CED of Lipoplatin™ or CHEMS liposomes or their “hollow” counterparts. Unexpectedly, Lipoplatin™ was highly neurotoxic when given i.c. by CED and resulted in death immediately following or within a few days after administration. Similarly “hollow” Lipoplatin™ liposomes showed similar neurotoxicity indicating that this was due to the liposomes themselves rather than the cisplatin. This was particularly surprising since Lipoplatin™ has been well tolerated when administered intravenously. In contrast, CHEMS liposomes and their “hollow” counterparts were clinically well tolerated. However, a variety of dose dependent neuropathologic changes from none to severe were seen at either 10 or 14 d following their administration. These findings suggest that further refinements in the design and formulation of cisplatin containing liposomes will be required before they can be administered i.c. by CED for the treatment of brain tumors and that a formulation that may be safe when given systemically may be highly neurotoxic when administered directly into the brain.     

Predicted sweat rates for group water planning in sport: accuracy and application

This study tested the accuracy of a novel, limited-availability web application (H₂Q™) for predicting sweat rates in a variety of sports using estimates of energy expenditure and air temperature only. The application of predictions for group water planning was investigated for soccer match play. Fourteen open literature studies were identified where group sweat rates were reported (n = 20 group means comprising 230 individual observations from 179 athletes) with fidelity. Sports represented included: walking, cycling, swimming, and soccer match play. The accuracy of H₂Q™ sweat rates was tested by comparing to measured group sweat rates using the concordance correlation coefficient (CCC) with 95% confidence interval [CI]. The relative absolute error (RAE) with 95% [CI] was also assessed, whereby the mean absolute error was expressed relative to an acceptance limit of 0.250 L/h. The CCC was 0.98 [0.95, 0.99] and the RAE was 0.449 [0.279, 0.620], indicating that the prediction error was on average 0.112 L/h. The RAE was < 1.0 for 19/20 observations (95%). Drink volumes modeled as a proxy for sweat losses during soccer match play prevented dehydration (< 1% loss of body mass). The H₂Q™ web application demonstrated high group sweat prediction accuracy for the variety of sports activities tested. Water planning for soccer match play suggests the feasibility of easily and accurately predicting sweat rates to plan group water needs and promote optimal hydration in training and/or competition.     

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides.Chromatin is a highly organized and dynamic entity in cell nuclei, mostly composed of DNA and histone proteins. Its structure directly influences gene expression, DNA repair, and cell duplication events such as mitosis and meiosis (1). Histones are assembled in octamers named nucleosomes, wrapped by DNA every ∼200 base pairs. Histones are heavily modified by dynamic post-translational modifications (PTMs)¹, which affect chromatin structure because of their chemical properties and their ability to recruit chromatin modifier enzymes and binding proteins (2). Moreover, histone PTMs can be inherited through cell division and thus are crucial components of epigenetic memory (3). The function of histone PTMs has been extensively studied in the last 15–20 years, and several links have been found between aberrations of histone PTM levels and development of diseases (4, 5). Such discoveries revealed the importance of histone PTMs in fine-tuning cell phenotype. Because of this, technology has been rapidly evolving to investigate histone PTM relative abundance with higher accuracy and throughput.Mass spectrometry (MS)-based strategies have continuously evolved toward higher throughput and flexibility, allowing not only identification and quantification of single histone PTMs, but also their combinatorial patterns and even characterization of the intact proteins (reviewed in (6, 7)). For histone analysis, a widely adopted workflow for nano-liquid chromatography–tandem mass spectrometry (nLC-MS/MS) includes derivatization of lysine residue side chains with propionic anhydride, proteolytic digestion with trypsin, and subsequent derivatization of peptide N termini (8, 9). Such protocol leads to generation of ArgC-like peptides (only cleaved after arginine residues) after digestion. Moreover, propionylation of N termini increases peptide hydrophobicity, thereby improving LC retention of shorter ones, and thus the MS signal. Because of the high mass accuracy, sensitivity, and the possibility to perform label-free quantification MS has become the technique of choice, outperforming antibody-based strategies, to study both known and novel global histone PTMs.Several acquisition methods have been developed for MS analysis to accomplish different needs of identification and quantification (10). The most widely adopted in shotgun or discovery proteomics is the data-dependent acquisition (DDA) mode. Such acquisition method does not require any previous knowledge about the analyte, as it automatically selects precursor ions detectable at the full scan level in a given order (commonly from the most intense) to perform MS/MS fragmentation (11). Label-free quantification is performed at the full MS scan level by integrating the area of the LC peak from an extracted ion chromatogram of the precursor mass corresponding to the given peptide. On the other hand, the selected reaction monitoring (SRM) mode is the most widely used acquisition method in targeted proteomics. Such method performs cyclic precursor ion selection, MS/MS fragmentation, and product ion selection of a list of masses input by the user. Even though the method preparation is intuitively more complex than DDA, SRM is highly popular because of the high selectivity and sensitivity, which leads to more accurate label-free quantification (12). However, both methods have inevitable drawbacks; a DDA approach cannot perform accurate quantification of isobaric and co-eluting peptides, for example, KacQLATKAAR and KQLATKacAAR (histone H3 aa 9–17), as the fragment ions should be monitored through the entire peptide peak elution to define the ratio between the two similar analytes. On the contrary, an SRM experiment prevents future data mining of unpredicted peptides, and thus such method cannot be used for any classical PTM discovery. Therefore, LC-MS/MS analysis of histone peptides is commonly performed by integrating shotgun and targeted acquisition within the same MS method (13). This method requires previous knowledge about retention time and mass of co-eluting isobaric species, and tedious manual peak integration or dedicated software to deconvolute such complex raw data. Although this mixed MS mode is a powerful approach, the targeted sequences in the method reduce the duty cycle and number of DDA MS/MS spectra that can be acquired, making it far from ideal.Data independent acquisition (DIA) modes are a third option that recently gained popularity in proteomics (14, 15). Sequential window acquisition of all theoretical mass spectra (SWATH™)-MS is a data independent workflow that uses a first quadrupole isolation window to step across a mass range, collecting high resolution full scan composite MS/MS at each step and generating an ion map of fragments from all detectable precursor masses (15, 16). From such data set, a virtual SRM, or pseudo-SRM, can be performed by extracting the product ion chromatogram of a given peptide (17) with bioinformatics tools such as Peakview®, Skyline (18), or OpenSWATH™ (19). In order to define which fragment masses should be used to quantify a given peptide, a spectral library of identified peptides can be manually programmed, downloaded (if available), or generated by previous DDA experiments. In terms of quantification power, SWATH™ combines the advantages of both DDA and SRM, as it allows for MS/MS-based label-free quantification, discrimination of isobaric peptides, and subsequent data mining of unpredicted species.Histone proteins are an excellent target sample to test SWATH™, as the peptides are heavily modified by PTMs and often have isobaric proteoforms present. We analyzed with both DDA and SWATH™ two model systems: (1) extracted histones from untreated (pluripotent) and retinoic acid (RA) treated (differentiated) human embryonic stem cells (hESCs, strain H9), and (2) extracted histones from undifferentiated and differentiated mouse trophoblast stem cells (mTSCs). The results from the DDA experiment were used to evaluate the reproducibility of peptide retention time and the variety of species identified. For the SWATH™ analysis we focused on histone H3, as it is the histone with the highest variety of modified peptides (6). Results highlighted that such acquisition method provides sensitive and precise MS/MS-based quantification of both isobaric and nonisobaric peptides. Our data demonstrate that quantification at the MS/MS level is highly reproducible, and identification of the peptide elution profile is assisted by the high mass accuracy and the large number of overlapping elution profiles of the fragment ions. Moreover, we show that by using different fragment ions for MS/MS quantification we achieved similar quantification results. Thus, we used all unique fragment ions for a given species to provide a robust quantification method, where by unique is intended fragment ions that belong to only one of the possible isobaric peptide proteoforms. Taken together, we prove that SWATH™-MS is a reliable and simple-to-use acquisition method to perform epigenetic histone PTM analysis.