The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. It is likely that clinical testing of these agents will be in combination with standard therapies to harness the apoptotic potential of both the agents. To support this strategy, it has been widely observed that a number of chemotherapeutics stimulate the activation of several intracellular signalling cascades including Ras/RAF/MEK/ERK. The MEK1/2 inhibitor selumetinib has been shown to have anti-tumour activity and induce apoptotic cell death as a monotherapy.The aim of this study was to identify agents, which would be likely to offer clinical benefit when combined with selumetinib. Here, we used human tumour xenograft models and assessed the effects combining standard chemotherapeutic agents with selumetinib on tumour growth. In addition, we analysed tumour tissue to determine the mechanistic effects of these combinations.γH2A.X suggesting that selumetinib is able to enhance the DNA damage induced by TMZ alone. In several models we observed that continuous exposure to selumetinib in combination with docetaxel results in tumour regression. Scheduling of docetaxel before selumetinib was more beneficial than when selumetinib was dosed before docetaxel and demonstrated a pro-apoptotic phenotype. Similar results were seen when selumetinib was combined with the Aurora B inhibitor barasertib.Combining selumetinib with the DNA-alkylating agent, temozolomide (TMZ), resulted in enhanced tumour growth inhibition compared with monotherapies. Biomarker studies highlighted an increase in The data presented suggests that MEK inhibition in combination with several standard chemotherapeutics or an Aurora B kinase inhibitor is a promising clinical strategy. Members of the Ras/RAF/MEK/ERK pathway, in particular KRAS and BRAF, are frequently deregulated in several cancers including melanoma, colorectal (CRC), non-small cell lung cancer (NSCLC) and pancreatic . ActivatThe MAPK signalling cascades are activated by several stimuli, including growth factors and hormones, which regulate gene expression and cell survival. Three major subfamilies of the MAPK pathways have been identified; JNK/SAPK's, p38 MAPK and MEK/ERK. Primarily in oncology, several MEK inhibitors have been developed and are undergoing clinical testing . p90RSK Once activated, ERK, along with RAF and MEK, migrate to the nucleus where they activate cyclin D1 and downregulate p27 thus driving cell proliferation .Selumetinib (AZD6244) and barasertib (AZD1152) were prepared as previously reported +1% glutamine or RPMI 1640+20% FCS+1% glutamine+1% sodium pyruvate. All cells were obtained from the American Type Culture Collection.nu/nu:Alpk; AstraZeneca, Macclesfield, UK) were housed in negative pressure isolators . Experiments were conducted in 8–12-week-old animals in full accordance with the United Kingdom Home Office Animal (Scientific Procedures) Act 1986. Human tumour xenografts were established by subcutaneous (s.c.) injection of 1 × 107, 5 × 106, 1 × 106 and 3 × 106 cells per mouse for HCT-116 (CRC), SW-620 (CRC), CaLu-6 (NSCLC) and AsPc-1 (Pancreatic), respectively. Animals were randomised into treatment groups (n=8 to 14) when tumours reached a defined palpable size (0.2 or 0.4 cm3). Selumetinib and TMZ were administered by oral gavage, docetaxel by intravenous injecton. All compounds were prepared at 0.1 ml per 10 g. Barasertib was prepared in Tris buffer (pH 9) and administerd as a continuous 48 h infusion via s.c. implanted osmotic mini-pumps . Tumour volume , animal body weight and tumour condition were recorded twice weekly during the study. Growth inhibition from the start of treatment was assessed by comparison of the differences in tumour volume between control and treated groups. Because the variance in mean tumour volume data increases proportionally with volume (and is therefore disproportionate between groups), data were log transformed to remove any size dependency before statistical evaluation. Statistical significance was evaluated using a one-tailed, two sample t-test.Female nude mice  staining.Protein lysates were prepared from frozen tumours using the FastPrep-24  in 10 × lysis buffer  diluted to 1 × in PBS containing phosphatase inhibitor cocktail I and II  and protease inhibitor cocktail . Protein concentration was determined by BCA assay . Total protein of 20 μg per sample were resolved on 12% Bis-Tris SDS–PAGE gels (Invitrogen), transferred to nitrocellulose membranes  and incubated with anti-Bim , total MAPK , phospho MAPK (Thr202/Tyr204)  and subsequently horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG . Immunoreactive proteins were detected by enhanced chemiluminescence (Pierce) and bands were detected and quantitated on a Chemigenius .M citrate (pH6.0) for 5 min at 110°C in a Milestone Rapid Microwaves Histoprocessor (model RHS-2) to retrieve antigen. Sections were placed in 0.01 ml l–1 citrate (pH 6) and incubated for 5 min at 110°C. Sections were cooled and transferred to a Lab Vision Autostainer. Following incubation with Dako serum-free protein block  for 20 min, sections were incubated with anti-phospho-Histone H3, pHH3 (Ser10), , Phospho-Histone Histone H2A.X, γH2A.X (ser139), (Cat # 2577) and Cleaved Caspase-3 (CC3) (Asp175), (Cat #9661)  antibodies at room temperature for 60 min. Sections were subsequently incubated with mouse/rabbit-labelled polymer from mouse EnVision kit (Dako) for 30 min at room temperature, and signal was detected using the En Vision kit 3.3′-diamino benzidine . Sections were counterstained with Carazzi's haematoxylin and mounted in Histomount . Cleaved caspase 3 and pHH3-positive cells were scored using algorithms developed for scoring percentage positive nuclei on an ACIS II image analyzer  using standard threshold settings for each marker. Representative images were taken using an Aperio Image Scope 10.2 using the × 20 objective and a subsequent zoom of × 20.Sections were dewaxed and endogenous peroxidase activity was blocked with 3% (v/v) hydrogen peroxide for 10 min. Heat-mediated antigen retrieval was achieved by incubating the sections in 0.01 Tumour tissues snap frozen in liquid nitrogen were disaggregated processed for propidium iodide staining (Sigma) as previously described (–1 per bid for 14 doses) in HCT-116 xenografts increased the levels of the pro-apoptotic BH3 protein Bim-EL (∼4-fold increase) compared with no change with the level of this protein following a shorter dosing period (three doses)  . In ordeThe effects of selumetinib in combination with a number of key standard of care agents were tested pre-clinically in human tumour xenograft models and resulted in enhanced anti-tumour activity. A number of compounds including the DNA-alkylating agent TMZ and the anti-mitotic drug docetaxel resulted in a greatly enhanced tumour growth inhibition compared with monotherapies . HoweverP<0.0005), compared with selumetinib  and TMZ  alone  alone . At the γH2A.X), apoptosis (cleaved caspase 3) and the cell cycle (pHH3) (scoring data not shown). The sampling timepoint, which gave us the greatest insight into the mechanistic effects of these agents was that taken when we started to see the TMZ and combination groups diverge (PD2). As expected, in response to selumetinib or TMZ alone we saw changes in the mechanistic biomarkers pERK1/2 (decrease) and γH2A.X (increase), respectively, and a reduction in mitotic cells as shown by pHH3 in the selumetinib group compared with an increase in the TMZ tissues  followed 24 h later by selumetinib (25 mg kg–1 per bid) for 7 days (schedule 1) or selumetinib was administered as above for 7 days followed 24 h later with docetaxel (schedule 2) (P<0.0005) and 50% (P<0.005) tumour growth inhibition, respectively (P<0.0005) compared with 61% (P<0.005) tumour growth inhibition when docetaxel was administered after selumetinib (schedule 2). These results suggest that selumetinib could enhance the efficacy of docetaxel when administered in the sequence of docetaxel followed by selumetinib.Continuous, concurrent combinations of selumetinib and docetaxel resulted in significant anti-tumour effects in several models . Howeveredule 2) . As monoectively . InteresP<0.0005). In schedule 2, pHH3 levels were seen to increase following docetaxel (PD7)   consistent with the inhibition of the MEK/ERK pathway resulting in a G1/S arrest. Interestingly, in both sequences in the combination there is a reduction in pHH3 compared with the docetaxel alone group at that timepoint (PD2 and PD7) .In order to determine the mechanism of action in the scheduling studies with docetaxel, a series of tumour tissue samples were taken at different time points following exposure to the two dosing regimens . To asse<0.0005) . When seP<0.0005) compared with the single agents alone    PD2. In ctively) . Represectively) . The dat–1 per qd for 2 consecutive days through a mini-pump (MP) with a 24 h gap followed by selumetinib 25 mg kg–1 per bid for 14 consecutive days (schedule 1) or the reverse of this schedule where barasertib was dosed following selumetinib treatment (schedule 2) (P<0.005) and 95% (P<0.0005) tumour growth inhibition compared with the vehicle-treated controls at day 21 after the start of dosing. In comparison when selumetinib was dosed before barasertib the anti-tumour efficacy was 74% (P<0.0005) in contrast to 106% (P<0.0005) observed when selumetinib was dosed following barasertib. At 21 days after the start of dosing the control, animals had to be culled due to tumour size; however, the monotherapy and combination-treated groups were kept on study for a further 14 days. During this time, the tumours in the monotherapy and schedule 2 groups started to re-grow. Interestingly, the tumours in the group where barasertib was administered before selumetinib (schedule 1) tumour re-growth was delayed  (16). We designed two regimes in which barasertib was dosed at 150 mg kgedule 2) . The CaL delayed .P<0.05) in the PD1 samples consistent with the mechanism of this agent  and at the end of the selumetinib-dosing period (PD2) . In scheis agent  (WilkinsP<0.0005) in the sub G1 population in the combination compared with the vehicle-treated controls and selumetinib monotherapy , there was a significant increase  results in an enhanced anti-tumourigenic phenotype . Work prAlthough concurrent dosing of selumetinib and docetaxel is highly effective in pre-clinical models . ATM-dependent signalling through the MEK/ERK pathway is critical for efficient HRR and for radiation-induced ATM activation and is suggestive of a regulatory feedback loop between ERK and ATM (In addition to mitotic-targeting agents, we also investigated the benefit of combining selumetinib with the DNA-alkylating agent TMZ/DTIC, a standard of care treatment for melanoma patients. In our studies we used TMZ, as opposed to the registered agent DTIC, as it has more reproducible pharmacokinetics and does not require enzymatic conversion in the liver (In summary, concurrent combination of the MEK1/2 inhibitor selumetinib with a number of conventional chemotherapeutic agents, or barasertib, results in enhanced anti-tumour efficacy in human tumour xenograft models. The role of Ras/RAF/MEK/ERK cascade in key cellular events such as the cell cycle, apoptosis and DNA damage, highlights the potential of combining MEK inhibitors with chemotherapeutics belonging to several functional classes. The work presented here has been able to link the enhanced anti-tumour effects seen when selumetinib is combined with TMZ or docetaxel to enhanced DNA damage and apoptosis, respectively. Furthermore, our studies have also explored the rationale for sequence scheduling when MEK inhibitors and agents targeting mitosis are combined. The work presented in this study does offer a greater mechanistic understanding of these combinations to help support clinical trial designs. Overall, our pre-clinical observations demonstrate mechanistic rationale that combinations of selumetinib with several standard chemotherapeutics or an Aurora B inhibitor may offer clinical benefit.


Neutrophil gelatinase-associated lipocalin (NGAL), measured early after cardiac surgery, has been demonstrated to predict postoperative acute kidney injury (AKI). Fluid overload potentially masks a subsequent acute renal function loss through dilution of serum creatinine and maintenance of urine output just above AKI-defining criteria.We investigated the early postoperative value of NGAL versus that of simultaneously measured serum creatinine to predict subsequent fluid overload. We studied 100 adult cardiac surgery patients in the control arm of a RCT (NCT00672334). Severe postoperative fluid overload was defined as positive fluid balance >10% of preoperative body weight within 48 hours after surgery.Severe postoperative fluid overload was present in 5% of patients with a mean positive fluid balance of 15.8 ± 9.5 l. At ICU admission, urine NGAL predicted severe fluid overload (AUC-ROC 0.82 (95% CI = 0.70 to 0.94)) (Figure Early NGAL-guided adjustments to fluid management may reduce organ edema after cardiac surgery. Findings should be validated in larger cohorts.


There is an error in the National Science Council grant number. The correct grant number is, "NSC 99-2314-B-532-002-MY2."


We are the first to report one-staged resection of a spinal metastasis from malignant cranial hemangiopericytoma after preoperative Onyx™-20 embolization by direct percutaneous puncture.Spinal metastases from cranial hemangiopericytoma are extremely rare. Surgical morbidity of these highly vascularized tumours results mainly from excessive blood loss. Preoperative embolization of hyper vascular tumours has been used to reduce intraoperative blood loss for a long time. To avoid complications from arterial catheter intervention, direct percutaneous puncture has been advocated as a safe and effective alternative.A 46-year-old man with a history of malignant cranial hemangiopericytoma deriving from the left frontal skull base presented with a short history of lower back pain. A magnetic resonance imaging scan revealed an intra- and extra spinal mass lesion of the thoracic spine at Th 12. Indication for tumour resection was made and the patient’s written consent was obtained. Preoperatively, arterial catheter angiography was performed to reveal the tumour’s angioarchitecture, revealing high-flow arteriovenous shunts. In order to impede the expected perioperative blood loss, tumour embolization by direct percutaneous puncture and application of Onyx™-20 was performed prior to surgery.After percutaneous Onyx™-20 embolization, complete and safe resection of the lesion could be achieved. There was only minimal blood loss perioperatively. A pathohistological report confirmed malignant, anaplastic hemangiopericytoma.In our case Onyx™-20 embolization via direct percutaneous puncture of a highly vascularized tumour was shown to be a safe and efficient tool prior to surgery. Despite high-flow arteriovenous shunts, direct percutaneous administration of non-adhesive ethanol liquid was an efficient alternative to transarterial catheter embolization. The perioperative blood loss could be substantially diminished. Hemangiopericytoma (HPC) is a rare mesenchymal tumour that is predominantly found in the pelvis, retro peritoneum and lower extremities, occasionally occurring in the larynx, spleen or bones of the thorax, or the meninges. HPC locTo the best of our knowledge, this is the first report of an interdisciplinary treatment of a HPC by percutaneous Onyx™ embolization and microsurgical resection. We present our result of safe excision after near complete preoperative devascularisation of a spinal metastasis of malignant HPC using a percutaneous embolization technique with Onyx™.A 46-year-old man suffering from cranial HPC initially resected in 1996 presented with a four-month history of lower back pain. A neurological examination on admission revealed local pain at the thoracolumbar junction and decreased response to pin prick and light touch at the right-sided L1 dermatome. A magnetic resonance imaging (MRI) scan of the thoracolumbar spine displayed an extensive, contrast-enhancing mass of the L1 vertebral body, originating from the right vertebral arch, extending extra- and intraspinally with a substantial mass effect to the spinal cord  and a resection of the tumour was performed under neurophysiological monitoring. Resection of the tumour was facilitated by the intratumoural Onyx™-20 cast and complete tumour removals were achieved. Perioperative blood loss was kept to a minimum (<50 ml).The spinal metastasis of the HPC could be resected completely and safely with an interdisciplinary approach. Histopathology workup confirmed metastasis of anaplastic (WHO °III) HPC. A postoperative MRI scan confirmed complete removal of the tumour Figure A, B. AdvSpinal metastases of HPC are rare, yet highly vascularized, tumours. Hence, sole surgical removal without preoperatively diminishing the blood supply of the tumour might lead to a significant blood loss associated with a high peri- and postoperative morbidity and incomplete tumour resection, which in turn is associated with a lower progression-free survival period. TherefoPreoperative embolization of a spinal metastasis of a meningeal HPC has been only reported on a single occasion so far. Direct Here, we report the preoperative embolization of a spinal metastasis of malignant cranial HPC by direct percutaneous puncture prior to surgical removal. Complete obliteration could be achieved by using nonadhesive Onyx™-20 as the sole embolic agent. This procedure was indicated as the extended and dilated vessels of the venous drainage consecutively leading to high arterio-venous shunts hindered transarterial embolization due to the risk of uncontrolled distribution of Onyx™-20 beyond the tumour bed. Moreover, tiny feeding arteries, not feasible for selective catheterization were amenable to percutaneous injections of Onyx™-20, hence leading to gradual devascularisation of the complete tumour. There are several embolic agents comprising mechanical devices, particles and fluids. According to the literature, the use of Onyx™-20 as a nonadhesive liquid embolic agent proved to be safe because the injection rate chosen can be very slow, resembling a lava-like flow. Thus, tIn the presented case, the preoperative embolization of a hyper vascular spinal metastasis of hemangiopericytoma with Onyx™-20 by percutaneous puncture was shown to be a safe and efficient technique. In cases with high AV shunt, direct puncture and embolization seems to be superior to transarterial catheter embolization, reducing the risks of uncontrollable outflow of embolic agents via the pathological angioarchitecture of the tumour. Percutaneous tumour embolization with immediate surgical intervention should be considered for large hyper vascular spinal tumours with high AV shunts as it may decrease the risk of intraoperative haemorrhage and facilitate gross total resection. However, we believe that more prospective studies have to be initiated to evaluate the possible beneficial effect of the direct percutaneous embolization of hypervascularized tumours prior to surgery.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.CNS: Central nervous system; CT: Computed tomography; HPC: Hemangiopericytoma; MRI: Magnetic resonance imaging.The authors declare that they have no competing interests.NE, OM, and AR carried out the procedures. US and MF participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


Mycobacterium immunogenum, a newly identified member of the Mycobacterium chelonae_M. abscessus complex is considered a potential etiological agent for hypersensitivity pneumonitis (HP) in machine workers exposed to contaminated metalworking fluid (MWF). This study investigated the biocidal efficacy of the frequently applied commercial formaldehyde-releasing (HCHO) biocides Grotan and Bioban CS 1135 and non-HCHO type biocides Kathon 886 MW (isothiazolone) and Preventol CMK 40 (phenolic) toward this emerging mycobacterial species (M. immunogenum) in HP-linked MWFs, alone and in presence of a representative of the Gram-negative bacterial contaminants, Pseudomonas fluorescens, using two semi-synthetic MWF matrices (designated Fluid A and Fluid B). Relative biocide susceptibility analysis indicated M immunogenum to be comparatively more resistant (2–1600 fold) than P. fluorescens to the tested biocides under the varied test conditions. In terms of minimum inhibitory concentration, Kathon was the most effective biocide against M. immunogenum. Fluid factors had a major effect on the biocide susceptibility. Fluid A formulation provided greater protective advantage to the test organisms than Fluid B. Fluid dialysis (Fluid A) led to an increased biocidal efficacy of Grotan, Kathon and Preventol against M. immunogenum further implying the role of native fluid components. Used fluid matrix, in general, increased the resistance of the two test organisms against the biocides, with certain exceptions. M. immunogenum resistance increased in presence of the co-contaminant P. fluorescens. Collectively, the results show a multifactorial nature of the biocide susceptibility of MWF-colonizing mycobacteria and highlight the importance of more rigorous efficacy testing and validation of biocides prior to and during their application in metalworking fluid operations. Modern water-based metalworking fluids (MWFs) used as coolants and lubricants in many metalworking industries are available as synthetic or semi-synthetic formulations. These fluids are vulnerable to microbial colonization, the extent of which may depend on the fluid composition. Microbial growth reduces the quality and effectiveness of these fluids and may cause occupational health hazards in the exposed machinists –4. DilutMycobacterium chelonae_M. abscessus complex in fluids associated with occupational respiratory illnesses [M. immunogenum has been reported as the predominant or one of the predominant species colonizing MWF [Several studies have suggested a possible relationship between MWF microflora and respiratory health hazards in metal workers particularly hypersensitivity pneumonitis (HP) ,3,5–7. Illnesses –17. In pzing MWF –23 and czing MWF ,15,18.M. immunogenum, an isolate from metalworking fluids associated with occupational HP, and P. fluorescens, a representative of the Gram-negative organisms in these fluids. These test organisms were studied individually and in mixed suspensions with an aim to determine the effect of fluid types, fluid dialysis, fluid use, and microbial mixture on biocide efficacy in MWF matrix. To our knowledge this is the first comprehensive biocide evaluation study in commercial semi-synthetic MWF formulations against the MWF-isolated Mycobacterium species alone and in presence of a common Gram-negative contaminant.Limited controlled studies have been undertaken on evaluation of the effect of commercially available biocides on specific microorganisms of occupational health significance in industrial MWF formulations and effect of various fluid factors affecting their biocide efficacy –27. ReceM. immunogenum and P. fluorescens, showed increased estimated MIC of the HCHO-releasing biocides (2.7–3.6 fold) in Fluid A as compared to Fluid B matrix  in Fluid B compared to Fluid A  in Fluid A was observed with non-HCHO biocides  against unogenum . This waunogenum . Howeverbiocides .versus Fluid B). For M. immunogenum, HCHO-biocides (Grotan and Bioban) were effective at lower concentrations in Fluid B as compared to Fluid A. On the other hand, non-HCHO biocides (Kathon and Preventol) were effective in Fluid A at lower concentrations. This could be because of a role of compatibility between the biocide and the fluid composition. In this context, earlier studies had suggested that interaction between corrosion inhibitors (ingredients in MWF) and added biocides was crucial in the biocidal efficacy outcome [P. fluorescens showed a different pattern of biocidal activity as all four biocides (both HCHO- and non-HCHO) were in general effective at lower concentrations in the Fluid B matrix as compared to Fluid A. This indicates the role of organism type as an additional factor in the biocide-MWF interaction phenomenon in determining the overall biocidal efficacy. In other words, the biocidal activity depends on the interaction among the active ingredient of the biocide, the native inhibitory components of the fluid, and the target organism.Taken together, our results revealed a differential biocidal activity of HCHO- and non-HCHO biocides in the two fluid formulations  Fluid A was selected to study the effect of dialysis on the efficacy of the test biocides. The dialysis step was meant to investigate the role of fluid constituents less than 3500 molecular weight size in biocide performance.M. immunogenum showed a mixed trend on the effect of fluid dialysis on the biocide efficacy either singly or in mixed suspension. For instance, Grotan showed increased MIC estimates (P. fluorescens showed an increased MIC estimate (1.1–2 fold) for both the HCHO-biocides in the undialyzed fluid compared to dialyzed matrix  in dialyzed fluid  against ed fluid , 4C. P. ed fluid , 4D. ColM. immunogenum against Grotan, Kathon and Preventol. This trend might be either because of removal of certain native fluid components potentially showing antagonism toward the biocide or due to a protective effect of <3500 molecular weight (M.W.) size components of the fluid that got removed by the dialysis process.Taken together, the fluid dialysis pre-step did seem to increase the biocide susceptibility of P. fluorescens. This protection may be due to removal of certain native components (<3500 M.W.) inhibitory to this organism. In contrast, HCHO-biocides showed increased biocidal activity in dialyzed as compared to undialyzed matrix, showing a possible protective effect of the native components toward the test organism. In an earlier study, specific MWF components including borate esters, triethanolamine, dicarboxylic acid, phosphate ester and polyglycol were shown to serve as sole carbon and/or nitrogen sources for the bacterial isolates and were indicated to be possibly responsible for the observed protective effect toward biocide action [On the other hand, a protective effect of the dialyzed fluid toward non-HCHO biocides (Kathon and Preventol) was observed for e action . Grotan e action  could bee action .M. immunogenum and P. fluorescens) in either (single or mixed) culture suspension. A 2.5–3.4 fold increase in MIC estimate was observed in the used fluid compared to unused fluid when the two HCHO-biocides were tested against M. immunogenum  was required to kill the organisms in the used fluid (Both the HCHO-biocides were less effective in the used fluid matrix than unused (pristine) fluid matrix against the two test organisms (unogenum . In caseed fluid .M. immunogenum showed a mixed trend in the used versus unused matrix. Kathon efficacy toward the single culture (pure) suspension was higher in the used fluid matrix  when compared to the unused matrix  as compared to the used fluid  whereas a reverse pattern was true for the mixed culture suspension (refined MICs of 6000 ppm in unused versus 4000 ppm in used).Interestingly, the efficacy of the non-HCHO biocides toward unogenum  in singlM. immunogenum, P. fluorescens showed a clear trend of increased MIC estimate (3–20 fold) for non-HCHO biocides in used fluid compared to unused fluid matrix , drop in pH, and formation of enzymes could cause the resistance development and in turn lead to the enhanced survival of microorganisms [Taken together, the results showed that the fluid use, in general, increased the antimicrobial resistance of the two test organisms, rganisms . PotentiM. immunogenum was comparatively more resistant (2–1600 fold) than P. fluorescens with the tested biocides under the varied test conditions as also indicated by a much higher MIC range for the former than the latter  than the HCHO-biocides  with some exceptions. Likewise, for P. fluorescens, the non-HCHO biocides required lower doses (MICs < 100 ppm) as compared to the HCHO- (>100 ppm) biocides  against chemical biocides. Overall, the results of this study indicated that the non-HCHO biocides (Kathon and Preventol) are more efficient in controlling the test organisms, M. immunogenum and P. fluorescens, compared to the HCHO-releasing biocides (Grotan and Bioban). Between the two non-HCHO biocides, Kathon (an Isothiazolone biocide) was the better performing biocide than Preventol (a phenolic biocide). In contrast, no such clear difference was observed between the efficacy of the two HCHO-releasing biocides (Grotan and Bioban). Although Kathon appeared to be the most effective among the four tested commercial biocides against Mycobacterium, there was some degree of Kathon resistance observed in this organism as compared to Pseudomonas. Isothiozolone (the active component in Kathon) is known to be inactivated by thiols. Mycobacteria produce mycothiol, a unique thiol compound comprised of N-acetylcysteine amide [M. immunogenum to Preventol [e latter . For M. biocides . More bine amide . The mycne amide  thereby istant 2–600 fold ne amide . Howeverreventol .M. immunogenum could be partially attributed to their mode of action, which involves surface activity of the released active component  on bacterial cells [The relatively lower effectiveness (in terms of MIC) of HCHO-releasing biocides, Grotan and Bioban, against al cells . The hydal cells .M. immunogenum was found to be more resistant in mixed suspension than in pure suspension, to both HCHO- and non-HCHO biocides in all matrices; the exceptions being Bioban and Preventol in used Fluid B matrix. The relative biocidal resistance of P. fluorescens in mixed suspension toward different test biocides in all matrices was also increased, except for Grotan in dialyzed Fluid A. An increased biocidal resistance of both M. immunogenum and P. fluorescens in presence of a co-contaminant suggests a possible protective mechanism among the microbial communities in MWF environment leading to their enhanced ability to resist biocide. The extent of protection varied with the biocide possibly due to differential distribution of the individual biocides between the two organisms and a consequent dilution effect. Reasons for the exceptions (Bioban and Preventol for M. immunogenum and Grotan for P. fluorescens) are however not clear.In general, both the test organisms showed increased biocide resistance (0–4 fold) in mixed culture suspensions compared to single culture (pure) suspensions except in a few instances. Mycobacterium immunogenum (ATCC 700506), an MWF-isolate, and Pseudomonas fluorescens (ATCC 13525), a representative of the Gram-negative organisms in MWF, were used in this study. The organisms were cultured using M7H9 broth and M7H10 agar  supplemented with Oleic acid-Albumin-Dextrose-Catalase enrichment . The cultures of M. immunogenum and P. fluorescens were grown in 40 mL of broth with continuous shaking (150 rpm) at 37 °C for 120 h and at 25 °C for 24 h, respectively.Two test organisms, namely, Two commercial semi-synthetic metalworking fluid formulations, arbitrarily designated for proprietary reasons as Fluid A (pristine) and Fluid B (pristine or in-use), were obtained from an industrial setting for use in this study. According to the manufacturer’s specifications, Fluid A is a “biocide free” formulation whereas Fluid B is a “biocide added” formulation and the two have some basic compositional differences as well. Initially, we used a 5% (v/v) dilution of these MWF formulations but no bacterial growth was observed in either of the matrices indicating the presence of certain inhibitory constituents in these formulations. This warranted the selection and use of a noninhibitory fluid concentration (2% v/v), standardized in our hands for the purpose, for the two test organisms (data not shown). A field used version of Fluid B obtained from the same commercial source that supplied the pristine fluid was used for comparison in this study.Four commercial biocides were evaluated in this study. Of these, two biocides were HCHO-releasing type, namely Grotan  and Bioban CS 1135 . The active ingredients were hexahydro-1,3,5-tris(2- hydroxyethyl)-s-triazine (78.9%) for Grotan and 4,4-Dimethyloxazolidine-3,4,4-trimethyloxazolidine (76%) for Bioban. An isothiazolone group biocide, Kathon 886 MW  with 14.1% 5-chloro-2-methyl-4-isothiazolin-3-one as the active ingredient and a phenolic biocide, Preventol CMK 40  with 4-chloro-3-methylphenol sodium salt (46.13%) as the active ingredient, were also tested.Dialysis strategy (using 3500 M.W. cut-off membrane tubing) was used to get rid of the native inhibitory components in Fluid A matrix and to remove the blended biocide as well as native inhibitory components in Fluid B matrix. To study the effect of fluid dialysis process on inhibitory potential of a test biocide, 2%-undialyzed and 2%-dialyzed forms of pristine Fluid A were compared. To study the effect of fluid usage, dialyzed version of the pristine and used Fluid B matrices were compared.8 CFU/mL) in MWF matrix for the biocide efficacy experiments. To understand the effect of co-contaminantion on biocide sensitivity of individual test organisms, the test fluid was spiked with the test organisms, individually or in mixture (1:1 ratio), and the minimum inhibitory concentration (MIC), in terms of concentration needed to kill all cells, was determined for individual biocides.Same batch of the culture for a given test organism was used in all treatments with a given test biocide to minimize the experimental variations. The bacterial cultures were grown in 40 mL of Middlebrook (MB) broth at corresponding temperature-time conditions to a cell density equivalent to 120 Klett reading measured using Klett Photoelectric Colorimeter . The cell counts corresponding to this Klett reading were determined to estimate the dilution requirements for achieving a constant initial number of cells  for different contact times  to determine the MIC estimates. In the subsequent step, a refined MIC value was determined by varying the concentrations between the estimated MIC concentration and its nearest lower concentration, at the estimated contact time. For instance, if the MIC estimate observed in the first step was 10,000 ppm, then we tested 1000 to 10,000 ppm range using 1000 ppm increments in the second step. In case of M. immunogenum) and 25 °C for 24 h (P. fluorescens) as described earlier (24). In case of mixed suspension treatments, selective counts for the individual test organisms were obtained by differentially incubating the two different sets of M7H10 agar plates at either temperature of incubation (37 °C for M. immunogenum and 25 °C for P. fluorescens), as above.Viability of the test suspensions, after biocide treatment and serial dilution in neutral phosphate buffered saline (PBS), was estimated by plating on the M7H10 agar followed by incubation at 37 °C for 120 h  were needed as compared to the non-HCHO biocides (Kathon and Preventol) to completely inactivate M. immunogenum in semi-synthetic MWF matrix. The observations pointed to the limitation of these existing commonly applied commercial MWF biocides in terms of their efficacy to control mycobacteria in these fluids. Hence, in addition to the need for development and evaluation of novel biocides, screening of a library of existing non-HCHO biocides against MWF-relevant test strains may be pursued to identify potentially more effective biocides for MWF-colonizing mycobacteria. On the other hand, the study highlights the role of fluid factors in understanding the efficacy of biocides toward the test organisms in metalworking fluids. The results showed a fluid type-dependent differential biocidal efficacy, indicating the importance of compatibility between the biocide and MWF matrix. Fluid dialysis studies indicated varying interaction of the inherent components of MWF with individual biocide groups thereby affecting the biocide efficacies. Used fluid matrix conferred increased resistance to the test organisms. This implies that prior fluid colonization activities including microbial degradation of MWF and biocide and further metabolic activities of microbes on the degraded products may lead to enhanced survival and growth of microbial contaminants and poor longevity of a biocide. The observed mutual protection against biocide killing between P. fluorescens and M. immunogenum in a mixture indicates that once the microbial communities containing Gram-negative and acid-fast bacteria are developed in the metalworking fluid environment they are able to tolerate relatively higher concentrations of biocides possibly due to biocide distribution and dilution effect. The above observations on multifactorial nature of the biocide susceptibility of mycobacteria in MWF suggest that efficient biocide control of microorganisms in these fluids would require proper fluid management practices including regular monitoring of the critical fluid factors in an industrial setting.


The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [18F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [18F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [18F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3′-deoxy-3′-[ However, single agent clinical activity with these inhibitors has in general been modest, and hence combinations are being evaluated in vivo efficacy, with dose-dependent activity over the range 25-150 mg/kg/day in the U87MG glioblastoma xenograft model PIK3CA mutant, PTEN null, EGFR mutant or wild type, with an associated decrease in AKT and S6 phosphorylation As a single agent, the pan PI3K inhibitor GDC-0941 has modest preclinical in vivo as a single agent, doses of 10–25 mg/kg causing significant tumour growth inhibition and in many cases regression, in a range of murine and human tumour xenograft models, including those which were BRAF or KRAS wild type or mutant The allosteric MEK inhibitor PD 0325901 also exhibited promising selective pre-clinical anti-cancer efficacy As the MEK inhibitor PD 0325901 appeared promising as a single agent but showed toxicity in clinical trials, and tumour growth inhibition was modest with the PI3K inhibitor GDC-0941 even at high doses, these and other PI3K and MEK inhibitors are now being investigated clinically in combination studies In vivo pre-clinical studies have shown that combinations of PI3K and MEK inhibitors consistently result in improved tumour growth inhibition compared to either single agent, and in many cases cause regression in a variety of human tumour xenograft and mouse tumour models with a range of genetic backgrounds, including those with KRAS, BRAF and/or PIK3CA mutations, and/or PTEN deletions Pharmacodynamic biomarkers of MAPK and PI3K pathway modulation, such as those mentioned above, require repeated invasive biopsies and hence may not be clinically feasible. Furthermore, changes in tumour size or disease stabilisation, as measured by volumetric imaging methods such as CT and MRI, may not become apparent until after many weeks of therapy, which can delay clinical decision making and potentially result in patients inappropriately remaining on ineffective and toxic treatments for prolonged periods of time. To address the limitations of conventional volumetric imaging, positron emission tomography (PET) is being used in pre-clinical studies and clinical trials as a functional surrogate response imaging biomarker 18F]-fluorothymidine ([18F]-FLT) is a PET radiotracer that is used for detecting anti-proliferative effects, as accumulation in cells is determined by the expression and activity of the enzyme thymidine kinase 1 and specific nucleoside transporters, both of which are under the control of S phase cell cycle regulators 18F]-FLT has been shown to correlate with standard proliferation markers, such as Ki67, TK1 and BrdU uptake 18F]-FLT PET, changes in proliferation compared to baseline have been demonstrated in a variety of human tumour xenografts as early as 18, 24 and 120 hours using either single agent GDC-0941 or PD 0325901 18F]-FLT PET has already been used to predict the efficacy of chemotherapy and radiotherapy 18F]-FLT PET The fluorine-modified thymidine analogue, 3′-deoxy-3′-[18F]-FLT PET can be used as a surrogate response biomarker for combined MEK and PI3K inhibitor therapy, as a prelude to clinical trials.The aim of the studies described in this report was to determine whether -FLT was obtained from PETNET , and radioactivity levels were measured using a Capintec well counter . Approximately 10 MBq of [18F]-FLT was administered intravenously and a 1 hour dynamic PET scan was performed, consisting of ten 1 minute, six 5 minute and two 10 minute time frames. Mice were recovered after scanning and continued to receive treatment for the remainder of the study. Data were analysed using Imalytics software ; a 3D region of interest was drawn around the tumour and the standardised uptake value (SUV) was calculated by dividing the tissue concentration (MBq/ml) by the injected dose (MBq)/g body weight. Mean SUV values were then plotted against time, and the area under the curve (AUC) and the percentage change in the area under the curve relative to the baseline scan was calculated. Paired t tests were used to compare the day 2 versus the baseline SUV AUC values for each individual mouse within each treatment group and differences with a p value <0.05 were considered statistically significant.Before treatment and after 2 days of treatment as described above, mice bearing HCT116 human tumour xenografts (7–9 mice/group) were anaesthetised, cannulated versus 10 mg/kg, achieved plasma AUC values that were 7-fold higher. In the case of PD 0325901, a comparable dose increase, i.e. 10 versus 1 mg/kg, resulted in a 15-fold increase in plasma AUC , the PK data do not necessarily indicate that pharmacologically active drug concentrations were not achieved.After a single dose of 100 mg/kg GDC-0941, alone or in combination with 1 mg/kg PD 0325901, concentrations of GDC-0941 in the plasma and the tumour tissue consistently exceeded that of the Although, following 1 mg/kg PD 0325901, concentrations were below the limit of detection in the tumour (<0.4 nmoles/g), this dose was able to reduce (1 hour) and completely ablate (3 and 6 hours) the phosphorylation of ERK1/2, with very little recovery by 24 hours. As expected, there was no marked effect of PD 0325901 on AKT, S6 or 4EBP1 phosphorylation . GDC-094in vitro conditions under which synergy had been demonstrated previously in these cell lines Based on the results of the PK/PD study, the efficacy of 100 mg/kg of the PI3K inhibitor GDC-0941 and 1 mg/kg of the MEK inhibitor PD 0325901 given orally, as single agents and in combination, was assessed in HCT116 and HT29 human tumour xenograft-bearing mice . The indThe time for the tumours to quadruple in size (time to RTV4) was calculated , and sta18F]-FLT PET can be used as a surrogate biomarker for tumour response to therapy, and dynamic PET scans were therefore performed after two days of treatment, at which time there were no significant differences in tumour volume between the control or any of the treated groups. Unfortunately, [18F]-FLT uptake by HT29 tumours was low and this tumour could not be used for [18F]-FLT PET studies. In contrast, HCT116 tumours were [18F]-FLT avid and 18F]-FLT tumour uptake after treatment with control vehicle, 1 mg/kg PD 0325901 alone or 100 mg/kg GDC-0941 alone, compared to baseline. However, there was a significant decrease in [18F]-FLT HCT116 tumour uptake after 2 days of PI3K/MEK inhibitor combination treatment (It has been proposed that [reatment .18F]-FLT SUV versus time curve (AUC) in HCT116 tumours was calculated for each individual mouse. 18F]-FLT uptake at baseline and after 2 days of treatment in the control or single agent PD 0325901- or GDC-0941-treated mice. In contrast, there was a statistically significant decrease of 18% in the tumour [18F]-FLT uptake after 2 days in the PI3K/MEK inhibitor combination treated mice (p<0.005). These data demonstrate that changes in [18F]-FLT uptake precede effects on tumour volume, and that [18F]-FLT PET is a valid early surrogate response biomarker for detecting the improved efficacy of combined PI3K and MEK inhibitor treatment.Based on the data in BRAF-mutant melanoma, single agent clinical activity with targeted therapies is modest, presumably due to the presence of multiple driver genetic lesions and the rapid development of resistance mechanisms. Combinations of targeted therapies are therefore being widely investigated. However, in developing optimal combinations, conventional clinical trial methodology has significant limitations as the large number of drugs, patient numbers required and the time taken for response and survival endpoints to be reached precludes the timely evaluation of all potential combinations. Consequently, surrogate response biomarkers, such as [18F]-FLT PET, are being investigated in an attempt to generate early proof-of-concept data for the activity of specific combinations. The studies described here have shown that the combination of the PI3K inhibitor GDC-0941 with the MEK inhibitor PD 0325901 is more efficacious in vivo than either single agent given at the same doses, and importantly that the increased efficacy of the combination treatment correlates with a subsequent decrease in tumour [18F]-FLT uptake measured by PET after just 2 days of treatment.With some notable exceptions, e.g. imatinib in the treatment of chronic myeloid leukaemia and vemurafenib in vitro growth inhibition and cytotoxicity studies As reported elsewhere in vitro studies in HCT116 and HT29 cells in vivo after treatment with 100 mg/kg GDC-0941. In particular, the decrease in pERK1/2 levels is in contrast to previous reports that PI3K inhibition may cause the opposite effect as a result of the cross-activation of the MAPK pathway KRAS mutant MDA-MB-231 breast tumour xenografts despite pronounced inhibition of AKT phosphorylation The efficacy of GDC-0941 has previously been shown to be associated with decreased AKT and S6 phosphorylation in vitro GI50 value of GDC-0941 .In pre-clinical models, combinations of PI3K and MEK inhibitors have consistently shown improved efficacy compared to either single agent alone, causing striking regressions in some cases, in a range of human tumour xenograft and mouse models in vivo combination studies have demonstrated that, whereas the phosphorylation of components of the MAPK and PI3K pathways was restored within 72 hours, effects on downstream markers of proliferation and apoptosis were sustained for over 72 hours, suggesting that intermittent dosing of combinations of PI3K and MEK inhibitors may be preferable rd or 4th day with high doses of the PI3K inhibitor GDC-0941, in combination with the MEK inhibitors PD 0325901 or GDC-0973, resulted in marked tumour growth inhibition, and potentially reduced toxicity In HCT116 tumours, the combination of 100 mg/kg GDC-0941 and 1 mg/kg PD 0325901 was shown to inhibit both the MAPK and PI3K pathways, and enhance the inhibition of ERK1/2, S6 and 4EBP1 phosphorylation, compared to either agent alone. Previous studies have not only reported an inhibition of ERK1/2, AKT and S6 phosphorylation, but have also observed effects on downstream determinants of proliferation and apoptosis, such as a decrease in cyclin D1 and Mcl-1, and an increase in Bim1 accumulation and caspase 3 cleavage 18F]-FDG PET has been shown to be a surrogate marker of sensitivity to PI3K inhibition by NVP-BEZ235 and NVP-BKM120 in human HNSCC (FaDu) and mouse mammary (EMT6) 3D tumour spheroids in vitroin vivo18F]-FDG PET has been shown to be of value in monitoring the activity of the MEK inhibitor GDC-0973 in combination with the BRaf inhibitor vemurafenib in A375 and vemurafenib-resistant A375R1 melanoma xenografts To investigate the potential utility of PET scanning as an early surrogate biomarker of tumour response to PI3K and/or MEK inhibitor therapy, PET scanning has been incorporated into efficacy studies in a small number of pre-clinical and clinical studies. For example, [18F]-FLT also represents a promising proof of concept anti-proliferative PD and surrogate response biomarker for PI3K and/or MEK inhibitor therapy. [18F]-FLT PET can measure anti-proliferative effects, as it is a thymidine analogue whose accumulation in cells is determined by the expression and activity of thymidine kinase 1 and specific nucleoside transporters, which are under the control of S phase cell cycle regulators 18F]-FLT PET scans were therefore incorporated into the HCT116 efficacy studies described in this paper. The tumour uptake of [18F]-FLT was monitored over 1 hour at baseline and on day 2 of treatment with the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901, alone and in combination. As has been reported by other studies, [18F]-FLT uptake by the HT29 tumours was low 18F]-FLT PET studies.The PET radiotracer [versus pre-treatment dynamic PET scans showed that there was no significant difference in [18F]-FLT tumour uptake in HCT116 xenograft-bearing mice treated with drug vehicle or with either single agent, whereas there was a significant decrease in [18F]-FLT tumour uptake after PI3K/MEK inhibitor combination treatment, which correlated with the enhanced efficacy observed later in the study. There have been no published pre-clinical or clinical studies measuring [18F]-FLT uptake after PI3K and MEK inhibitor combination treatment. There are previous reports that the PI3K inhibitors GDC-0941 or NVP-BEZ235, or the MEK inhibitor PD 0325901, given as single agents caused significant decreases in [18F]-FLT uptake as early as 18, 24, 48 or 120 hours, associated with subsequent tumour growth inhibition in a variety of human tumour xenograft models 18F]-FLT uptake following combination therapy preceded HCT116 tumour growth inhibition, suggesting that [18F]-FLT PET could be used as an early surrogate response biomarker for combined PI3K and MEK inhibitor treatment. Clinical trials involving combinations of PI3K and MEK inhibitors should therefore be extended to include the use of [18F]-FLT PET, in parallel to other common proliferation markers, as a biomarker of early response to combination treatment.The day 2 in vivo, and that this anti-tumour effect can be predicted by measuring [18F]-FLT uptake at baseline and after 2 days of treatment. Pharmacodynamic analyses following the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Together these results suggest that in studies of PI3K and MEK inhibitor combinations [18F]-FLT PET can be used as an early proof of concept PD and surrogate response biomarker for detecting enhanced anti-proliferative and antitumour effects in a pre-clinical setting, and therefore warrants further testing in clinical trials.Overall, these studies confirm that dual targeting of PI3K and MEK can induce marked tumour growth inhibition The authors are very grateful to Paul Bevan, Dagmar Ewald, Carola Mala and Wolfgang Schmalix, Wilex AG, Munich, and Colin Stubberfield, UCB Celltech, Slough, for informative and stimulating discussions. Also, the authors are indebted to Celine Cano, Bernard Golding and Roger Griffin for supervising the synthesis of GDC-0941.


Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant.Breast cancer cell lines were cultured under either moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.Exposure of three different breast cancer cell lines to moderate (1% OThese data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion. Exosomes are biological nanovesicles (30 – 100 nm in diameter) constitutively released by cells through the fusion of multivesicular endosomes with the plasma membrane and subsequent release of intraluminal vesicles into the extracellular environment . Exosome2) by the action of specific O2-, iron- and 2-oxoglutarate dependent prolyl hydroxylases . (PDF 29 kb).Click here for file


P3, generated by class I PI3Ks, functions as tumour promotor by recruiting effectors involved in cell survival, proliferation, growth and motility. Conversely, there is evidence that PtdIns3P, generated by class III PI3K, functions in tumour suppression. Three subunits of the class III PI3K complex  have been independently identified as tumour suppressors in mice and humans, and their mechanism of action in this context has been proposed to entail activation of autophagy, a catabolic pathway that is considered to mediate tumour suppression by scavenging damaged organelles that would otherwise cause DNA instability through the production of reactive oxygen species. Recent studies have revealed two additional functions of PtdIns3P that might contribute to its tumour suppressor activity. The first involves endosomal sorting and lysosomal downregulation of mitogenic receptors. The second involves regulation of cytokinesis, which is the final stage of cell division. Further elucidation of the mechanisms of tumour suppression mediated by class III PI3K and PtdIns3P will identify novel Achilles’ heels of the cell’s defence against tumourigenesis and will be useful in the search for prognostic and diagnostic biomarkers in cancer.Phosphorylated derivatives of the membrane lipid phosphatidylinositol (PtdIns), known as phosphoinositides (PIs), regulate membrane-proximal cellular processes by recruiting specific protein effectors involved in cell signalling, membrane trafficking and cytoskeletal dynamics. Two PIs that are generated through the activities of distinct PI 3-kinases (PI3Ks) are of special interest in cancer research. PtdIns Eukaryotic cells contain very extensive intracellular membrane systems, and many vital cellular processes, such as metabolic reactions, signal transduction, cytoskeletal rearrangements, protein sorting and regulation of membrane dynamics, occur partially or entirely at membrane–cytosol interfaces. The main advantages of executing biochemical reactions on membranes include the limitation of substrate diffusion (i.e. limited to two dimensions instead of three) and the possibility of confining biochemical processes to restricted subcellular locations.P3, recruits several proteins involved in cell signalling to the plasma membrane, the most important one being the protein kinase AKT, which orchestrates various signalling cascades that promote cell growth and survival. On the other hand, class III PI 3-kinase (PI3K-III) is considered to be a tumour suppressor based on the findings that three of its accessory subunits, Beclin 1, UVRAG and BIF-1, have been independently identified as tumour suppressors whose partial or complete inactivation causes the increased occurrence of spontaneous tumours in mice and (probably) humans  through a WD40-repeat-containing β-propeller structure [A breakthrough in our understanding of how PI3K-III and its catalytic product control cellular functions came with the identification of the FYVE  domain, and the demonstration that this domain binds PtdIns3ndosomes . The finndosomes , and biocificity –23. Furtg domain –27. Alth domains . The hummains [P . Additiotructure ,31, and tructure –34.P-binding domains offered the possibility to design probes that reveal the intracellular distribution of this lipid. Early work revealed that a single FYVE domain from the endosomal protein HRS binds PtdIns3P with too low affinity to be useful as a probe. Remarkably, however, when two FYVE domains from HRS were fused in tandem (2xFYVE), the resulting construct could be used for imaging of cellular PtdIns3P with high sensitivity and specificity, presumably as a result of an avidity effect [P. Subsequently, other probes have been used for monitoring PtdIns3P, including the FYVE domain of SARA, which has an intrinsic ability to dimerize and therefore does not need to be expressed as tandem fusion [P is associated with the limiting and intraluminal membranes of endosomes at steady-state [P can also be detected at the plasma membrane of cells stimulated with insulin or lysophosphatidic acid [P is generated through the activity of PI3K-II [P on autophagosomes, especially on the inner membranes [P is formed on membranes of the endoplasmic reticulum (ER) [The identification of PtdIns3y effect . The facm fusion , and cerm fusion . In genedy-state . Subsequdic acid ,39. Beca PI3K-II , which hembranes , and, dulum (ER) . The impP were those associated with endosomal trafficking  machinery ,59. ThisP . Vps27/HESCRT-II ,34. The P during the early phase of autophagosome formation [P is enriched on inner side of the phagophore during autophagosome formation [Macroautophagy (referred to here as autophagy) is a process that involves the sequestration of cytoplasm by a double membrane called phagophore or isolation membrane, followed by fusion of the resulting autophagosome with endosomes and lysosomes  Fig. 2BFig. 2B. ormation ,66. Seveormation .Studies in yeast have revealed that a complex consisting of Vps34, Vps15, Vps30 and Atg14 is required for autophagy , and subP mediate autophagy? The only known PtdIns3P effector in autophagy in yeast is the Proppin protein Atg18 [P is required for autophagy [P-containing structures positive for DFCP1 (double FYVE domain-containing protein 1) that are considered to act as platforms for autophagosome formation. This suggests a role for WIPI2 in the progression from omegasomes into autophagosomes.How does PtdIns3in Atg18 , whose butophagy . Althougutophagy . A mammautophagy . InteresP accumulates in the bridge separating two dividing cells, the so-called midbody. PtdIns3P is frequently associated with the central, electron-dense part of the midbody, referred to as the midbody ring or the Flemming body, but can also be observed on small vesicles throughout the midbody region [P raises the question of whether its formation is required during cytokinesis, the final stage of cell division (P effector in cytokinesis. FYVE-CENT localizes to the centrosome in interphase cells and translocates to the midbody during telophase. This translocation appears to be mediated by the microtubule-based motor protein KIF13A. The precise function of FYVE-CENT during cytokinesis remains to be characterized, although one clue arises from the finding that TTC19, a protein that associates with FYVE-CENT and accompanies it on its translocation from the centrosome to the midbody, interacts with the ESCRT-III subunit CHMP4B [A surprising finding when using a green fluorescent protein-tagged version of the 2xFYVE probe was that PtdIns3y region . This lodivision . Indeed,t CHMP4B . This ist CHMP4B ,75, and t CHMP4B , and TTCbeclin 1 knockout mice develop spontaneous mammary tumours [The PI3K-III subunit Beclin 1 is monoallelically deleted in a large proportion of breast and ovarian cancers, and heterozygous  tumours . These f tumours . Further tumours ,7. Even  tumours . BecauseP-binding endosomal fusion regulator, Rabenosyn, is a tumour suppressor in flies [P in the endosomal sorting of mitogenic receptors [P binding ESCRT-0 protein that initiates further ESCRT recruitment to membranes, is not a tumour suppressor in Drosophila.Given the importance of endocytosis and lysosomal downregulation of growth factor receptors in attenuation of mitogenic cell signalling , one disin flies , and theeceptors –83. ArguP effector FYVE-CENT is functionally ablated, may represent a step in oncogenesis [P effector is a genuine tumour suppressor.The recent discovery that PI3K-III regulates cytokinesis has offered a third possible explanation for the tumour suppressor activity of this enzyme complex . Inhibitogenesis . It is iogenesis ,86, althP binding for its function [P binding subunits mediate membrane association of the retromer complex [As discussed in the present review, PI3K-III may theoretically function as a tumour suppressor via at least three different mechanisms . The invfunction , and thefunction . Further complex , which s complex ,90. EvenThe involvement of PI3K-III in diverse cellular processes raises the question of how this complex is recruited to the correct membranes at the right time. There is evidence that PI3K-III is recruited to early and late endosomal membranes through interactions with the small GTPases RAB5 and RAB7, respectively –93. LessP is formed by PI3K-III, we are also beginning to learn about the metabolism of this lipid. Three known routes for PtdIns3P metabolism have been described: degradation by lysosomal lipases, phosphorylation by the PtdIns3P 5-kinase Fab1/PIKfyve, and dephosphorylation by 3-phosphatases [P binding protein [P, can associate with PI3K-III on endosomal membranes [P formation and turnover, and it will be interesting to determine whether PIKfyve and MTM1/MTMR2 may play any role in tumourigenesis.Although considerable efforts have been made to understand how PtdIns3phatases . It is w protein , and thaembranes ,97. ThisP at the correct intracellular membranes, this has not been formally demonstrated and, to date, we do not know whether the catalytic subunit of PI3K-III, VPS34, is a tumour suppressor. Further studies should clarify this, and it will also be important to establish whether the catalytic function of VPS34 is required for its eventual tumour suppressor function.Even though it is assumed that PI3K-III functions as tumour suppressor through its ability to produce PtdIns3P generation, can this be exploited in cancer diagnosis and therapy? Because it is much easier to inhibit an enzyme pharmacologically than to boost its function, the tumour promotor PI3K-I is a more attractive drug target than the tumour suppressor PI3K-III. On the other hand, we know that PtdIns3P can be dephosphorylated and that PI3K-III undergoes negative regulation [P effectors in various cancers. Such studies should reveal mutational and expression-based signatures that can be used to predict the outcome of disease, and to guide the choice of therapeutic regimens.If we nevertheless accept that PI3K-III acts as tumour suppressor through PtdIns3gulation ,12, and gulation ,67,98,99


Key issues in early clinical trials of targeted agents include the determination of target inhibition, rational patient selection based on pre-treatment tumour characteristics, and assessment of tumour response in the absence of actual shrinkage. There is accumulating evidence that functional imaging using advanced techniques such as dynamic contrast enhanced (DCE)-magnetic resonance imaging (MRI), DCE-computerised tomography (CT) and DCE-ultrasound, diffusion weighted-MRI, magnetic resonance spectroscopy and positron emission tomography-CT using various labelled radioactive tracers has the potential to address all three. This article reviews this evidence with examples from trials using targeted agents with established clinical efficacy and summarises the clinical utility of the various techniques. We therefore recommend that input from specialist radiologists is sought at the early stages of trial design, in order to ensure that functional imaging is incorporated appropriately for the agent under study. There is an urgent need to strengthen the evidence base for these techniques as they evolve, and to ensure standardisation of the methodology. An increasing knowledge about the major pathways dysregulated in cancer has resulted in a large array of targeted pathway inhibitors submitted for phase I trials. Recent figures suggest that <10% of new molecules reach the market primarily because of a lack of demonstrable clinical activity . Other cvs metastatic lesions may be biologically different and these characteristics may change with treatment. This article reviews currently available functional imaging methods and discusses their potential for stratifying patients for targeted therapies and assessment of tumour response by measuring on-target and off-target effects. PubMed searches for the terms phase I trial, RECIST, antiangiogenic, cytostatic, molecular imaging, dynamic contrast enhanced (DCE)-magnetic resonance imaging (MRI), diffusion weighted (DW)-MRI, DCE-ultrasound (US), FDG-positron emission tomography (PET), treatment response and derivative references, were performed. Abstracts and articles judged most relevant to the goals of this report were reviewed with emphasis on limitations and strengths of the imaging approaches to treatment response assessment.Functional imaging biomarkers have the potential to quantify biological characteristics of tumours and measure on-target and off-target effects that indicate early likelihood of response to a specific therapy, which can then be used to guide the optimal biological dose and drug schedule. Serial, non-invasive assessments of whole tumour are possible. This is particularly important in the context of inter and intra-patient tumour heterogeneity as different parts of the tumour and primary The biological features of a tumour, for example, vascularity, hypoxia, cell turnover determine its characteristics on functional imaging. The currently available imaging techniques available in the clinic for identifying tumour characteristics are MRI, PET, computerised tomography (CT) and US. Most of the functional techniques use an additional external contrast agent or radioactive tracer. Perfusion imaging techniques exploit pathophysiological differences between ‘leaky’, disorganised, tumour neovessels and normal, well-organised vasculature. Serial images are acquired dynamically, through a volume of interest before, during and after administration of a contrast agent: gadolinium for MRI, iodinated contrast for CT and microbubbles for US. The acquired data are fitted to mathematical models to obtain quantitative parameters. The techniques are relatively simple to perform on standard clinical systems  but require strict protocols, careful acquisition, accurate contrast agent dosing and injection rate, image timing, and image analysis for quantification.Ktrans), size of the EES (ve) and IAUGC (area under the Gadolinium curve) for assessing antiangiogenic and vascular disrupting agents in clinical trials (Ktrans is often used as a marker for the permeability of tumour vasculature. Related measures such as the rate constant (kep), which describes the ratio of Ktrans/ve, have also been used. For DSC-MRI, the degree of signal loss observed is dependent on the vascular concentration of the contrast agent and tumour perfusion; the most common used parameters are relative cerebral blood volume (BV), relative cerebral blood flow (BF) (The most common methods of perfusion imaging are dynamic contrast-enhanced (DCE) and dynamic susceptibility contrast (DSC) MRI, the latter being used almost exclusively in brain imaging. Magnetic resonance imaging sequences are designed to be sensitive to the presence of contrast medium in the extracellular space (EES) on DCE-MRI (T1 or relaxivity-based methods) or to the vascular phase of contrast medium delivery (T2 or susceptibility-based methods) on DSC-MRI. For DCE-MRI, the resulting signal intensity within the tumour reflects a composite of overall perfusion, vessel permeability or ‘leakiness’ and the volume of EES. A recent consensus meeting has recommended the following readouts: volume transfer constant of contrast between the blood plasma and the EES  is relatively high, resulting in a limited use of CT for repeated scanning. The commonly used parameters include: BF, BV, MTT (mean transit time) and permeability -PET.Amongst, positron-emitting tracers, H18F-labelled fluoromisonidazole (18F-FMISO) is the most extensively studied PET tracer for imaging tissue oxygenation. Its use in clinical practice has been hindered by its relatively poor signal to noise ratio because of slow specific accumulation as well as slow clearance from normoxic tissues . The technique is sensitive to pO2 within and in tissues adjacent to perfused vessels and can be easily quantified but does not measure pO2 directly. pH, carbon dioxide tension and haematocrit can affect R2* readings and for correct interpretation of ISW-MRI the BV in the tissues of interest must be known (Intrinsic susceptibility weighted (ISW)-MRI also known as BOLD (blood oxygenation level dependent)-MRI relies on the paramagnetic properties of deoxyhaemoglobin, to generate contrast because of the increased MR transverse relaxation rate , has been correlated with cellular density, presence of necrosis and in the therapy setting, tumour cell apoptosis and proliferation indices (18F]fluorothymidine (18F-FLT) PET had initial encouraging results as a potential biomarker for tumour proliferation. Currently, there is not enough data to support its use for reliable assessment of therapy response  studies have demonstrated that proliferating cells and many tumours display elevated choline signals. The method has been successfully used in characterisation and detection of brain and prostate cancer and there is some evidence towards its use as a response biomarker following chemotherapy in breast cancer  of a combrestatin A4 derivative, AVE8062 in melanoma-bearing nude mice (which causes a rapid and selective shutdown of tumour neovasculature resulting in ischaemia and extensive haemorrhagic necrosis in xenografts) showed that intratumoural vessel depletion started 15-min post injection and was maximal at 6 h. This enabled planning of a clinical phase I assessment with DCE-US at 6- and 24-h post drug   of 12 months were seen in a small study  to EGFR inhibitors with a reduction in SUVmax of >10.4% at 7 days post gefitinib therapy predicting RECIST response on CT at 6 weeks  and a prolonged PFS – median, 33.1 vs 8.6 weeks  uptake at baseline is predictive of responsiveness to endocrine therapy  with a high tumour metabolism has been linked with aggressive cancer phenotypes in breast  is the most advanced such drug in development and the relatively limited benefit in tumour control in >1100 patients was likely partially due to poor patient stratification with inclusion of patients with better-oxygenated tumours in the trials. A recent report comparing cisplatin/5-FU Ktrans change following treatment with sorafenib  and brivanib  was 6.2% and 9.7% for serial measurements of NAA and choline, respectively . To account for differential response in phase I trials , identifADC obtained 3 weeks and PRMrBV and PRMrBF at 1 and 3 weeks into conventional therapy was prognostic for later radiographic response, time to progression and overall survival (OS) whereas percentage change in mean parameters  was less significant  are novel methods of image analysis using a voxel-by-voxel approach that can be applied to various functional imaging techniques such as perfusion or ADC maps. Two imaging volume data sets  are co-registered and computationally analysed to yield PRM, which are generally colour coded based on the magnitude and direction of the parameter change. The changes can be displayed as histograms or scatter plots. In malignant brain tumours, PRMKtrans, CBV and plasma collagen IV after one dosing of cediranib has been shown to correlate with PFS and OS in patients with recurrent glioblastoma  for each technique need to be set in order to measure treatment-induced changes that specifically relate to drug activity. Although available studies are small and confounded by inter-patient heterogeneity, generally data show that patients whose tumours undergo at least a 50% reduction in DCE-MRI parameters attain stable disease or response by RECIST; also, DCE-MRI can differentiate tumour progression from treatment-induced changes in the brain with 95% sensitivity and 78% specificity  of treatment will be indicative into tumour response , mature they will prove invaluable for patient selection and treatment monitoring.We therefore recommend that perfusion imaging should be routinely incorporated into trials of agents targeting tumour vasculature as a biomarker of antiangiogenic activity and techniques such as Currently, the use of functional imaging in large-scale multicentre clinical trials is prohibited by the limited technique reproducibility between centres and scanners, inadequate standardisation of data acquisition and analysis techniques and lack of necessary skills outside large centres. These obstacles are being addressed by programs such as the EU innovative medicine initiative in imaging, which should achieve standardisation of acquisition and analysis methodology for functional imaging biomarkers. There is an urgent need to strengthen the evidence base and increase familiarity with functional imaging techniques, through both single and multicentre studies.


There was information missing in the Funding. The complete Funding information is: This work was supported by NCI/NIH grant CA133449, CA 158895, GI SPORE CA95060, and NIH Arizona Cancer Center Support Grant CA023074. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Transthoracic echocardiography (TTE) is now widely used in the ICU to assess hemodynamic status. Combined mitral index measured by TTE, as the mitral Doppler inflow E wave velocity to annular tissue Doppler Ea wave velocity ratio (E/Ea), is a reliable diastolic indicator in cardiologic patients. In ICU, E/Ea has only been investigated as a pulmonary arterial occlusion pressure surrogate which poorly reflects fluid responsiveness (FR). Therefore, the aim of this study was to evaluate the reliability of E/Ea to FR in the setting of ICU ventilated patients.t test or chi-square test, ROC analysis and LHR method).We carried out a TTE prospective observational study in mechanically ventilated patients receiving fluid challenge for circulatory failure. Complete TTE examination involving stroke volume (SV) estimation, mitral and tissue Doppler measurements  were performed at end-expiratory time, before and after a 500 ml saline solution over 15 minutes of fluid challenge. A positive hemodynamic response was defined as a 15% minimal increment of SV. General characteristics, mitral parameters and combined index (E/A and E/Ea) were compared between responders (R) and nonresponders (NR)  n = 69, with no difference (R vs. NR). E/Ea values before fluid loading were not statistically different between R and non-NR for which we observed a huge overlap . The results were similar when considering the population with baseline under the median value; that is, E/Ea <8: 28 R versus 24 NR, E/Ea = 6.0 ± 1.5 versus 5.6 ± 1.5 R versus NR, P = 0.28. The E/A index was significantly lower in R  but poorly predicted FR: ROC curve AUC = 0.64 (0.54 to 0.74), best cut-off: 0.8 . Extreme values were predictive in our population: R was likely with E/A <0.6  and unlikely with E/A >1.8 .Ninety-four case-mix patients were enrolled: 43 R and 51 NR, with similar baseline characteristics. LV ejection fraction was: altered (< 50%) The E/Ea ratio is not statistically different between responders and nonresponders in the ICU and no low discriminant threshold value of E/Ea could identify patients likely to respond to fluid expansion. While E/A is statistically significant, only extreme values could be clinically relevant (< 0.6 or >1.8).


In the Funding statement, an additional grant from the Korea National Cancer Center is incorrectly omitted. The Funding statement should read: "This study was supported by grant-in-aids from the Korea National Cancer Center  and a grant (2009-022) from the Asan Institute for Life Sciences, Seoul, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."


Massive fluid resuscitation followed by hypoperfusion abnormalities in the ICU is a risk factor for development of intra-abdominal hypertension (IAH). The aim of our study was to determine what influence would have the control of daily fluid balance on the incidence of IAH in patients after extensive abdominal surgery or after abdominal trauma.n = 22) or after abdominal surgery (n = 60). During first 7 days the fluid intake and balance was monitored and corrected at 6-hour intervals not to exceed 1,500 ml positivity over 24 hours . IAP was measured from admission twice daily . A sustained elevation of the IAP above 12 mmHg in two consecutive measurements was considered as IAH.A prospective observational study included a total of 82 adult patients , number of patients with intra-abdominal pressure (IAP) above 12 at admission: 23 (28%), admitted to a single ICU with the diagnosis of abdominal trauma (The incidence of IAH in relation to daily fluid intake and daily fluid balance is shown in Figure The incidence of IAH in patients after abdominal surgery or abdominal trauma may exceed the value of 40%, especially in situations associated with massive fluid resuscitation . We have


TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling. Necc1 and its ortholog, Necc2, are preferentially expressed in neuronal, neuroendocrine, and endocrine tissues  and HEK-293 AD cells derived from human embryonic kidney 293 cells [ATCC (CRL-157)] were grown to 90% confluence and exposed for 4 h to low-serum differentiation media . Cells were then stimulated with NGF  for several time intervals, as indicated. For differentiation experiments, PC12 cells were seeded onto 25-mm poly L-lysine coated glass coverslips (25.000 cells/coverslip) and incubated in differentiation media containing 0,76 mmol/L of NGF for 4 days.Necc2. Twenty-four h later, transfected cells were selected with 1,4 µmol/L of geneticin. Selection efficiency was tested by flow cytometry for GFP expression, which was detected in 51% of the transfected cellsTransient transfection of cells was performed using Lipofectamine 2000. Transfected cells were used for experiments 24 h later. For short-term NGF treatments of cells overexpressing NECC2, T25 flasks of transiently transfected-PC12 cells were plated 1 day after transfection at a density of 100.000 cells onto 12-well plates. Cell viability was assessed by MTT assay according to the manufacturer’s instructions. To generate stably transfected cells, PC12 cells were transfected with 1 µg of pEGFP-RNAi or pEGFP-RNAi-Necc2 using specific primers . Nested PCR was carried out to amplify alternative spliced Necc2 isoforms using internal reverse primers . Non-DNA controls were run to control for exogenous contamination. PCR products were purified , and sequenced  to confirm identities of amplicons.Total RNA from PC12 cells was isolated using RNA-easy Mini Kit columns , and a 2-µg aliquot of total RNA was reverse transcribed with the cDNA First-Strand Synthesis kit using random primers . The cDNA was used as a template for standard PCR amplification of rat PC12 and HEK-293 AD cells grown on glass coverslips were processed for immunofluorescence and examined by confocal microscopy as described previously . In a seWhole-cell protein extracts were obtained by homogenization in RIPA buffer containing protease inhibitors. NGF-stimulated cells were lysed in SDS-dithiothreitol buffer . 60 µg oCytosolic (S) and crude membrane (P) fractions of PC12 cells were obtained by subcellular fractionation . ProteinCaveolin-enriched membrane fractions were isolated from PC12 cells grown to confluency . After uData from PC12 cells were obtained from a minimum of three independent experiments. Statistical analysis was performed using GraphPad Prism 4 . A one-way ANOVA followed by a statistical test for multiple comparisons (Newman-Keuls Multiple Comparison test) was applied to compare experimental treatments. For the analysis of the effects of NGF on TrkA signaling in the short-term, a Student’s t –test was used. Differences were considered statistically significant if p < 0.05. Data are expressed as mean ± S.E.M.The general features of the NECC2 sequence have been already depicted . FurtherNECC2 contains four predicted CRAC (cholesterol recognition/interaction amino acid consensus) domains , five diNecc2 allowed us to identify a novel Necc2 gene variant. This variant would originate by alternative intra-exonic splicing of exon 21, which codes for most of the C-terminal HR (residues 825–843). Alternatively spliced mRNAs would generate a short isoform of 844 amino acids containing the HR , and a 915 amino acids-isoform (106 kDa) containing an alternative C-terminus  resulted in no staining  revealed that NECC2 distributed diffusely throughout the cytoplasm and in close apposition to the plasma membrane . Preadsostaining . The abigenously . Moreovegenously . The antgenously . SimilarAD cells . The antAD cells .Given the presence of two overlapping CBDs at the C-terminus of NECC2, we carried out double immunofluorescence in PC12 cells using antibodies against the caveolae marker, caveolin-1, and the anti-NECC2 antibody. Examination by confocal microscopy revealed a high degree of overlap between NECC2 and caveolin-1 at the cell surface . ImmunolNecc2 ortholog, Necc1, which forms protein aggregates when transfected in PC12 cells [Necc2 and CFP-cav1 revealed the colocalization between the two proteins  transfected cells (see below). The anti-NECC2 antibody strongly immunolabeled exogenous NECC2 in cMyc-NECC2-transfected HEK-293 AD cells, whereas it did not recognize exogenous NECC1 in cMyc-NECC1-transfected cells  increased the association of NECC2 to the cell surface . SpecifiIn line with the microscopic data, immunoblot analysis revealed that NECC2 protein levels increased progressively during NGF-induced differentiation of PC12 cells .Necc2 silencing and overexpression on the phosphorylation rate of the main TrkA-activated effectors, ERK1/2 and Akt, in NGF-treated cells. To reduce NECC2 expression, PC12 cells were stably transfected with an expression vector encoding a shRNA for Necc2 (siRNA) (Necc2 siRNA-expressing cells). Decreased concentrations of NECC2 did not affect Akt phosphorylation at any time point analyzed  . Down-reanalyzed . In contFinally, we examined the effect of NECC2 overexpression on the phosphorylation of Akt and ERK1/2. To this end, PC12 cells were transfected with the NECC2 isoform lacking the HR domain, which colocalizes with TrkA, both at the cell surface and intracellularly , or withCaveolae are cholesterol- and sphingolipid-rich membrane microdomains, which, among other key functions, constitute specialized signaling platforms that allow physical localization and direct interaction of multiple types of receptors with their intracellular mediators . These sFluorescence microscopy using two different anti-NECC2 antibodies revealed that NECC2 co-localized with caveolin-1 at the cell surface of PC12 cells. NECC2 immunosignal was also closely associated with cortical actin, which has been shown to regulate the dynamics and localization of caveolin-1 in caveolae . AccordiSubcellular fractionation studies showed that endogenous NECC2 distributed mainly in the cytosol and, to a lesser extent, in membrane fractions. Detergent-free extraction of caveolae-enriched membranes and sucrose gradient fractionation analysis confirmed that both the endogenous protein and the NECC2 variant lacking the HR were located in non-buoyant fractions. However, full-length NECC2 containing the HR migrated to caveolae-enriched membrane fractions. These observations, which are consistent with the expression of several NECC2 variants in PC12 cells, suggest that NECC2 associates to caveolae membranes as an integral or a peripheral membrane protein depending on the presence or absence of the HR domain, respectively. In line with these findings, the family of long coiled-coil proteins ERC comprises two distinct members generated by alternative splicing, ERC1a and ERC1b, that differ in both their expression pattern and intracellular distribution . Thus, wNeurotrophic factor receptors have been found associated with lipid rafts in several cell types ,37–40. IOur microscopic and biochemical findings suggested that NECC2 might regulate TrkA-mediated NGF signaling in caveolae. In support of this proposal are our functional studies showing that decreasing NECC2 content by shRNA-mediated (siRNA) silencing altered NGF-induced phosphorylation of the TrkA downstream effector ERK1/2. Remarkably, NECC2 silencing had no effect on NGF-induced activation of Akt, which is also mediated via NGF binding to TrkA ,44. OverTrafficking, sorting, and ubiquitination of ligand-activated TrkA receptors regulate the magnitude and duration of the intracellular response evoked by NGF . It is wIn sum, our data are the first to demonstrate the presence of a long coiled-coil protein, NECC2, in caveolae, and to identify a potential regulatory role for this protein in the control of the NGF-TrkA system in PC12 cells.Figure S1Associated with Analysis of the specificity of anti-NECC2 and anti-NECC2-II antibodies by immunostainig. A. HEK293 AD cells were transfected with full-length cMyc-Necc2 or cMyc-Necc1 and doubled-stained with anti-cMyc (green) and anti-NECC2 antibodies (red). As shown in the binary mask (right panels), the anti-NECC2 antibody recognized exogenous NECC2, but not NECC1. Scale bars, 10 µm B. PC12 cells were immunolabeled with anti-NECC2-II (red) and anti-caveolin1 (green) antibodies. NECC2 distributes at the plasma membrane (left panel). Significant colocalization between NECC2 and caveolin-1 (green) is shown in the binary mask at the rightmost panels. C. cMyc-Necc2- or cMyc-Necc1-transfected HEK293 AD cells were immunolabeled with anti-cMyc (green) and anti-NECC2-II (red) antibodies. Anti-NECC2-II antibody recognized exogenous NECC2 but not NECC1. D. Immunoblot analysis of PC12 cell lysates using the anti-NECC-II antibody revealed a 110-kDa immunoreactive band along with two other bands of 140 and 95 kDa.(TIF)Click here for additional data file.Figure S2Associated with Intracellular distribution of exogenous NECC2 and Caveolin1. A. PC12 were transfected with cMyc-Necc2, GFP-Necc2, or untagged Necc2 and stained with anti-cMyc (leftmost panel) or anti-NECC2 antibodies (rightmost panel). Regardless of the reporter sequence used, NECC2 exhibited a juxtanuclear distribution in transfected PC12 cells. B. Representative confocal images of PC12 cells transfected with CFP-Caveolin-1 (GFP-cav1). CFP-cav1 located to the plasma membrane and also accumulated close to the nucleus, as has been previously reported for other cell types [ll types ,27. Scal(TIF)Click here for additional data file.Figure S3Associated with Analysis of the specificity of anti-NECC2 and anti-NECC2-II antibodies by immunoblotting. A. HEK293 AD cells were transfected with cMyc-Necc2 or cMyc-Necc1 and analyzed by immunoblotting using anti-cMyc, anti-NECC2, and anti-NECC2-II antibodies. Non-transfected HEK293 AD cells were used as controls. Exogenous NECC2 protein, but not NECC1, was detected with the anti-NECC2 and the anti-NECC-II antibodies. B. Immunoblot analysis of whole cell lysates from cMyc-Necc1- or cMyc-Necc2Δ285- transfected HEK293 AD cells. Anti-NECC-II antibody did not recognize cMyc-Necc2Δ285, the truncated form of NECC2 lacking the peptide sequence employed to obtain the anti-NECC2-II antibody. C. HEK293 AD cells were transfected with cMyc-Necc2Δ285 and double-stained with anti-cMyc (green) and anti-NECC2 antibodies (red). As shown in the images, anti-NECC2-II antibody did not recognize the truncated form, cMyc-NECC2Δ285. Scale bars, 10 µm.(TIF)Click here for additional data file.


A novel spectral transmittance approach for measuring the extinction coefficient of magnetic fluids is proposed. The measuring principle and accuracy of the approach are analysed. Experiments are conducted to measure the extinction coefficient of magnetic fluids with different particle volume fractions. The relative uncertainty of experimental data is less than 1.8%. The experimental results indicate that the extinction coefficient of magnetic fluids increases with increase of the volume fraction of suspended magnetic nanoparticles and the optical properties of the particle material have a significant effect on the extinction coefficient of the magnetic fluids. Magnetic fluid is a kind of colloidal suspension containing surfactant-coated magnetic nanoparticles dispersed in carrier liquids such as water and oil. Since the emergence of magnetic fluid in the 1960s, researchers have conducted many investigations on the characters of magnetic fluids. When light is incident into the magnetic fluid, the magnetic nanoparticles suspended in the base fluid will interact with the incident light and affect the propagation of the incident light. Magnetic fluid presents some special optical characters such as birefringence  and magnWith the development of the manufacturing technique, the spectral transmittance of a translucent film can be accurately measured by a spectrophotometer. Based on the film transmission principle, the method for measuring the extinction coefficient of magnetic fluids can be established. The procedure of our method is as follows: first, two magnetic fluid films with different thicknesses are prepared. The spectral transmittance of these films can be accurately measured using a spectrophotometer. Then, based on the film transmission principle, two transmittivity equations of the magnetic fluid films with regard to the optical constant of the magnetic fluid are established. Finally, by solving the two equations, the extinction coefficient of the magnetic fluid can be obtained.For fabricating a magnetic fluid film, two JGS3 glasses and a polyester film are needed. The glass has 50-mm width, 50-mm length and 1-mm thickness. The polyester film has a 30-mm-diameter circular hole in the central section. The polyester film is sandwiched in between the two glasses, and the magnetic fluid is injected into the circular hole. Then, the three-layer film is clamped by an attachment. We can obtain magnetic fluid film of different thicknesses by choosing polyester films of different thicknesses.R and the transmittivity T of the slab areFor a light beam with normal incidence on a slab (6) is a powerful method to predict the optical properties of composite materials. One of the most popular EMT is the Maxwell--Garnett theory (MGT) . In thisr,w is the dielectric function of water, εr,p is the dielectric function of Fe3O4 and ϕ is the particle volume fraction.where εn + ik and the dielectric function The relationship between the complex refractive index NM = tion  is (13)(14)(3O4 can be obtained from the research of Schlegel [3O4 were calculated, and the results are shown in Figure The dielectric function of FeSchlegel . Using E3O4 are both available. Based on the MGT formula, the dielectric function of magnetic fluid is calculated. Then, according to Equations 15 and 16, the extinction coefficients of magnetic fluid were predicted. The predicted extinction coefficients of magnetic fluids with different particle volume fractions are shown in Figure Since the complex refractive index of water can be obtained from Ref. , the diek = 1.25 × 10-7 from Ref. [k = 2.06 × 10-4. The latter is 1647 times than the former. This is because the size of suspended magnetic nanoparticles is much smaller than the incident wavelength. According to the Mie's scattering theory, the absorption of the incident light is the main optical character of these magnetic nanoparticles. The presence of the magnetic nanoparticles in water greatly enhances the total absorption of light. With an increase of the particle volume fraction, the number of particles in the unit volume increases, and the total absorption of light further increases.From the curves in Figure rom Ref.  and the 3O4 particles decreases with the increase of the wavelength (see Figure From Figure In this study, a simple spectral transmittance method was proposed for measuring the extinction coefficient of magnetic fluids. The extinction coefficients of magnetic fluids with different particle volume fractions in the continuous wavelength range of 500-800 nm were measured. The experimental data have revealed that the extinction coefficient of magnetic fluids increases with increase of the particle volume fraction, and the optical properties of the particle material have a significant effect on the extinction coefficient of the magnetic fluids. It is feasible to control the optical character of the magnetic fluids by changing the optical properties of the nanoparticles.EMT: Effective medium theory; MGT: Maxwell-Garnett theory.The authors declare that they have no competing interests.The experimental measurement and the theoretical calculation of this study were mainly done by the first author, XF. YX contributed to both the experimental and theoretical work and gave some useful suggestions to XF, which helped him finish the work. QL contributed to the experimental work and gave XF some help. This article was written by XF and revised by YX.