Orobanche minor seeds grown with tobacco. On the basis of the structure of abamine, several chemicals have been designed to specifically inhibit CCDs during SL synthesis. Cytochrome P450 monooxygenase is another target enzyme in the development of SL biosynthesis inhibitors, and the triazole-derived TIS series of chemicals is known to include SL biosynthesis inhibitors, although their target enzyme has not been identified. Recently, DWARF14 (D14) has been shown to be a receptor for SLs, and the D-ring moiety of SL is essential for its recognition by D14. A variety of SL agonists are currently under development and most agonists commonly contain the D-ring or a D-ring-like moiety. Several research groups have also resolved the crystal structure of D14 in the last two years. It is expected that this information on the D14 structure will be invaluable not only for developing SL agonists with novel structures but also in the design of inhibitors of SL receptors.Demands for plant growth regulators  are increasing globally, especially in developing countries. Both positive and negative PGRs are widely used to enhance crop production and to suppress unwanted shoot growth, respectively. Strigolactones (SLs) are multifunctional molecules that function as phytohormones, inhibiting shoot branching and also functioning in the rhizospheric communication with symbiotic fungi and parasitic weeds. Therefore, it is anticipated that chemicals that regulate the functions of SLs will be widely used in agricultural applications. Although the SL biosynthetic pathway is not fully understood, it has been demonstrated that β-carotene isomerases, carotenoid cleavage dioxygenases (CCDs), and a cytochrome P450 monooxygenase are involved in strigolactone biosynthesis. A CCD inhibitor, abamine, which is also an inhibitor of abscisic acid biosynthesis, reduces the levels of SL in several plant species and reduces the germination rate of  It is likely that PGRs will be utilized for large numbers of species and cultivars.Chemicals are widely used in agriculture to increase the yields of crops. For example, pesticides, including herbicides, fungicides, insecticides, and/or insect growth regulators, protect crops from the attack of pests that damage them, such as weeds, fungal diseases, and insects. Because pesticides usually protect crops by killing these pests, they are thought of as negative regulators of pests. However, because plant growth regulators (PGRs) are chemicals that control plant growth and benefit crop production by enhancing crop quantities and quality and by improving the postproduction quality of some plants, they are thought of as positive regulators of plants. In developing countries, such as China, the plant growth regulator industry has seen remarkable progress and shows attractive future market pot ential  biosynthesis. In this case, PGRs are considered plant growth retardants and are applied to agronomic and horticultural crops to reduce unwanted longitudinal shoot growth without lowering plant productivity . Their tlutarate . EnzymesThe importance of the chemicals that control plant function has recently been increasing, not only in agriculture but also in the plant sciences. The primary advantage of using bioactive chemicals to analyze the roles of plant hormones in plants, rather than plant-hormone-deficient mutants, is that they can be applied regardless of the plant species. The phenotypic changes induced by chemical treatments can reveal the physiological functions associated with the target proteins. Furthermore, genetic redundancy does not significantly influence the effects of these inhibitors. Plants treated with an antagonist or an inhibitor of biosynthesis show phenotypes almost identical to those of untreated plants, as is seen in multiple mutants when the target protein is redundant. Finally, chemicals easily regulate the functions of their target proteins only transiently . Therefobzr1 to identify the novel protein BZR1, which functions in the brassinosteroid signal transduction pathway . They are multifunctional molecules, acting as germination stimulants in root parasitic weeds, root-derived signals that induce hyphal branching in arbuscular mycorrhizal fungi, and plant hormones that regulate various phenomena, such as shoot branching, root morphology, secondary growth, and so on (Arabidopsis. AtMAX2 and AtDWARF14 (AtD14), an F-box protein and an α/β hydrolase, respectively, act in SL signaling  by As described above, chemicals that regulate the functions of SLs will be very useful, so several approaches have been used to develop chemically synthesized SL agonists. As a consequence, several chemicals have been reported that are germination stimulants of root parasitic weeds, regulators of shoot branching, and inducers of hyphal branching in arbuscular mycorrhizal fungi . One of Arabidopsis) in SL biosynthesis has been demonstrated (trans-β-carotene (3) to 9-cis-β-carotene (4) by D27 and cleavage of 9-cis-β-carotene (4) by CCD7 and CCD8 generates carlactone (5), which has a carbon skeleton similar to that of the SLs, including a methylbutenolide ring, a characteristic part of the SL structure . Because all of these enzymes include an iron atom in their molecules and nitrogen has a lone pair electrons that can form a coordinated bond with the 3D orbital of the iron atom, chemicals that include a nitrogen atom(s) in their molecules and have binding affinity for the substrate-binding pocket of these enzymes could be inhibitors of SL biosynthesis. For example, abamine (6), the first CCD inhibitor, which includes a nitrogen in its molecule, inhibits 9-cis-epoxycarotenoid dioxygenase (NCED) in the abscisic acid biosynthetic pathway and reduces abscisic acid levels in abamine (6)-treated Arabidopsis , CCD7, CCD8, and cytochrome P450 monooxygenase . Han et al. synthesized several analogs of stilbene and found that several types of lignostilbene analogs that contain nitrogen in the C–C bond are inhibitors of lignostilbene-alpha,beta-dioxygenase . Abamine (6) and abamineSG (7) contributed significantly to the recent findings that abscisic acid plays a role in controlling the number of nodules on the roots of leguminous plants . Because carotenoid biosynthesis is necessary for the biosynthesis of normal levels of SLs .Lignostilbene-alpha,beta-dioxygenase cleaves the olefinic double bond of phenolic stilbenes with a mechanism similar to that of NCED, a key enzyme in abscisic acid biosynthesis , which were designed based on the structures of abamine (6) and abamineSG (7), inhibited the activities of many CCDs, including AtCCD7, and increased the number of branches in inhibitor-treated Arabidopsis at a concentration of 100 μM  have been designed based on the structure of abamine . We selected TIS13 (11) as a candidate inhibitor of SL biosynthesis (Striga hermonthica than those of the control plants. These results strongly suggest that TIS13 (11) inhibits SL biosynthesis in rice. Because we considered TIS13 (11) a useful lead compound for developing specific and potent SL biosynthesis inhibitors, we performed a structure–activity relationship study of TIS13 (11) using chemical modification, which led to the identification of the more potent SL biosynthesis inhibitor, TIS108  is probably involved in the SL biosynthetic pathway in bidopsis  and therbidopsis . We screbidopsis , 2002, tynthesis . TIS13 . However, several biosynthetic steps in the SL biosynthetic pathway have still to be clarified and other P450s may be involved in SL biosynthesis. The target site of the TIS series will be identified in future studies.Because the TIS series (11 and 12) and tebuconazole (13) are triazole-type inhibitors and have potential affinity for the cytochrome P450s, their target could be CYP711A (Arabidopsis (Arabidopsis (branching inhibitor).Precise knowledge of the mechanism of molecular SL recognition will greatly assist chemists in designing and developing SL agonists and antagonists. Numerous studies have revealed that SL is received by D14 class of α/β hydrolase proteins in rice , Arabidobidopsis  and petubidopsis . Karrikibidopsis . Karrikibidopsis . While nbidopsis , and havbidopsis . Since DFigure 4A; On the basis of the results discussed above, it is assumed that the structural requirements for active SL are as follows: (1) it must be hydrolyzable by D14; and (2) it must produce D-OH after hydrolysis. These requirements are consistent with the hypothesis proposed by trans-β-carotene (3) by D27, CCD7 and CCD8 generates carlactone (5), which has a carbon skeleton similar to that of the SLs, including a methylbutenolide ring, a characteristic part of the SL structure . When applied exogenously, carlactone (5) rescued the shoot-branching phenotype of d27 and d10, but not that of d3, suggesting that carlactone is a biosynthetic intermediate of the SLs (epi-5-deoxystrigol (1) when 13C-labeled carlactone is fed to rice . However, it is still possible that carlactone (5) itself is recognized and hydrolyzed by D14 to produce D-OH and elicit SL activity, because carlactone (5) has a methylbutenolide ring connected to a carbon chain via an enolether moiety, which can be hydrolyzed to yield D-OH, as shown above. Recently, avenaol (18) was reported to mimic SLs in stimulating the germination of root parasitic plants , which have a dimethylbutenolide motif instead of the D-ring of SL. 3′-methyl-GR24 (20) and AR36 (21) show hormonal activity in pea, but not in parasitic weed germination or fungal hyphal growth, indicating that the dimethylbutenolide structure is recognized by the SL receptor involved in its branching-inhibition activity, but not by the receptor involved in the action of SL in the rhizosphere  was firstly identified as a molecule that increases SL synthesis and regulates light adapted growth through AtMAX2 in Arabidopsis  bind KAI2 and promote interaction between KAI2 and AtMAX2. These CTL compounds inhibits hypocotyl growth of Arabidopsis. These results indicate that they are SL agonist. Figure 4C). These three compounds showed SL activity in Arabidopsis hypocotyls and Striga seed germination assay. Although the germination stimulation activity of these lead compounds was much weaker than that of GR24, this approach can be useful to identify novel SL agonists.Cotylimide (CTL) compound . Using the LigandScout software (in silico screening method.With the recent increase in computer power and the development of bioinformatics algorithms, computer-assisted drug design, which utilizes the 3D structures of proteins determined with X-ray crystallography, has become a common method of drug discovery. Because we now have considerable information on the 3D structure of D14, we are planning to use an software , we haveIn this review, we have described recent attempts to design inhibitors of SL biosynthesis based on the recently accumulating knowledge of the enzymes involved in SL biosynthesis, and to design SL agonists and SL antagonists based on our recent model of SL recognition in plants. These chemicals have potential utility in both agricultural applications and in basic science, to dissect the mechanisms underlying the wide spectrum of SL functions. The stability of these chemicals is an important feature of their agricultural use. In addition to the trials described above, we anticipate that the protein–protein interactions necessary for SL signaling may be alternative and efficient targets for the design of novel inhibitors of SL functions. There are many examples of the pharmacological screening of inhibitors of protein–protein interactions , althougArabidopsis MAX2 interacts with BES1 and BZR1 (It has been reported that petunia DAD2, a petunia homolog of D14, interacts with petunia MAX2  and thatand BZR1 , which aThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Correlations between MET gene copy number increase, Chromosome 7 gain and MET protein overexpression were analyzed statistically. A highly significant correlation was observed between the percentage of tumor cells with MET gene copy number ≥3 and CEP7 copy number ≥3  across two subtypes of PRCC. In addition, the percentage of tumor cells with MET gene copy number ≥3 was found to increase along with increases in MET IHC score. This correlation was further confirmed in those PRCC tumor cells with average MET gene copy number >5 using combined IF and FISH methodology. Overall, this study provides evidence that Chromosome 7 gain drives MET gene copy number increase in PRCC tumors, and appears to subsequently lead to an increase in MET protein overexpression in these tumor cells. This supports MET activation as a potential therapeutic target in sporadic PRCC.To investigate the relationships between Chromosome 7 gain,  Papillary renal cell carcinoma (PRCC) is the second most common subtype of renal cell carcinoma (RCC) and accounts for 10% ~ 15% of all RCC in the West, with clear cell renal cell carcinoma (CRCC) accounting for 80% of all RCC . PreviouMET lead to the development of hereditary Type 1 PRCC [https://clinicaltrials.gov/ct2/show/NCT02127710. Accessed May 26, 2015.). In sporadic PRCC patients, MET gene mutation [et al reported MET gene copy number increases accompanied with high MET mRNA expression in a large cohort of 220 French PRCC patients [MET gene resides, frequently exhibits trisomy in PRCC [MET gene mutations commonly show trisomy 7 with non-random duplicated mutant MET genes and one wildtype MET gene [Mesenchymal-epithelial transition factor (MET) protein functions as a transmembrane tyrosine kinase receptor . When boe 1 PRCC –8, sparke 1 PRCC , and a pmutation  were als in PRCC –21, alsoMET gene copy number variation and MET protein expression level in PRCC tumor tissues from a cohort of Chinese patients.Nevertheless, the etiology of sporadic PRCC is still largely unknown especially in Asian patients, possibly due to the lower prevalence of the disease in Asia . Thus, oTumor samples were collected from 98 PRCC patients who underwent surgery between 2010 and 2013 at Ren Ji Hospital, Shanghai, China. Prior written informed consent was obtained from all patients and the study protocol was approved by the ethics committee at Ren Ji hospital. Adjuvant chemotherapy was administered to 6 patients, while 46 patients did not receive chemotherapy. Chemotherapy status for the rest of the 65 patients was not available. Survival data was only available for 54 patients and therefore overall survival was not included in the data analysis due to the low follow-up response rate (55.1%). Histological subtypes (Type 1 and Type 2) were determined after review of tumor sections by two independent pathologists. This study was approved by Renji Hospital Institutional Review Board.MET probe was derived by labeling BAC  DNA with SpectrumRed-dUTP . CEP7-SpectrumAqua probe, specifically targeting the centromere of chromosome 7, was purchased from Vysis  and used as an internal control. FISH signals were observed using a fluorescence microscope equipped with the appropriate filters to allow visualization of the intense red/aqua signals and the blue counterstained nuclei. MET gene and CEP7 copy number was scored on 100 tumor cells for each case. Cases with MET gene average copy number ≥6 were defined as MET amplification. The percentage of tumor cells with MET gene copy number increase (≥3) (%MET) and CEP7 copy number increase (≥3) (%CEP7) was further calculated to statistically assess the correlation between MET and chromosome 7 copy number variation by Spearman’s correlation analysis.Dual-color FISH assay was performed as previously described  on tissuIHC analysis was performed on a 4 μm thick TMA. MET immunostaining was performed using CONFIRM anti-total c-MET (SP44) rabbit monoclonal antibody  on an automatic immunostainer  following standard staining protocol from Ventana Medical Systems. Ki67 IHC was performed as follows: slides were dried at 37°C overnight and then baked at 56°C for 30min, then deparaffinized in xylene and rehydrated through a graded series of ethanol concentrations. Antigen retrieval was performed in PTlink using high pH target retrieval solution at 97°C, 15 minutes . Slides were covered with primary antibody solution  and incubated at room temperature for 60 minutes, then washed twice in TBS-T and incubated with the EnVision+ system-HRP labelled polymer anti-rabbit secondary antibody  for 30 minutes. Following two additional washes in TBS-T, slides were visualized using DAB substrate-chromagen . All slides were independently evaluated and results were agreed by two pathologists who were blinded to patient information.MET gene and CEP7 were applied and slides were co-denatured at 79°C for 6 minutes, and then incubated at 37°C for more than 30 hours. After overnight incubation, slides were washed with 0.3% NP40/2 x SSC (pH 7.0–7.5) at 72°C for 2 minutes, and then in 2 x SSC at room temperature for 2 minutes. Slides were finally dehydrated in ethanol series, air-dried, and mounted with mounting medium with DAPI . Slides were analyzed using fluorescent microscopy.4 μm thick PRCC sections were firstly de-waxed and pretreated in high pH antigen retrieval solution  at 95°C for 30 minutes. Sections were then treated with protease 3  for 6 minutes and then incubated in CONFIRM anti-total c-MET (SP44) rabbit monoclonal antibody for 1 hour. Goat anti-rabbit IgG FITC  was added and sections were incubated for 30 minutes at room temperature. After post-IF fixation in neutrally-buffered formalin for 10 minutes, sections were dehydrated in ethanol series and air-dried. Probes for p>0.05 was defined as statistically significant.Statistical analysis was performed using R statistical software, and p<0.001) and clinical stages (p = 0.01), when compared with Type 1 PRCC.Detailed patient demographics and tumor characteristics are listed in MET gene and chromosome 7 copy numbers were analyzed in 91 PRCC samples using FISH. Tumor cells with MET gene copy number ≥3 were observed in 90.1% (82/91) patients, while tumor cells with CEP7 copy number ≥3 were observed in 92.3% (84/91) patients. The percentage of tumor cells with MET or CEP7 copy number ≥3 was variable among patients. One case (1/91) of MET gene amplification (as defined by gene copy number ≥6), was observed in our cohort. Ki67 index, defined as the number of Ki67 positive cells per 1000 tumor cells, was evaluated in 98 PRCC samples. The overall Ki67 index was 0.65±1.72. Comparisons between Type 1 and Type 2 PRCC revealed similar biomarker prevalences (p<0.001).For biomarker evaluation, samples which failed quality control (no tumor tissue found or failed assays) were excluded. MET protein expression was analyzed in 94 PRCC samples using IHC staining. The MET protein overexpression rate, as defined by IHC 2+ and 3+, was 73.4% (69/94). valences . All bioMET gene copy number and CEP7 copy number in 91 PRCC samples. After evaluation of MET and CEP7 copy numbers in the tumor cells, a significant correlation was found between the percentage of tumor cells with MET gene copy number increase (≥3) (%MET) and the percentage of tumor cells with CEP7 gene copy number increase (≥3) (%CEP7) .FISH assays were performed to determine the MET was significantly higher in Type 1 PRCC samples, as compared to Type 2 (p = 0.01). No significant relationship was found between %MET and clinical stage (p = 0.82), indicating that the increase in the percentage of tumor cells with MET gene copy number increase may not be directly related to the severity of the disease or tumor metastasis. After stratification of the tumor subtypes, no significant relationship was found between %MET and tumor grades  or clinical stages . Similar results were observed in %CEP7 (data not shown).As shown in MET and increasing IHC score (MET gene copy number increase and protein overexpression in PRCC tumor cells. Further studies using IF combined with FISH showed that MET protein overexpression (IF 3+) was colocalized in tumor tissues with average MET gene copy number >5  and %CEP7 gene copy number increase (≥3) in PRCC tumor cells. Only a single case of MET gene amplification (≥6) was found in our PRCC sample set. These results suggest a role for Chromosome 7 trisomy or polysomy during the pathogenesis of sporadic PRCC. Chromosome 7 gain (trisomy 7) has also been observed in hereditary PRCC, and causes duplication of a mutated MET gene [MET gene amplification, without Chromosome 7 gain, was found to be a potent activator of MET pathway signaling [In this study, we used FISH and IHC techniques to investigate MET gene . Howeverignaling –30, undeMET gene copy number ≥3 together with higher MET IHC score, suggestive of a relationship between MET gene copy number increase and MET protein overexpression. This confirms previous findings observed between MET gene copy number variation and MET mRNA expression [MET gene copy number increase (>5), but not in those tumor cells with normal MET gene copy number.Furthermore, we also found an increasing trend in the percentage of tumor cells with pression . FinallyMET gene variation and MET protein expression across the two subtypes of PRCC in a Chinese patient cohort. Our results clearly demonstrate that MET gene copy number increases in PRCC tumors are highly associated with increases in the copy number of Chromosome 7, which differs from previous observations in lung and gastric cancer. Such increases in MET gene copy number likely lead to increases in MET protein overexpression in PRCC tumor cells, especially in those with a high increase in the average MET gene copy number (>5). This study lends additional support to the concept of therapeutically targeting MET in sporadic PRCC patients.Herein, we have studied the relationships between Chromosome 7 gain,


Tivantinib, a c-MET inhibitor, is investigated as a second-line treatment of HCC. It was shown that c-MET overexpression predicts its efficacy. Therefore, a phase-3 trial of tivantinib has been initiated to recruit “c-MET-high”patients only. However, recent evidence indicates that the anticancer activity of tivantinib is not due to c-MET inhibition, suggesting that c-MET is a predictor of response to this compound rather than its actual target. By assessing the mechanisms underlying the anticancer properties of tivantinib we showed that this agent causes apoptosis and cell cycle arrest by inhibiting the anti-apoptotic molecules Mcl-1 and Bcl-xl, and by increasing Cyclin B1 expression regardless of c-MET status. However, we found that tivantinib might antagonize the antiapoptotic effects of c-MET activation since HGF enhanced the expression of Mcl-1 and Bcl-xl. In summary, we show that the activity of tivantinib is independent of c-MET and describe Mcl-1, Bcl-xl and Cyclin B1 as effectors of its antineoplastic effects in HCC cells. We suggest that the predictive effect of c-MET expression in part reflects the c-MET-driven overexpression of Mcl-1 and Bcl-xl in c-MET-high patients and that these molecules are considered as possible response predictors. Consequently, patients' selection in the ongoing phase 3 METIV-HCC trial of tivantinib is based on the detection of high expression of c-MET in tumor biopsies. The predictive value of c-MET in determining survival improvement in patients on tivaninib was recently confirmed by a subgroup analysis of a randomized controlled trial in patients with non-squamous, non-small cell lung cancer [in vitro studies published very recently challenged this notion by showing that this compound exerts a remarkable cytotoxic effect in several cell lines without affecting the kinase activity of this receptor. These studies questioned the rationale for the use of this compound in c-MET-high patients [Tivantinib ARQ 197) is being investigated in clinical trials as selective orally available c-MET inhibitor 97 is bei, 3. Analg cancer , 5. Althpatients –9 and rapatients , 11. In In the attempt to provide an answer to this question, we decided to investigate the so far still unclear intracellular mechanisms of action of tivantinib on cell death and cell cycle progression, and to assess how their regulation is influenced by this compound in cell lines exhibiting different c-MET expression status , 13.50 values comprised between 9.9 nM (Huh7) and 448 nM (Hep3B). These results were clearly confirmed by colony forming assays showing a reduction in the number and size of colonies in cells treated with tivantinib  and to increased Mcl-1 and Bcl-xl. However, a slight increase of Cyclin B1 could also be observed upon c-MET stimulation Fig. . These dc-MET inhibitors are considered a promising therapeutic option for tumours exhibiting c-MET-status alterations . TivantiSince tivantinib was previously shown to induce apoptosis and G2/M-cell cycle arrest, we first assessed its effect on crucial mechanisms of apoptosis and cell cycle control. Assessment of apoptosis showed that tivantinb causes cleavage of Procaspase-8 followed by the cleavage of Bid. Cleavage of Caspase 8 is a hallmark of the activation of the extrinsic apoptotic pathway, which is initiated at the cell membrane in consequence of the stimulation of the “death-receptors” such as TRAIL-R1 and –R2 or CD95. Accordingly, administration of tivantinib sensitized several cancer cell lines to the action of the TRAIL-R2 agonistic antibody tigatuzumab . Neverthin vivo e.g. by affecting the response to endogenous TRAIL, tivantinib causes apoptosis principally by shifting the balance between the mediators of the mitochondrial pathway by inhibiting Bcl-xl and Mcl-1.As we assessed the effect of tivantinib on the intrinsic signaling pathway we observed a strong decrease of the antiapoptotic proteins Bcl-xl and Mcl-1. The intrinsic pathway is regulated by the balance between pro-apoptotic molecules  and antiapoptotic molecules . Mcl-1 and Bcl-xl are Bcl-2-related proteins which bind to Bax and Bak, thereby blocking their pro-apoptotic interaction with the outer surface of the mitochondria . The funIn addition, we observed a strong increase of Cyclin B1 as soon as 3 hours after administration of tivantinib. Cyclin B1 controls the G2-M cell cycle transition , 21 and c-MET exon 16 KO DLD1 cell lines, which lack the tivantinib-binding site of c-MET, showed that the effect of tivantinib in these cell lines was indistinguishable from that observed in parental-Wild Type DLD1 cell lines. Accordingly, tivantinib led to the downregulation of Bcl-xl, Mcl-1 and to the upregulation of Cyclin B1 independently of the c-MET status of these cell clones. Moreover, silencing of c-MET by specific siRNA failed to reproduce the effect of tivantinib on apoptosis and cell cycle. Administration of tivantinib caused a decrement of total c-MET and, to the same extent, of its phosphorylated form, indicating that the decrease of overall c-MET largely accounts for the observed decrease of c-MET phosphorylation. Nevertheless, our results show that the antineoplastic activity of tivantinib is largely independent of its effect as c-MET inhibitor.Experiments conducted with in vivo, e.g. those underlying invasion and metastasis formation [This discrepancy between our data and the predictive effect of c-MET expression in the clinical setting might be due to differences related to the higher complexity of the mechanisms of action of tivantinib ormation , and to ormation , 25. NevTo find possible explanations for the predictive relevance of c-MET in clinical trials, we hypothesized that Bcl-xl, Mcl-1 and Cyclin B1 are downstream targets of c-MET, and that the efficacy of tivantinib on tumors overexpressing c-MET may reflect the effect of this compound on c-MET-driven overexpression of these molecules. Stimulation of c-MET by HGF showed a clear increase of Bcl-xl and Mcl-1 suggesting that tivantinib might antagonize the effect of c-MET-dependent expression of these molecules in cells overexpressing this receptor regardless of the inhibitory effect of tivantinib on the kinase activity of c-MET.Although the mechanisms by which tivantinib causes such molecular alterations still remains elusive, a possible answer to this issue might be provided by the recent works published by Basilico , KatayamIn summary, we identified Bcl-xl, Mcl-1 and Cyclin B1 as mediators of the antineoplastic effects of tivantinib. Our study provides a possible explanation for the preferential effect of tivantinib in c-MET-high patients but indicates that the beneficial clinical effect of tivantinib may not be predictable solely by c-MET, and prompts for the evaluation of the expression of other molecules, such as Mcl-1, Bcl-xl and Cyclin B1 as possible and potentially more reliable markers for patients' selection.in vitro experiments. DLD1 colorectal cancer cells and two different syngenic c-MET exon 16 knock-out DLD-1 clones were provided by A. Bardelli and S. Arena and have been previously described [Huh7, HepG2, Hep3B, Chang cells (hepatocellular carcinoma), TFK1 (human cholangiocarcinoma), DLD1 cells (human colon carcinoma), PL5 and PANC1 cells (human pancreatic carcinoma) were used for escribed . All cel3/well) and were treated with increasing concentrations of tivantinib or vehicle. To investigate the effect of tivantinib on cell viability at low concentrations, cells were kept in culture for 6 days. At day 6 cells were washed with PBS, underwent osmotic-lysis in 100 μl ddH2O, and then incubated in 5% CO2 incubator for 45 minutes. Fluorescence was measured after addition of 0.2% Sybr green .Cells were seeded onto 96-well plates at different cell densities to avoid overgrowth . Total cell colonies in each well were counted after being photographed.4–5 × 104−1.5 × 105 cells were seeded in 12-well plates and treated after overnight incubation. Fluorescence activated cell sorting  was performed to detect the sub-G1 cell fraction to determine apoptosis and the respective different phases of cell cycle after propidium iodide (PI) staining as previously described [8.0 × 10escribed . In addi2EDTA, 1 mmol/L EGDA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 μg/ml leupeptin, and 1 mmol/l phenylmethane sulphonyl fluoride. Membranes were incubated overnight at 4°C with specific antibodies in TBS-T (TBS-0.1% Tween20) and subsequently for one hour with the appropriate horseradish-peroxidase-conjugated anti-rabbit/mouse secondary antibody. The following mouse monoclonal antibodies were used: Anti-CyclinB1 , anti-Caspase-8, anti-β-actin . The following rabbit polyclonal antibodies were used: anti-Caspase-3, anti-PARP, anti-Bid, anti-Bad, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Mcl-1 , anti-c-MET, anti-phosphorylated c-MET (Tyr1349 and Tyr1234/1235). For Caspase-3/7 activity assays the Apo-ONE® Reagent kit  was used according to the instructions of the manufacturer. Actin was used for control of appropriate protein load for each membrane. In figures, one actin loading control has been exemplarily shown.Cells growing in Petri dishes were collected and lysed in cell lysis buffer  containing 20 mmol/L Tris-HCL (PH7.5), 150 mmol/L NaCl, 1 mmol/L NaCyclin B1, Bcl-xl, Mcl-1 and c-MET were silenced by small interfering RNA (siRNA) or non-coding siRNA. Specific siRNA SMARTpool® sequences consist of 4 pooled 21-nucleotide RNA oligonucleotides forming a 19-bp duplex core with 2-nucleotide 3 overhang and were purchased from Dharmacon, Inc. . For transfection the DharmaFECT4 reagent  was used according to the manufacturer's instructions.P values < 0.05 were regarded as statistically significant.Statistical analysis was performed by using IBM SPSS Statistics . Comparison of means was performed by two-tailed Student's test. Results are expressed as mean and standard deviation (SD) of at least 3 independent experiments.


Academia.edu, Google Scholar Citations, ResearcherID and ORCID. The data set is enriched by demographic information including age, gender, position and affiliation, which are provided by the national CRIS-system in Norway. We find that approximately 37% of researchers at the University of Bergen have at least one profile, the prevalence being highest (> 40%) for members at the Faculty of Psychology and the Faculty of Social Sciences. Across all disciplines, ResearchGate is the most widely used platform. However, within Faculty of Humanities, Academia.edu is the preferred one. Researchers are reluctant to maintain multiple profiles, and there is little overlap between different services. Age turns out to be a poor indicator for presence in the investigated profiling sites, women are underrepresented and professors together with PhD students are the most likely profile holders. We next investigated the correlation between bibliometric measures, such as publications and citations, and user activities, such as downloads and followers. We find different bibliometric indicators to correlate strongly within individual platforms and across platforms. There is however less agreement between the traditional bibliometric and social activity indicators.The use of academic profiling sites is becoming more common, and emerging technologies boost researchers’ visibility and exchange of ideas. In our study we compared profiles at five different profiling sites. These five sites are ResearchGate,  There is little written about profiling services in an academic context and the goal of this paper is to contribute to the understanding of the impact of academic network sites. This is done by exploring the population of different types of services and the relationship between traditional bibliometric indicators and activity measures such as downloads, views and followers. This study has been inspired by the University of Utrecht Library and their comprehensive information on research impact and visibility Utrecht University Library , and by From a library point of view, and working at the University Library ourselves, it is important to know who and where the users are, what kind of tools they facilitate, and how they communicate inside the scholarly community. This knowledge will contribute to how the library services are further developed.Before the digital transition, the discoverability of scholarly content has been limited to library catalogues and their subject indexing routines. Since the invention of the Science Citation Index a new way of navigating the pool of information has evolved  and contScholarly online profiles have embedded features adapted for academic use. The profiles may often be pre-fabricated based on existent Web content, such as uploaded or database-indexed papers. The only effort required—to make the profile active—is to confirm the dormant information, eventually adding personal details and editing the list of indexed publications. Even though creating and maintaining profiles is often considered as a distraction, these platforms provide researchers the possibility to control and shape their online presence .Academic social network sites have become more prominent since the mid 2000s and have developed enhanced features for connecting users and viewing various activities. Still a critical mass isn’t yet achieved, and interface harmonization is needed (e.g. via APIs) for a smooth data exchange between different services .Academia.edu and 35% on ORCID. Also the number of profiles listed at the Web page of the University of Utrecht Library indicates a growing popularity of scholarly profiling sites. Within one year (2013 to 2014) the number of profiles increased by 43% on Google Scholar Citations and 27% on ResearcherID [A recent study by Ortega  examinede year 203 to 2014Academia.edu. LinkedIn, the only non-academic platform investigated, was the most popular service (27%). The authors report further that scientists having one type of Web presence were more likely to have another. There seems to be a reluctance to engage in wider debates behind established networks and outside own communities [Academia.edu (5%).The success of academic network sites is dependent on their simplicity and their adaptivity to the researchers’ need. Mas-Bleda, Thelwall  assessedmunities . Based omunities  reports Academia.edu faculty members contribute significantly more than post-docs, who again contribute more than graduate students [Despite the potential for communicating across established borders and being a democratic arena, social network sites still reflect the same hierarchical structures, imposed by position and seniority, as in real-life , 12, 13.Academic and social network sites come with different features for monitoring activities. The most common metrics provided are based on number of publications, citations, views, downloads, following, followers and co-authors. Traditionally, performance indicators are built upon peer reviewed articles and their uptake in the scholarly community, measured by number of citations. The main providers of these bibliometric data are Thomson Reuters and Elsevier through their proprietary databases Web of Science and Scopus, respectively. However, with the digital transition and a change in communication patterns, these traditional metrics can only reflect a limited aspect of impact . The impEngagement beyond the scholarly context is advocated by research funders, governments and higher educational institutions. The US National Science Foundation recently requested applicants’ “contribution to science” instead of “peer reviewed” publications, which marks a change of evaluation practice towards new channels of distribution . Also thaltmetric.com .–24.altmeom . However, user activity and bibliometric indicators on dedicated academic network sites have rarely been object to investigation. This paper follows up the problems addressed in previous research concerning attendance of researchers on academic platforms, demographic distribution and correlation of available indicators. In particular, this paper answers the following research questions:The scientific literature has so far mainly shed light on publication uptakes available through Which services are the most popular among researchers at the University of Bergen?How many profiles is a researcher willing to maintain?Who uses which services? Is there a difference between gender, age, position and faculty?How representative are academic sites when it comes to number of publications?How do bibliometric indicators such as publications or citations correlate with new metrics? Could the new metrics be used as a proxy for future publications and citations?A list of researchers at the University of Bergen was retrieved from the database CRIStin . CRIStinAs funding of Norwegian research institutions and universities is partly based on number of publications in CRIStin, severe control mechanisms are applied to ensure high quality and completeness . The datBased on this data set, the author names and related attributes are extracted and serve as a reference value for our investigation. Data included areAuthor names of researchers, affiliated to the University of Bergen, who have at least one publication during 2011–2014Author attributes: Number of publications, Faculty, Positon, Age, GenderBetween 2011 and 2014, 4307 unique authors, affiliated to the University of Bergen were retrieved. They authored a total of 11422 publications. This author set is used to match names with names in the investigated profiling services.Academia.edu. Researcher profiles are similar to enhanced business cards, available in a pre-structured environment including contact information, publication lists and bibliometric and activity measures [Academia.edu emphasize on the social character of network sites and are sometimes considered as a Facebook for researchers [We analyzed data from five different  academic network sites that allow researchers to create their own profile. These are ORCID, ResearcherID, Google Scholar Citations, ResearchGate and measures . The fivmeasures  but for earchers , 6, 11. Below we give a more detailed overview about the different services and the analyzed indicators are listed in publications, see ORCID (Open Researcher and Contributor ID) is a platform-independent identifier established in October 2012. As of May 2015 it has 1.3 million registered users . ORCID ppublications, citations and the h-index, see As ORCID, ResearcherID provides a digital identifier that distinguishes each researcher from any other. ResearcherID however is not platform independent, but integrated with Web of Science and owned by Thomson Reuters. It was established 2008 and according to its own webpage it has more than 270 thousand users . From Rpublications, the number of citations and the h-index is calculated and has been investigated here, see Google Scholar is a free search engine for scholarly literature, covering approximately 80–90% of all scientific papers . The asspublications and citations as ‘bibliometric indicators’; the number of downloads, publications views, the ResearchGate Score (RGScore) and the Impact Point as ‘publication scores’; and the number of profile views, followers and following researchers as ‘social activity’, see RGScore and Impact Points (indicators designed by the service itself) are not particularly discussed in this study, but categorized under ‘publication scores’.ResearchGate is a social networking site for scientists that was established in 2008. According to its own webpage , ResearcAcademia.edu is, as ResearchGate, a social network site also established in 2008. Alongside with ORCID, Academia.edu offers an unique identifier independent of the researcher’s publication record. According to its own webpage, Academia.edu has over 20 million members and over 5 million uploaded papers as of May 2015. Also on Academia.edu it is possible to create a profile, upload publications, follow peers and get engaged in discussions. From Academia.edu we investigated the number of publications, profile views, followers and following, see Data is collected using open source Java libraries such as Selenium WebDriver, HtmlUnit and Apache POI by searching for the researchers that are affiliated with the University of Bergen. Selenium Webdriver and HtmlUnit are tools that automate a Web browser by providing APIs to emulate browsing. In our context, this includes selecting parts of the page, filling out search forms, clicking buttons and following links. Apache POI is a Java library that provides APIs for reading and writing data in Microsoft Office formats (such as spreadsheets). We used Apache POI to programmatically store our data to spreadsheets for further analysis. Data collection was done by analyzing the page structure of every academic site and then building up scripts using a set of selectors and expressions so that we could navigate the page and extract the potential indicators that we were interested in.Academia.edu offer an organizational starting page, and users have to select an affiliation during registration. For ResearcherId and ORCID we searched by affiliation, using both “University of Bergen” and “Universitetet i Bergen” as both language forms are in use. For Google Scholar Citations we combined the search with email domains suffixes containing “uib”.This study applies a method for collecting data different from the one described in . Since tAcademia.edu changed its structure after we had completed the data extraction. To repeat the process in future, we will have to rewrite our scripts to keep them in sync with the changes of the Webpage structure.Data was extracted between April and May 2015. We experienced some issues during this period which made the data extraction process slower. For one of the indicators, “publication count”, Google Scholar Citations counted duplicates, which caused the number of publications to fluctuate between each attempt. This problem was solved by counting the unique outgoing links for publications listed on the profile page. In addition, Coupling of profiles and researchers indexed in CRIStin is done by matching last names in the different datasets and manually comparing first names when last names were equal. Unidentified names have then been checked by possibly different spellings due to transcription of special characters , use of nicknames, and use of different order of first, middle and last names appearing in the name string. Homonyms are checked by age and discipline, and duplicates have been removed.However, this cleansing procedure has its limitations and some profiles might not have been recognized. In Between 2011 and 2014, 4361 authors affiliated with the University of Bergen have been identified in CRIStin. Even though CRIStin data are highly controlled, we recognized 54 researchers with multiple accounts.Academia.edu (670), Google Scholar Citations (382), ORCID (223) and ResearcherID (154).As of May 2015, the results show that ResearchGate is the largest with 2207 members followed by Academia.edu . Google Scholar Citations had the lowest rate . A possible explanation may be problems to memorize passwords or various ways to sign in.Relative to the total amount of retrieved profiles, ResearchGate  had the most duplicates followed by Academia.edu the recognition rate is lowest (26%). Members on Academia.edu constitute a diverse group of scholars, many of them students, who haven’t published yet. In contrast to the profiles at ResearcherID, Google Scholar Citations are built upon self-authored publications in the respective services and recognition consequently is high.The recognition rate of authors in CRIStin is given in the last row of The small recognition rate for ORCID (48%) can be explained by the retrieval method used. We searched ORCID by a combination of English and Norwegian institutional names which researchers might have used in their profile (“University of Bergen” and “Universitetet i Bergen”). As many scientists have uploaded a full CV, we retrieved researchers that at some point have been employed or have studied at the University of Bergen. The set of researchers in CRIStin, however, is comprised of only researchers that have been recently publishing.Members on ResearchGate constitute a diverse group of scholars. Profiles in this service are prefabricated and incomplete but still retrievable by our scraping method. Prefabricated and not-verified profiles could therefore explain the relatively small rate of recognition (62%).Cleansing and de-duplication has only been done on author level. The extracted indicators given on the various platforms were not verified. I.e. we did not investigate whether the number of publications is falsified by duplicates. We further did not filter the publications to those published between 2011 and 2014. Since the CRIStin dataset only constitutes of researchers who have authored a publication between 2011 and 2014, it also only counts publications within this timeframe. The retrieved numbers from the network sites include potentially all publications during a researcher’s career.Below we answer the questions stated in the introduction and compare our results to previous studies.Academia.edu and ResearchGate, the latter is the most popular among researchers affiliated with the University of Bergen. 30% of the researchers have a profile at this service , ResearcherID (130) and ORCID (108). These findings correspond to earlier findings showing a similar popularity of the three first mentioned [Of the investigated academic network sites, ResearchGate is the largest with 1307 members followed by Google Scholar Citations (333), entioned , 9, 11.The success of academic (network) sites depends mainly on their adaptiveness to researchers’ need , 9 and eAcademia.edu also had a profile in ResearchGate, while this overlap accounts for only 6% of the identified ResearchGate profiles.The overlap between profiles that researchers have in the five different services is shown in None of the researchers have only a profile in ORCID. This might be because the service enables exchange of data between other services, and profiles might be created based on these. Additionally, ORCID still is a young service (founded in 2012) and not as established as the others. However, it has a great potential as an umbrella platform, linking different author identifiers and providing easy data exchange via APIs , 36.Academia.edu and ORCID (5) and ResearcherID (8). The two last mentioned offer primarily a digital identifier.The largest overlap is found between ResearchGate and Google Scholar Citations, which also are the services with the largest number of profiles from researchers affiliated with the University of Bergen. For these two services researchers seem more likely to maintain multiple profiles. Least overlap is observed between It seems that as long as minimal effort is needed to create a profile, scholars are more likely willing to maintain several services. In this regard, ResearchGate and Google Scholar Citations seem to meet most effectively the researchers’ need by automatically fabricating profiles. In addition, the offensive and smart marketing strategy of ResearchGate is identified as one of the reasons why people sign up . CriticaThe dataset from CRIStin allows us to analyze the use of the investigated network sites in more details with respect to age, gender, faculty and position.Academia.edu has the highest percentage for the group of age 55–66. Our presumption that foremost junior researchers are inclined to establish their presence on the digital space was wrong. We actually find that senior researchers are well aware of these services and seem to use them more often than younger researchers. However, the high percentage of ResearchGate among >64 year old researchers might be due to the automatic creation of profiles through co-authors instead of active use of the platform; the older a researcher, the higher the number of papers and co-authors and thus the higher the chance that one of their papers has been added to trigger an automatic profile. Nevertheless, our results are in accordance to earlier findings by Rowlands, Nicholas [Nicholas  and ProcNicholas .Academia.edu the female share is relatively high, however, we could not confirm that this site is particular popular among women as found by Thelwall and Kousha [altmetric.com and seems to persist on academic network sites as well.d Kousha . We findd Kousha , investiAcademia.edu. The success of Academia.edu among humanity scholars has earlier been reported [reported  and may The last row in Finally, we examined which positions the profile owners hold, see Academia.edu and ResearchGate higher.The distribution of profiles by position somewhat reflects a hierarchical structure, with professors at the top of the hierarchy, PhD students, researchers and associate professors in the middle and students at the bottom. This has earlier been reported by Menendez, de Angeli  and Ortepublications, citations and h-index. Then we look at the number of downloads and the site dependent scores, which are linked to online activities related to publications (‘publication scores’). At the right (bottom) we list the indicators related to ‘social activity’: how much other scholars used the profile (profileViews and followers) and how the researchers themselves are using the site for networking (following).We investigated the representation of publications on academic profiling sites by correlating the number of publications with the number of reported publications in CRIStin.We find a high correlation coefficient between the number of publications reported in CRISTin and the number of publications in ResearcherID (0.69), Google Scholar Citations (0.83), ResearchGate (0.7). However, the correlation coefficient is rather low for the other two services (<0.5). Similarly, we find a high correlation coefficient between Google Scholar Citations, ResearchGate, and ResearcherID  but a weak correlation with the other services . Also the h-index is strongly correlated between the two services Google Scholar Citations and ReasearchGate (0.77).For many years researchers have been evaluated by their publication productivity and citation impact. The academic profiling sites with their alternative metrics can provide a new way to evaluate the impact. We divided the alternative metrics into two groups: Publication score and Social activity. The first group is related to publications, and their impact measured by e.g. downloads, views. The second is related to the users’ social activity and online network engagement.The indicators in the category ‘publication scores’ can only be analyzed for ResearchGate. For ReasearchGate the correlation coefficient between the publication scores and the number of publications & citations is above 0.8, but lower for the h-index (>0.6). The most interesting finding is hereby the strong correlation between the number of citations and the number of downloads (0.81). As citations usually take 2–5 years’ time  several Academia.edu. For ResearchGate, the correlation coefficient between the number of profileViews & followers and number of publications & citations is also quite high with values above 0.6 and 0.7. The correlation coefficient between the number of following and number of publications & citations in turn is below 0.35. For Academia.edu the correlation coefficient between the number of profileViews and publications is at the same range as for ResearchGate with 0.67 but lower between publications and number of followers (0.43). As for ResearchGate the correlation coefficient between the number of peers researchers are following and number of publications is below 0.35The indicators in the category ‘social activity’ have been analyzed for ResearchGate and Our results are consistent with previous findings that social network sites reflect the same hierarchical structures as in real life . ScientiAcademia.edu to be most popular for scholars in the Humanities.Engagement in public, beyond the scholarly context is increasingly advocated by research funders, governments and higher educational institutions, and there is growing pressure on scholars to document societal impact. We find that academic network sites have indeed become a part of the researchers’ professional life. With a prevalence of 37% of University of Bergen scholars on various platforms, these services seem to have reached a critical member mass and will increasingly contribute to the researchers’ work flow. More interface harmonization is needed (e.g. via APIs) for a smooth data exchange between different services, but taking into account their short existence and usefulness in a scholarly context, their future seem to look bright. While researchers at the University of Bergen are reluctant to maintain multiple profiles, they will more likely be represented on several platforms when harmonization is achieved. For now we find ResearchGate to be most widespread throughout all faculties, and Our study confirms established hierarchical patterns in regard to age, gender and position, showing the elder, male professors are best represented. However, this result is not unambiguous. We find that post doc fellows to a large extent embrace network sites, which reflects their stronger need to be visible on the job market.We investigated available metrics and find traditional bibliometric indicators strongly correlated. However, there seems to be less agreement between bibliometric and social activity indicators. Although network sites gain ground, their uptake is far from universal, and available metrics should be used carefully in an evaluation context e.g. , 46, 47), 4746, 4Even though this study is limited to the University of Bergen, we believe our results to be universal, confirming a growing attendance on digital arenas, being worth monitoring.S1 TableThe file includes all data used in our analysis.(CSV)Click here for additional data file.S2 TableThe file contains the numbers visualized in (CSV)Click here for additional data file.


Staphylococcus caprae for one patient and methicillin-sensitive Staphylococcus aureus for the other). The probabilistic antibiotic treatment started during the first side (after harvesting intraoperative samples) did not prevent the culture of the bacteriologic harvested during the intervention of the second side. Cultures were positive for the same bacteria for both sides in the two cases presented herein. After results of intraoperative cultures, patients received culture-guided antibiotic therapy for three months and were considered cured at the end of a two-year follow-up. Our results suggest one-stage bilateral change of infected THA is a viable option and that early intraoperative antibiotic, started during the first-side exchange, does not jeopardize microbiological documentation of the second side. This work brings indirect arguments, in favor of the use of prophylactic antibiotics during revision of infected THA.Recommendations for the management of chronic and bilateral total hip arthroplasty (THA) infection are lacking. However, this type of infection involves medical problems concerning the management of the antibiotic therapy. We report two cases of such infections operated as one-stage revision. For each case, both hips were infected with the same bacteria ( Total hip arthroplasty (THA) infections occur in 0.5 to 3% of patients, and may account for a third of reoperations and require multidisciplinary management –3. Bilat Staphylococcus caprae in both hips. The patient underwent a bilateral change in one stage with the administration of broad-spectrum empiric antibiotic therapy as soon as surgical samples were taken on the first side. Replacement of the second side was conducted with the same protocol. Microbiological cultures of both hips were positive with the same bacteria (Staphylococcus caprae sensitive to methicillin) found on four samples out of six, on standard and Rosenow's growth medium enriched cultures [The first case was a sixty-year-old male, with an American Society of Anesthesiology (ASA) score of 2 and history of bilateral arthroplasty in 2009 and 2010 done at another institution. He was refereed to our center in 2011 for bilateral hip pain and was suspected of presenting chronic infection, because of radiological signs of intense femoral periosteal reaction . Joint acultures  (Table 1 Staphylococcus aureus (MSSA). Three weeks later, right hip pain occurred that justified a joint aspiration from the hip in which the same MSSA strain according to the antibiotype was identified. The patient underwent bilateral hip change in one stage according to same surgical and antibiotic therapy protocols as the first case. Cultures of the two hips were positive with the same bacteria (MSSA) on four samples out of six, on standard and Rosenow's growth medium enriched cultures [The second case was a sixty-year-old female, with an ASA score of 2 and history of bilateral arthroplasty in 2006 and 2009, who consulted in 2012 for left hip pain associated with systemic inflammatory reaction . Microbicultures .The pathogens identified during hip aspiration were sensitive to the probabilistic antibiotics. Both cases received the same antibiotic treatment adapted to their antibiograms (rifampicin + levofloxacin) introduced on day 5 for five days intravenously and then for three months per os regimen. At the end of a three-year (case one) and two-year (case two) follow-up, the functional Oxford score  was, resBoth cases suggest that it seems possible, with a satisfactory microbiological documentation and therefore culture-guided appropriate antibiotic therapy, to perform bilateral one-stage replacement of infected THA. Former studies were in favor of a two-stage revision to manage bilateral infected hip arthroplasties , but repIn the two cases presented above, there are some practical problems. Firstly, the one-stage revision option implies potential surgical risks. However, our patients had low morbidity with an ASA score of 2 and our surgery protocol was strict . One-stage strategy allows sterilizing all septic sites and reduces cost  and faster rehabilitation. Secondly, we believe there is a risk of contamination of the nonreplaced side, when the replacement is unilateral. Thirdly, the probabilistic antibiotic therapy could have resulted in false negative culture of the second hip, though it was not reported in our cases. The two cases of the present study advocate the use of antibiotic prophylaxis in case of THA revision for infection , 9. It i


Second, concrete samples made with 350 kg/m3 of cement with a Superplasticizer admixture made with drinking water and treated wastewater and then cured with treated wastewater. The compressive strength of all the concrete samples made with treated wastewater had a high coefficient of determination with the control concrete samples. A 28-day tensile strength of all the samples was 96–100% of the tensile strength of the control samples and the setting time was reduced by 30 min which was consistent with a ASTMC191 standard. All samples produced and cured with treated waste water did not have a significant effect on water absorption, slump and surface electrical resistivity tests. However, compressive strength at 21 days of concrete samples using 300 kg/m3 of cement in rapid freezing and thawing conditions was about 11% lower than concrete samples made with drinking water.We tested the setting time of cement, slump and compressive and tensile strength of 54 triplicate cubic samples and 9 cylindrical samples of concrete with and without a Super plasticizer admixture. We produced concrete samples made with drinking water and treated domestic wastewater containing 300, 400 kg/m Specification Table1•The data indicate the suitability of treated domestic wastewater for producing concrete.•The data illustrate the initial setting time of cement made with treated domestic wastewater is higher than the cement made with drinking water.•A high coefficient of determination exists between the compressive strength data of concrete produced with drinking water and concrete produced by the treated domestic wastewater.•The water absorption and surface electrical resistivity data of the concrete samples made with treated domestic wastewater and drinking water had approximately similar results.•3 of cement which made with treated wastewater at 21 days was 10.11% lower than concrete samples made of drinking waterThe compressive strength of concrete samples, under rapid freezing and thawing, with 300 kg/m2The strength and durability of concrete is very dependent on the chemical characteristics of cement. According to ACI 201 3 of cement in one cubic meter of concrete. B300, B350, B400 labels were used for the concrete samples produced with drinking water and cured with treated wastewater. C300, C350 and C400 labels were used to illustrate the concrete samples produced and cured with treated wastewater. A300, A350 and A400 labels were used to indicate the concrete samples produced and cured by drinking water as the control samples, and 300, 350 and 400 indicate the kg/m3 of cement which made with treated wastewater at 21 days was 10.11% lower than concrete samples made of drinking water. For concrete samples with 350 kg/m3 of cement without using microsilica, the compressive strength at 21 days for concrete made with the treated wastewater was 11.7% lower than concrete samples made with drinking water.As indicated in 3As illustrate in 3 and 400 kg/m3 of cement without Super plasticizer admixtures and 350 kg/m3 of cement with an added Super plasticizer admixture. As indicated in the figures, a high coefficient of determinations exists among the various types of concrete samples that were made and cured with treated wastewater and drinking water.3 and 400 kg/m3 of cement using drinking water and cured with treated wastewater, respectively. In the figures, using treated wastewater for curing concrete samples made with drinking water did not affect the strength of the concrete samples when compared with concrete samples made and cured with drinking water.3 of cement and a super plasticizer admixture using drinking water and cured with both drinking water and treated waste water. We achieved the same results with concrete made with 300 and 400 kg/m3 of cement without a Super plasticizer admixture and concrete made with 350 kg/m3 of cement with a Super plasticizers admixture.3 of cement using drinking water and treated wastewater, respectively.3 of cement(B300) which was made of treated wastewater (3 of cement (A300) which made of drinking water illustrates concrete forming of subhedral to anhedral crystals and more dense and less void than concrete made with treated waste water.The Scanning Electron Microscopy (SEM) images of concrete sample with 300 kg/mstewater , Fig. 10stewater , Fig. 103 of cement using drinking water and treated wastewater.As indicated in 4We used effluent from the Lorstan domestic wastewater treatment plant to produce concrete samples. We examined the physical and chemical characteristics of treated wastewater, Portland cement specification, particle size analysis, cement setting time, slump, compressive strength, tensile strength, concrete water adsorption, surface electrical resistivity, resistance of concrete to rapid freezing and thawing and Scanning Electron Microscopy (SEM) combing energy-dispersive X-ray spectroscopy (EDX). The number of concrete samples produced was 54 triplicate cubic and 9 cylindrical samples with and without a Super plasticizer admixture for compressive and tensile strength tests.


Daubentonia) do not follow this pattern and show markedly less variability in HHO periodicity and lower correlation coefficients and slopes. Moreover, body mass is uncorrelated, and brain size and activity levels are more strongly correlated with HHO periodicity in these animals. We argue that lemurs evolved this pattern due to selection for risk-averse life histories driven by the unpredictability of the environment in Madagascar. These results reinforce the idea that HHO influences life history evolution differently in response to specific ecological selection regimes.Skeletal histology supports the hypothesis that primate life histories are regulated by a neuroendocrine rhythm, the Havers-Halberg Oscillation (HHO). Interestingly, subfossil lemurs are outliers in HHO scaling relationships that have been discovered for haplorhine primates and other mammals. We present new data to determine whether these species represent the general lemur or strepsirrhine condition and to inform models about neuroendocrine-mediated life history evolution. We gathered the largest sample to date of HHO data from histological sections of primate teeth (including the subfossil lemurs) to assess the relationship of these chronobiological measures with life history-related variables including body mass, brain size, age at first female reproduction, and activity level. For anthropoids, these variables show strong correlations with HHO conforming to predictions, though body mass and endocranial volume are strongly correlated with HHO periodicity in this group. However, lemurs (possibly excepting  Growth, metabolism, and reproductive physiology all have a role to play in the allocation of resources over individual lifespans, and have all been implicated in multiple explanatory models of life history evolution e.g. –6). Whil. Whil6])in situ fibroblasts scale with body mass across species, whereas metabolic rates of explanted fibroblasts from the same species do not scale. This suggests that a central physiological mechanism is responsible for coordinating metabolic function among the cells that make up an organism. It has also been demonstrated that all primate tissue and organ masses necessarily covary with body mass, and thus also scale with HHO periodicity in a manner similar to one another [Although bones and teeth are both mineralized tissues, they have different cellular and developmental origins; dental tissues, in fact, have origins from both ectoderm-derived epithelium (enamel) and mesenchyme (dentine), and both express long-period growth lines  . The syn another ; this in another  have alsAccordingly, the HHO would act as a metronome around which life history physiology is organized. Following this model, high-frequency HHO oscillations e.g. 2–3 days) will foster more rapid cell proliferation and faster overall life histories linked to smaller body size, while low-frequency HHO oscillations  will have the opposite effect. A more detailed discussion of the physiology putatively underlying the HHO mechanism and the evidence to support it may be found in studies by Bromage et al. [–3 days wFollowing the HHO hypothesis, interspecific HHO periodicity should correlate positively with body mass, brain mass, and basal metabolic rate. Likewise, the HHO should also relate to tissue specific metabolic rates, inasmuch as they aggregate in rational proportion to whole-body metabolic rate. A proxy for bone tissue specific metabolic rate is osteocyte lacuna density (the number of osteocytes per unit volume of bone), which should correlate negatively with body mass, as smaller species should have higher-frequency HHO and therefore more cell proliferation rhythms per unit time. Prior studies have corroborated the HHO periodicity predictions for anthropoid primates and osteocyte densities predicted for mammals ,3,7.Daubentonia, Daubentonioidea), they also have reduced activity levels, smaller home ranges, and smaller brains compared to like-sized anthropoids [Nycticebus and the giant extinct lemurs Megaladapis and Palaeopropithecus, the latter two of which have a very short HHO periodicity relative to their body size as compared to all anthropoids. This anomaly suggests that the giant lemurs may differ from other primates in HHO biology. As a second possibility, it may be that the lemurs in general differ from other primates, or third that the strepsirrhines as a whole follow this pattern. On the basis of prior osteocyte density data [a priori. While an unusual lemur HHO biology is not entirely surprising given the unusual nature of life histories in this group, it behooves us to examine the strepsirrhines further to clarify their patterns of life history evolution. Doing so will also inform our understanding of life history evolution among primates and mammals in general, and possibly develop a more nuanced understanding of the conditions under which the HHO may evolve. Metabolic flux is key to an organism's life history adaptation to its ecology [The HHO mechanism holds great promise for the development of powerful explanatory models of life history evolution. However, the predictive power of HHO-based models is currently limited by a lack of information on how HHO periodicity varies among taxa, and a lack of information on how this variation may be influenced by differing ecological selection regimes and phylogenetic histories. The insular lemurs of Madagascar  provide an excellent opportunity to fill some of these gaps, since they exhibit major life history differences from their relatives, the monkeys and apes  . With thhropoids . Howeverhropoids . Importaity data , we pred ecology ; thus whThis study examines the relationships among HHO and life history-related variables for the largest sample of primate taxa analyzed to date, including a greatly expanded strepsirrhine dataset. We predict that HHO periodicity values seen previously in giant lemurs will extend to the lemurs in general, which will differ from anthropoids in the expression of their HHO biology due to differences in their physiology, life history, and ecology. Since strepsirrhines as a whole may share aspects of their life history space that are absent in anthropoids, as stated above it is alternatively possible that differences extend to the non-lemur strepsirrhine superfamilies (Lorisoidea and Adapoidea). We also expect that when HHO periodicity is regressed against metabolic rate, differences seen among taxa will be less pronounced than they are for body mass and brain mass. This is due to the fact that metabolism is a more direct putative target for HHO regulation than is body or brain size, both of which may be subject to more diverse physiological influences.Avahi laniger, Jonah Ratsimbazafy Collection, Manombo Reserve; Duke Lemur Center: DLC 11832, "Poe", 6514m, 4564m, 6845m, 5628f; Great Divide Basin Project: WMU VP 359, 654826, 6541103, 4587, 371, 363, g501589; University of Massachusetts Anthropological Primate & Natural History Collection: UM-APC 27, 9, 253, 170, 163; Collection of Anthony DiFiore, PhD, University of Texas: 2 uncatalogued Lagothrix poeppiggii; Newcastle University School of Dental Sciences: HT 13–90, 16–90, 25–08, 01–10, 06–02, 07–02, 19–05, 12–90, 17–00; Arizona State University: S26; Montpelier II University: ACQ 6415; Universität Zürich-Irchel: Co.p 3. More detail on specimens and their sources is available in the supporting information  in teeth of 41 anthropoid and 21 strepsirrhine species. We are introducing the term RP as a synonym and replacement for “periodicity” and “repeat interval” (RI) used by authors in prior studies e.g., ,14–16) i–16 i14–1RP values were determined from histological images in one of two ways. First, cross-striations (24-hour growth increments) between successive, periodic striae of Retzius were counted directly at several locations within a tooth . Second,0.646) [Natural logarithm (ln) of RP mean (mode for species where n > 3) for each species were regressed against ln of data from the literature for body mass, endocranial volume , and age at first female reproduction  ,15,17–370.646) ,28 and ihttp://www.R-project.org/), in order to control for phylogenetic effects. Consensus trees for these analyses were generated using 10K Trees (URL: http://10ktrees.fas.harvard.edu/) except for lemurs, whose phylogeny was taken from Catlett et al. [The natural logarithms (ln) of body mass, ECV, SCR, and AFR were analyzed against ln RP using bivariate phylogenetic generalized least-squares (PGLS) regression  using tht et al.  and Kistt et al.  to inclut et al. .Our raw data table  and cladOverall, strepsirrhines have a high degree of variation in body mass, on a level comparable with the anthropoids . HoweverPhylogenetically corrected bivariate analysis results are available in Tables Among anthropoids, results are consistent with prior studies in that ln body mass and ln ECV both show strong and significant relationships with RP . Althoug2 values are not high  also falls well below the line.These weak correlations between our predictor variables and ln RP are best explained as a consequence of such low variation in ln RP among the strepsirrhines in general and the lemurs in particular. Or, in other words, given the same RP value for a strepsirrhine as for an anthropoid, we see a much greater range of variation in the predictor variable values among the strepsirrhines. This is especially the case for the lemurs, since the non-lemur strepsirrhine species in our analyses all have small body sizes and a low variation in their range of body sizes, and indeed fall near the intersection of the anthropoid and strepsirrhine regression lines. So while it is difficult to discern a pattern in the non-lemur strepsirrhines given the data we have, the plots make it very clear that the original, unusual RP pattern noted for lemurids , but theArchaeolemur, Hadropithecus, and Daubentonia consistently have the highest positive residuals and therefore resemble anthropoid patterns more than other lemurs do—particularly Daubentonia madagascariensis. This species is a particularly interesting case, as it is exceptional among lemurs due to its high encephalization and relative BMR, and unique life history profile. Since Daubentonia is unusually encephalized, we wanted to visualize the position of Daubentonia compared to the anthropoids and other strepsirrhines in a manner that would control for the effect of body size on brain size and ln RP. Therefore, we performed an analysis of ln RP residuals (from a regression against ln body mass) against index of cranial capacity  possessing an especially “hyper-strepsirrhine” pattern when assessed against ln body mass and ln ECV. Loris tardigradus is also interesting in that it generally falls well above all other similar-sized primates in all graphs, with the exception of ln AFR  have an HHO biology that is more constrained than that of anthropoids.Strepsirrhines consistently show scaling relationships for HHO that diverge from those of the anthropoids. The most obvious pattern is that anthropoids possess a broad range of mean RP values (and therefore HHOs) from 1 to 11 days, whereas strepsirrhines range only between 2 to 4 days. A comparison is useful: one of the largest extinct lemur species, Furthermore, the relationships between HHO periodicity and our predictor variables are markedly different between anthropoids and strepsirrhines. Anthropoids show a strong linear relationship between ln RP and all predictor variables, particularly ln ECV and ln body mass, as we would predict from prior studies. In sharp contrast, variation in ln RP is poorly explained in lemurs by any single variable, mostly because the variance in ln RP among lemurs is very limited. As a clade, lemurs have relatively small brains. They are also hypometabolic and RP is low, while other variables such as body mass and age at first reproduction vary tremendously. The Indriidae, for example, can delay first reproduction long after weaning, and species with remarkably late ages at first reproduction are among those taxa with the lowest values for RP. In other words, indriids can delay age at first reproduction with no effect on RP (which remains low). This is not the case for lemurids, where one sees an opposite pattern: age at first female reproduction is early, regardless of RP .Hapalemur griseus  has an early age at first female reproduction and low RP, while the largest-bodied extant lemurid, Varecia, which is highly frugivorous, has relatively high RP but also begins reproducing early. Propithecus diadema, probably the most frugivorous indriid, has higher RP values than the more folivorous Indri indri and P. verreauxi (which begin reproducing slightly to considerably later), and it has higher RP values than Avahi laniger (which begins reproducing considerably earlier). The daubentoniid Daubentonia madagascariensis has the highest RP value among extant lemurs (as well as relatively large brains and high activity levels), and a rich diet of structurally defended fruit and animal matter. Age at first reproduction is early in comparison to Indri, but delayed in comparison to lemurids.For both Indriidae and Lemuridae, there appears to be a dietary effect, wherein species that consume more fruit have higher RP values, regardless of age at first reproduction . Thus HaPalaeopropithecus especially but also Babakotia, Mesopropithecus, and Megaladapis all have RP values of 2 or 3 (regardless of body size), and diets that were highly folivorous. The extinct lemurs with the highest RP values (4) were those with the most unusual diets; Archaeolemur was highly frugivorous and likely able to exploit, like Daubentonia, structurally defended resources [Hadropithecus likely specialized on the leaves of succulent CAM plants [The dietary constraint that we have identified here may hold for the extinct lemurs, as esources , 23, whiM plants , which cpar excellence. Lemurids place a selective premium on being able to reproduce rapidly when conditions are right. They have higher adult mortality during resource crunches , but they begin reproducing earlier, are more likely to have twins, and have a higher maximum rate of population increase; thus population recovery is faster, when it is possible. RP values for lemurids tend to be higher than in indriids, but in comparison to other primates, they are still low.The point is that lemurs display a variety of life history strategies each of which, in its own way, enables them to survive in an unpredictable climate with limited resource availability during particular times of the year. Godfrey et al.  discuss Daubentonia, no analysis we performed makes the lemurs quantitatively or qualitatively resemble the anthropoids. Therefore, our end results confirm our first prediction that HHO characteristics seen previously in subfossil lemurs extend to lemurs in general. Also with regard to this prediction, we raised the possibility that strepsirrhines as a whole may share the RP patterns expressed in lemurs. While our statistics do show some differences between the whole strepsirrhine and lemur samples, it is difficult to extrapolate the non-lemur pattern in the overall picture since the species in this sample fall near the intersection of the anthropoid and strepsirrhine lines. Nevertheless, the differences we do see in our strepsirrhine vs. lemur analyses, the unusual RP pattern of Daubentonia  environments in Madagascar , which aAmong anthropoids, RP is highly and significantly associated with encephalization and metabolism, and what is more, the slopes of these relationships are well above those of all other tissue and organ systems : This meThe lemur species that colonized Madagascar is estimated to have weighed around 2000 g  althoughVoay, which likely arrived on Madagascar after the mid-Miocene), raptors belonging to the family Accipitridae (which likely arrived during the late Eocene or later), and the mammalian carnivorans, the Eupleridae (which likely arrived during the late Oligocene or early Miocene)  that have relatively long Retzius periodicities (low for anthropoids of similar body mass but well above the strepsirrhine regression line)  . These alar diet  but appeArchaeolemur and Hadropithecus emphasize that diet is important to energetic constraints. Having been somewhat loosened from the hold of the ecological parameters that define the evolutionary patterns of the lemuroid group as a whole by having likely evolved to exploit resources different from those of other lemurs (as Daubentonia does), these taxa seem to have been able to move into life history spaces that more closely resemble those of monkeys and apes [In sum, our results show that HHO evolves in a complex manner in response to ecology and evolutionary history. In particular, these forces seem to have driven an unusual pattern in the evolution of HHO within the lemuroids as compared to other primates. The central factor governing HHO evolution seems to be energetics, which is expected given that life history theory in large part revolves around the adaptive apportioning of energetic resources over time. However, the energetics picture is not as simple as we initially predicted, since ln SCR  correlates significantly but not strongly with ln RP in lemurs. Rather, lemurs seem to be risk averse, adaptively apportioning energy derived from limited, unpredictable resources to differing needs such as maximizing adult survival, maximizing population recovery rate, or increasing brain metabolism and activity levels. Differences in HHO expression between lemurs and anthropoids indicate that differing energetic needs among taxa can drive a variety of life history adaptations, even within a single order of mammals. Our study also shows that phylogenetic history is important in the evolution of specific HHO physiologies. However, the exceptions of and apes , althougS1 FigIncludes all extant primates in the study and subfossil lemurs. See the main paper for details & references. To incorporate subfossil lemurs, branch lengths were calibrated as a chronogram. Anthropoid/strepsirrhine divergence date calibrated following Kistler et al. .(PDF)Click here for additional data file.S1 TableAll specimens that were used to generate new data  are listed here. Sources are listed as follows: UMass = University of Massachusetts uncatalogued, Jonah Ratsimbazafy Collection, Manombo Reserve; DLC = Duke Lemur Center; GDBP = Great Divide Basin Project; UM-APC = University of Massachusetts Anthropological Primate and Natural History Collections; ASU = Arizona State University; Newcastle = Newcastle University School of Dental Sciences; M = Montpelier II University; UZI = Universität Zürich-Irchel.(PDF)Click here for additional data file.S2 TablePreference was given to modal values for Retzius periodicity (RP) data whenever possible, since RP is expressed in multiples of 1 day; whenever data were available for only two or three specimens, mean values were used. However, the majority of species samples consist only of a single individual. For species with high sexual dimorphism, values for both males and females were included separately where available. RP and osteocyte data lacking a citation are newly published values generated by our study. References given in main paper; wherever possible, RP, mass, and ECV data drawn from the compilation in Bromage et al.  and Isle† = Extinct species* = Modal value reported from literature** = Data are presented for more than one individual for these species, but too few individuals have been sampled to incorporate a reliable mode value. For these species, statistical analyses incorporate means of the reported values.(PDF)Click here for additional data file.S3 TableA. Regression statistics (least squares) for ln RP & ln OD relationships. Significant relationships highlighted in bold. B. Bivariate correlations matrices for all variables. Significant correlations for RP & OD highlighted in bold. * = no data available for subfossil lemurs, so no separate analysis excluding subfossil species conducted; ** = sample too small for analysis.(PDF)Click here for additional data file.


We also present an example to illustrate the feasibility of our results. The results of this paper are completely new and complementary to the previously known results even in both the case of differential equations (time scale In this paper, by using the existence of the exponential dichotomy of linear dynamic equations on time scales and the theory of calculus on time scales, we study the existence and global exponential stability of periodic solutions for a class of  The theory of dynamic equations on time scales was introduced by Hilger  in 1988 and Tien , the autPeterson , Graef aPeterson , Li and Peterson , Li and Peterson , LupulesPeterson , Su and Peterson , Wang etPeterson , Zhang ePeterson , b, 2014Peterson , 2012 anSince it is natural and important that systems will contain some information about the derivative of the past state to further describe and model the dynamics for such complex reactions, many authors have studied the existence of solutions of various neutral delay models  corresponding entries of B. For For convenience, we denote The initial condition of  is\documThroughout this paper, we assume that the following condition holds: In this section, we introduce some definitions and state some preliminary results.Let m, then m, then A point f is continuous at each right-dense point and each left-dense point, then f is said to be continuous on A function y(t), U of t such thatFor We denote the class of all y is continuous, then y is right-dense continuous, and if y is t, then y is continuous at t.If y be right-dense continuous. If Let A be an A is rd-continuous on A is rd-continuous on A is differentiable on A is differentiable on and(ii)(iii)(iv)(v)B(t) (Bohner and Peterson Ifandthenand(Bohner and Peterson LetandA(t) be amatrix-valued function onthe linear systemis said to admit an exponential dichotomy onif there exist positive constantsprojectionPand the fundamental solution matrixX(t) of  is the fundamental solution matrix of  is a uniformly boundedrd-continuousmatrix-valued function onand there is asuch thatthen  be an y(t) of (x(t) is said to be globally exponentially stable.Let mentclass2pt{minimmentclass2pt{minimSet Lethold. Suppose thatsystem  with the initial value y(t) is an arbitrary solution of  of (Case Three:  and 9)  9) are bmentclass2pt{minimLetandhold. Then (has a unique periodic solution, which is globally exponentially stable.mentclass2pt{minimIn , if we tmentclass2pt{minimBy a simple calculation, we have entclass1pt{minimaent}T=Z,  has a \dExample  shows thn-dimensional neutral dynamic equations on time scales.Our results of this paper are completely new and complementary to the previously known results even in both the case of differential equations (time scale In this paper, by using the existence of the exponential dichotomy of linear dynamic equations on time scales and the inequality techniques, we established the existence and global exponential stability of periodic solutions for a very general class of


There is an error in reference 24. The correct reference is: Obirikorang C, Selleh PK, Abledu JK and Fofie CO (2013) Predictors of Adherence to Antiretroviral Therapy among HIV/AIDS Patients in the Upper West Region of Ghana. ISRN AIDS 2013: 873939.In