A collagen prolyl 4-hydroxylase inhibitor reduces adhesions after tendon injury

Collagen synthesis inhibition potentially can reduce adhesion formation after tendon injury but also may affect cutaneous wound healing. We hypothesized that a novel orally administered collagen synthesis inhibitor (CPHI-I) would substantially reduce flexor tendon adhesions after injury, without any clinically important effect on cutaneous wound healing. The experiments were performed in a rat model with an in-continuity crush injury model in the rat hindfoot flexor tendon to provoke adhesion formation. Assays of dermal collagen production and the rate of healing of an excised wound were performed to assess cutaneous wound healing. Animals in the treatment groups received CPHI-I for 1, 2, or 6 weeks and were assessed at either 2 or 6 weeks. The work of flexion in the injured digit was reduced in the CPHI-I-treated animals compared with control animals, (0.188 J versus 0.0307 J at 2 weeks, and 0.0231 J versus 0.0331 J at 6 weeks) The cutaneous wound healing rate was similar in all animals, but dermal collagen synthesis was reduced in the treated animals. The CPHI-I seems to reduce tendon adhesion, and although collagen synthesis was reduced in cutaneous wounds, CPHI-I did not retard wound healing.

BRAF inhibitor therapy for melanoma, thyroid and colorectal cancers : development of resistance and future prospects

BRAF is a major oncoprotein and oncogenic mutations in BRAF are found in a significant number of cancers, including melanoma, thyroid cancer, colorectal cancer and others. Consequently, BRAF inhibitors have been developed as treatment options for cancers with BRAF mutations which have shown some success in improving patient outcomes in clinical trials. Development of resistance to BRAF kinase inhibitors is common, however, and overcoming this resistance is an area of significant concern for clinicians, patients and researchers alike. In this review, we identify the mechanisms of BRAF kinase inhibitor resistance and discuss the implications for strategies to overcome this resistance in the context of new approaches such as multi-kinase targeted therapies and emerging RNA interference based technologies.

Impact of nitrification inhibitor (DMPP) on soil nitrous oxide emissions from an intensive broccoli production system in sub-tropical Australia

Vegetable cropping systems are often characterised by high inputs of nitrogen fertiliser. Elevated emissions of nitrous oxide (N2O) can be expected as a consequence. In order to mitigate N2O emissions from fertilised agricultural fields, the use of nitrification inhibitors, in combination with ammonium based fertilisers, has been promoted. However, no data is currently available on the use of nitrification inhibitors in sub-tropical vegetable systems. A field experiment was conducted to investigate the effect of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on N2O emissions and yield from broccoli production in sub-tropical Australia. Soil N2O fluxes were monitored continuously (3 h sampling frequency) with fully automated, pneumatically operated measuring chambers linked to a sampling control system and a gas chromatograph. Cumulative N2O emissions over the 5 month observation period amounted to 298 g-N/ha, 324 g-N/ha, 411 g-N/ha and 463 g-N/ha in the conventional fertiliser (CONV), the DMPP treatment (DMPP), the DMMP treatment with a 10% reduced fertiliser rate (DMPP-red) and the zero fertiliser (0N), respectively. The temporal variation of N2O fluxes showed only low emissions over the broccoli cropping phase, but significantly elevated emissions were observed in all treatments following broccoli residues being incorporated into the soil. Overall 70–90% of the total emissions occurred in this 5 weeks fallow phase. There was a significant inhibition effect of DMPP on N2O emissions and soil mineral N content over the broccoli cropping phase where the application of DMPP reduced N2O emissions by 75% compared to the standard practice. However, there was no statistical difference between the treatments during the fallow phase or when the whole season was considered. This study shows that DMPP has the potential to reduce N2O emissions from intensive vegetable systems, but also highlights the importance of post-harvest emissions from incorporated vegetable residues. N2O mitigation strategies in vegetable systems need to target these post-harvest emissions and a better evaluation of the effect of nitrification inhibitors over the fallow phase is needed.

A FimH inhibitor prevents acute bladder infection and treats chronic cystitis caused by multidrug-resistant uropathogenic Escherichia coli ST131

Background. Escherichia coli O25b:H4-ST131 represents a predominant clone of multidrug-resistant uropathogens currently circulating worldwide in hospitals and the community. Urinary tract infections (UTIs) caused by E. coli ST131 are typically associated with limited treatment options and are often recurrent. Methods. Using established mouse models of acute and chronic UTI, we mapped the pathogenic trajectory of the reference E. coli ST131 UTI isolate, strain EC958. Results. We demonstrated that E. coli EC958 can invade bladder epithelial cells and form intracellular bacterial communities early during acute UTI. Moreover, E. coli EC958 persisted in the bladder and established chronic UTI. Prophylactic antibiotic administration failed to prevent E. coli EC958–mediated UTI. However, 1 oral dose of a small-molecular-weight compound that inhibits FimH, the type 1 fimbriae adhesin, significantly reduced bacterial colonization of the bladder and prevented acute UTI. Treatment of chronically infected mice with the same FimH inhibitor lowered their bladder bacterial burden by >1000-fold. Conclusions. In this study, we provide novel insight into the pathogenic mechanisms used by the globally disseminated E. coli ST131 clone during acute and chronic UTI and establish the potential of FimH inhibitors as an alternative treatment against multidrug-resistant E. coli.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 μM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Validation of an immunoassay to measure plasminogen-activator inhibitor-1 concentrations in human saliva

Introduction We have previously shown that the concentrations of D-dimer are significantly elevated in saliva compared with plasma. Saliva offers several advantages compared with blood analysis. We hypothesised that human saliva contains plasminogen activator inhibitor-1 (PAI-1) and that the concentrations are not affected by the time of saliva collection. The aim was to adopt and validate an immunoassay to quantify PAI-1 concentrations in saliva and to determine whether saliva collection time has an influence in the measurement. Materials and methods Two saliva samples (morning and afternoon) from the same day were collected from healthy subjects (N = 40) who have had no underlying heart conditions. A customized AlphaLISA® immunoassay (PerkinElmer®, MA, USA) was adopted and used to quantify PAI-1 concentrations. We validated the analytical performance of the customized immunoassay by calculating recovery of known amount of analyte spiked in saliva. Results: The recovery (95.03%), intra- (8.59%) and inter-assay (7.52%) variations were within the acceptable ranges. The median salivary PAI-1 concentrations were 394 pg/mL (interquartile ranges (IQR) 243.4-833.1 pg/mL) in the morning and 376 (129.1-615.4) pg/mL in the afternoon and the plasma concentration was 59,000 (24,000-110,000) pg/mL. Salivary PAI-1 did not correlate with plasma (P = 0.812). Conclusions The adopted immunoassay produced acceptable assay sensitivity and specificity. The data demonstrated that saliva contains PAI-1 and that its concentration is not affected by the time of saliva collection. There is no correlation between salivary and plasma PAI-1 concentrations. Further studies are required to demonstrate the utility of salivary PAI-1 in CVD risk factor studies.

Relationship between EGFR expression, EGFR mutation status, and the efficacy of chemotherapy plus cetuximab in FLEX study patients with advanced non-small-cell lung cancer

INTRODUCTION: The phase III FLEX study (NCT00148798) in advanced non-small-cell lung cancer indicated that the survival benefit associated with the addition of cetuximab to cisplatin and vinorelbine was limited to patients whose tumors expressed high levels of epidermal growth factor receptor (EGFR) (immunohistochemistry score of >/=200; scale 0-300). We assessed whether the treatment effect was also modulated in FLEX study patients by tumor EGFR mutation status. METHODS: A tumor mutation screen of EGFR exons 18 to 21 included 971 of 1125 (86%) FLEX study patients. Treatment outcome in low and high EGFR expression groups was analyzed across efficacy endpoints according to tumor EGFR mutation status. RESULTS: Mutations in EGFR exons 18 to 21 were detected in 133 of 971 tumors (14%), 970 of which were also evaluable for EGFR expression level. The most common mutations were exon 19 deletions and L858R (124 of 133 patients; 93%). In the high EGFR expression group (immunohistochemistry score of >/=200), a survival benefit for the addition of cetuximab to chemotherapy was demonstrated in patients with EGFR wild-type (including T790M mutant) tumors. Although patient numbers were small, those in the high EGFR expression group whose tumors carried EGFR mutations may also have derived a survival benefit from the addition of cetuximab to chemotherapy. Response data suggested a cetuximab benefit in the high EGFR expression group regardless of EGFR mutation status. CONCLUSIONS: The survival benefit associated with the addition of cetuximab to first-line chemotherapy for advanced non-small-cell lung cancer expressing high levels of EGFR is not limited by EGFR mutation status.

The fatty acid synthase inhibitor triclosan : repurposing an anti-microbial agent for targeting prostate cancer

Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

Molecular and functional characterizations of a Kunitz-type serine protease inhibitor FcKuSPI of the shrimp Fenneropenaeus chinensis

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44e53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNAwas expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays.

Determination of the effect of JO146, a CtHtrA inhibitor, on the chlamydial developmental cycle and persistence

The role of Chlamydia trachomatis HtrA (CtHtrA) for the growth and pathogenesis of this organism is presented. Inhibition of CtHtrA led to loss of infectious progeny in Chlamydia, particularly during heat stress or recovery from penicillin persistence. Isolation of CtHtrA inhibitor resistant mutants identified positive selection for mutants in fatty acid related pathways. Importantly, HtrA inhibition was effective against clinical isolates of C. trachomatis. Thus the findings combined indicate that CtHtrA is an important chlamydial growth factor that has the potential to be targeted for the development of anti-chlamydial drugs in the future.

Annual nitrogen dynamics and urea fertilizer recoveries from a dairy pasture using 15N; effect of nitrification inhibitor DMPP and reduced application rates

Direct nitrogen (N) losses from pastures contribute to the poor nitrogen use efficiency of the dairy industry, though the exact fate of applied N and the processes involved are largely unknown. Nitrification inhibitors such as DMPP can potentially increase fertilizer N use efficiency (NUE), though few studies globally have examined the effectiveness of DMPP coated urea in pastures. This study quantified the NUE of DMPP combined with reduced application rates, and the effect on N dynamics and plant–soil interactions over an annual ryegrass/kikuyu rotation in Queensland, Australia. Labeled 15N urea and DMPP was applied over 7 winter applications at standard farmer (45 kg N ha−1) and half (23 kg N ha−1) rates. Fertilizer recoveries and NUE were calculated over 13 harvests, and the contribution of fertilizer and soil N estimated. Up to 85% of the annual N harvested was from soil organic matter. DMPP at the lower rate increased annual yields by 31% compared to the equivalent urea treatment with no difference to the high N rates. Almost 40% of the N added at the conventional fertilizer application rate as urea was lost to the environment; 80 kg N ha−1 higher than the low DMPP. Combining the nitrification inhibitor DMPP with reduced fertilizer application rates shows substantial potential to reduce N losses to the environment while sustaining productivity in subtropical dairy pastures.

Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

Cilengitide combined with cetuximab and platinum-based chemotherapy as first-line treatment in advanced non-small-cell lung cancer (NSCLC) patients: Results of an open-label, randomized, controlled phase II study (CERTO)

Background: This multicentre, open-label, randomized, controlled phase II study evaluated cilengitide in combination with cetuximab and platinum-based chemotherapy, compared with cetuximab and chemotherapy alone, as first-line treatment of patients with advanced non-small-cell lung cancer (NSCLC). Patients and methods: Patients were randomized 1:1:1 to receive cetuximab plus platinum-based chemotherapy alone (control), or combined with cilengitide 2000 mg 1×/week i.v. (CIL-once) or 2×/week i.v. (CIL-twice). A protocol amendment limited enrolment to patients with epidermal growth factor receptor (EGFR) histoscore ≥200 and closed the CIL-twice arm for practical feasibility issues. Primary end point was progression-free survival (PFS; independent read); secondary end points included overall survival (OS), safety, and biomarker analyses. A comparison between the CIL-once and control arms is reported, both for the total cohorts, as well as for patients with EGFR histoscore ≥200. Results: There were 85 patients in the CIL-once group and 84 in the control group. The PFS (independent read) was 6.2 versus 5.0 months for CIL-once versus control [hazard ratio (HR) 0.72; P = 0.085]; for patients with EGFR histoscore ≥200, PFS was 6.8 versus 5.6 months, respectively (HR 0.57; P = 0.0446). Median OS was 13.6 for CIL-once versus 9.7 months for control (HR 0.81; P = 0.265). In patients with EGFR ≥200, OS was 13.2 versus 11.8 months, respectively (HR 0.95; P = 0.855). No major differences in adverse events between CIL-once and control were reported; nausea (59% versus 56%, respectively) and neutropenia (54% versus 46%, respectively) were the most frequent. There was no increased incidence of thromboembolic events or haemorrhage in cilengitide-treated patients. αvβ3 and αvβ5 expression was neither a predictive nor a prognostic indicator. Conclusions: The addition of cilengitide to cetuximab/chemotherapy indicated potential clinical activity, with a trend for PFS difference in the independent-read analysis. However, the observed inconsistencies across end points suggest additional investigations are required to substantiate a potential role of other integrin inhibitors in NSCLC treatment.

The genetic basis of human height : the role of estrogen

Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.