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2.  Isolation and Characterisation of Degradation Impurities in the Cefazolin Sodium Drug Substance 
Scientia Pharmaceutica  2013;81(4):933-950.
Two unknown impurities were detected in the cefazolin sodium bulk drug substance using gradient reversed-phase high-performance liquid chromategraphy (HPLC). These impurities were isolated by preparative HPLC and characterized by using spectroscopic techniques like LC-MS, LC-MS/MS, 1D, 2D NMR, and FT-IR. Based on the spectral data, the impurities have been characterized as N-(2,2-dihydroxyethyl)-2-(1H-tetrazol-1-yl)acetamide (Impurity-I) and 2-{carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl}-5-methylidene-5,6-dihydro-2H-1,3-thiazine-4-carboxylic acid (Impurity-II). The structures of these impurities were also established unambiguously by co-injection into HPLC to confirm the retention time. To the best of our knowledge, these two impurities were not reported elsewhere.
doi:10.3797/scipharm.1304-14
PMCID: PMC3867249  PMID: 24482765
Cefazolin sodium; Degradation Impurities; LC-MS/MS; NMR; β-Lactam
3.  Identification and Characterization of Process-Related Impurities of Trans-Resveratrol 
Scientia Pharmaceutica  2013;81(3):683-695.
This article deals with the identification and characterization of process-related impurities of trans-resveratrol (3,5,4′-trihydroxystilbene), which exhibits several health benefits, including cancer prevention. During the synthesis of the bulk drug resveratrol, three new impurities were observed. The impurities were detected using the high-performance liquid chromatographic (HPLC) method, whose area percentages ranged from 0.05 to 0.3%. A systematic study was carried out to characterize them. These impurities were isolated by preparative HPLC and characterized by spectral data, subjected to co-injection in HPLC, and were found to be matching with the impurities present in the sample. LC-MS was performed to identify the mass of these impurities. Based on their spectral data (IR, NMR, and Mass), these impurities were characterized as 2-benzyl-5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol [Impurity-B], 3-(benzyloxy)-5-[(E)-2-(4-hydroxyphenyl)ethenyl]phenol [Impurity-C], 5-{(E)-2-[4-(benzyloxy)phenyl]ethenyl}benzene-1,3-diol [Impurity-D). These compounds are not reported earlier as process-related impurities.
doi:10.3797/scipharm.1301-17
PMCID: PMC3791933  PMID: 24106667
Resveratrol; Impurities; Structure characterization; HPLC; Structural elucidation; NMR; LC-MS
4.  Antimicrobial Activity and Phytochemical Constituents of Leaf Extracts of Cassia auriculata 
Plants produce a wide variety of phytochemical constituents, which are secondary metabolites and are used either directly or indirectly in the pharmaceutical industry. ‘For centuries, man has effectively used various components of plants or their extracts for the treatment of many diseases, including bacterial infections. In the present study methanol, chloroform and aqueous extracts of Cassia auriculata leaf were subjected for antimicrobial activity by well-diffusion method against six bacterial strains namely Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis. The results revealed that the methanol and chloroform extracts exhibited strong inhibitory activity against all the tested organisms (zone of inhibition of 12-20 mm), except Pseudomonas aeruginosa (zone of inhibition 10 mm or nil). The aqueous extracts showed moderate activity by ‘Zone of inhibition ≤12 or nil). The extracts were screened for their phytochemical constituents by standard protocols’ and were shown to contain carbohydrates, proteins, alkaloids, flavonoids, steroids, saponins and tannins. The antibacterial activity of these extracts is possibly linked to the presence of flavonoids, steroid, saponins and/or tannins. Further studies are needed to determine the precise active principles from Cassia auriculata.
doi:10.4103/0250-474X.113546
PMCID: PMC3719143  PMID: 23901174
Antibacterial activity; Cassia auriculata; phytochemical constituents
6.  Molecular Basis for Unidirectional Scaffold Switching of Human Plk4 in Centriole Biogenesis 
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. Here we showed that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal structure analyses revealed that Cep192 - and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152-peptide bound to the CPB markedly better than the Cep192-peptide and effectively snatched the CPB away from a preformed CPB–Cep192-peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.
doi:10.1038/nsmb.2846
PMCID: PMC4125498  PMID: 24997597
7.  Spontaneous expectoration of a Blalock-Taussig shunt a decade after operation 
An eleven-year-old boy expectorated a foreign body in cough that was identified as the prosthetic graft used for a Blalock-Taussig shunt. The shunt procedure was done 10 years earlier, and a definitive repair for tetralogy of Fallot was done a year later. He had no other symptoms, and a computed tomography (CT) angiogram did not reveal any other significant anomaly. The reason for this extremely rare event is unclear.
doi:10.4103/0974-2069.149518
PMCID: PMC4322401
Blalock-Taussig Shunt; foreign body; tetralogy of Fallot
8.  Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability 
Neuron  2013;80(6):10.1016/j.neuron.2013.09.021.
Summary
Mutations of the FOXP2 gene impair speech and language development in humans and shRNA-mediated suppression of the avian orthologue FoxP2 disrupts song learning in juvenile zebra finches. How diminished FoxP2 levels affect vocal control and alter the function of neural circuits important to learned vocalizations remains unclear. Here we show that FoxP2 knockdown in the songbird striatum disrupts developmental and social modulation of song variability. Recordings in anaesthetized birds show that FoxP2 knockdown interferes with D1R-dependent modulation of activity propagation in a corticostriatal pathway important to song variability, an effect that may be partly attributable to reduced D1R and DARPP-32 protein levels. Furthermore, recordings in singing birds reveal that FoxP2 knockdown prevents social modulation of singing-related activity in this pathway. These findings show that reduced FoxP2 levels interfere with the dopaminergic modulation of vocal variability, which may impede song and speech development by disrupting reinforcement learning mechanisms.
doi:10.1016/j.neuron.2013.09.021
PMCID: PMC3881289  PMID: 24268418
9.  Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules 
Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation.
doi:10.3390/ijms151223418
PMCID: PMC4284775  PMID: 25526563
beta (β) cells; diabetes; differentiation; stem cells; pancreas
10.  Identification of RASAL1 as a Major Tumor Suppressor Gene in Thyroid Cancer 
Background
RAS-coupled MAPK and PI3K pathways play a fundamental role in thyroid tumorigenesis, and classical genetic alterations upregulating these pathways are well characterized. We hypothesized that gene abnormality of negative modulators of these signaling pathways might be an important alternative genetic background for thyroid cancer.
Methods
By examining gene expression patterns of negative modulators of RAS signaling, we attempted to identify potential tumor suppressor genes. We then analyzed the methylation and mutation patterns of the identified gene in 101 thyroid tumors and tested its functions in vitro and in vivo to establish the tumor suppressor role in thyroid cancer.
Results
Among 13 negative modulators of the RAS pathway screened, RASAL1, encoding a RAS GTPase-activating protein, was frequently hypermethylated in thyroid cancers, which was coupled to its silencing in thyroid cancer cells. We also, for the first time, identified the presence of RASAL1 mutations, with a prevalence of 4.88% (n = 2 of 41) in follicular thyroid cancer (FTC) and 16.67% (n = 5 of 30) in anaplastic thyroid cancer (ATC). RASAL1 displayed MAPK- and PI3K-suppressing and thyroid tumor–suppressing activities, which were all impaired by the mutations. Hypermethylation and mutations of RASAL1 were mutually exclusive and collectively found in zero of 20 benign thyroid tumors, 3.22% (n = 1 of 31) of papillary thyroid cancers, 31.70% (n = 13 of 41) of FTCs, and 33.33% (n = 10 of 30) of ATCs. A rate of 20.83% (n = 5 of 24) of tumors carrying RASAL1 mutation or methylation at high levels (>50%) vs 44.16% (n = 34 of 77) of tumors carrying no RASAL1 mutation or methylation at low levels (< 50%) harbored any of the classical mutations (two-sided P = .02, Fisher exact test) in RAS, BRAF, PTEN, and PIK3CA genes in the MAPK and PI3K pathways, revealing a largely mutually exclusive relationship.
Conclusions
We identified RASAL1 as a major tumor suppressor gene that is frequently inactivated by hypermethylation and mutations, providing a new alternative genetic background for thyroid cancer, particularly FTC and ATC.
doi:10.1093/jnci/djt249
PMCID: PMC3818169  PMID: 24136889
11.  Combined Therapeutic Use of Oral Alitretinoin and Narrowband Ultraviolet-B Therapy in the Treatment of Hailey-Hailey Disease 
Dermatology Reports  2014;6(1):5604.
Hailey-Hailey disease (HHD) is a chronic familial bullous disease characterized by recurrent blisters and erosions typically at friction-prone areas of the body accompanied by acantholysis upon histologic examination. There are a number of therapies used in the management of HHD. Its symptoms have been effectively treated with antimicrobial therapies, corticosteroids and other agents such as cyclosporine and prednisone. However, such treatments are not always effective. Therefore, there is a need for new treatments for the management of HHD. In this report, a patient with long-standing HHD responsive only to high levels of prednisone is described. After the successful tapering and cessation of oral prednisone the patient began a new combination therapy of complementary doses of oral alitretinoin, and narrowband UVB therapy, which yielded a favorable response within 2-3 weeks. After 6 weeks, a mono-therapy of daily (30 mg) oral alitretinoin was sufficient to maintain successful near-complete remission of the disease.
doi:10.4081/dr.2014.5604
PMCID: PMC4224006  PMID: 25386331
Hailey-Hailey disease; pemphigus; alitretinoin; narrowband UVB
12.  Alcohol exposure in utero increases susceptibility to prostate tumorigenesis in rat offspring 
Alcoholism, clinical and experimental research  2013;37(11):10.1111/acer.12171.
Background
Prenatal alcohol exposure has been shown to increase offspring susceptibility to some chemical carcinogens. Whether prenatal exposure to alcohol makes the offspring more susceptible to the development of prostate cancer is not known. Therefore, we determined if any functional abnormalities and increased cancer susceptibility exist in the prostate of fetal alcohol exposed male rats during the adult period.
Methods
Pregnant rats were fed with a liquid diet containing alcohol (alcohol-fed), pair-fed with isocaloric liquid diet (pair-fed), or ad libitum fed with rat chow (ad lib-fed). Male offspring of these rats were given N-Nitroso-N-methylurea and testosterone to induce prostate neoplasia or left untreated. Around 6 to 8 months of age, the prostate of these animals were processed for determination of biochemical changes and histopathologies.
Results
Prostates of non-carcinogen treated animals which were alcohol exposed during the prenatal period demonstrated inflammatory cell infiltration and epithelial atypia and increased number of proliferative cells in the ventral lobe of this gland, but the prostate of control animal showed normal cytoarchitecture. In addition, prenatally alcohol-exposed rats showed decreased levels of cell-cell adhesion marker and increased estrogenic activity in the ventral prostate. Prenatally ethanol-exposed rats, when treated with carcinogen and testosterone, showed histological evidence for high-grade prostatic intraepithelial neoplasia primarily in the ventral prostate, whereas control animals showed only low-grade prostatic intraepithelial neoplasia. Prenatally ethanol-exposed rats treated with carcinogen and testosterone also showed increased number of proliferative cells and androgen receptor with concomitant decreased levels of tumor suppressor proteins in the ventral prostate.
Conclusions
These results suggest for the first time that prenatal ethanol exposures induces histophysiological changes in the prostate as well as it increases the susceptibility of the prostate to develop neoplasia during adulthood.
doi:10.1111/acer.12171
PMCID: PMC3812315  PMID: 23889735
Aromatase; estrogen receptor; fetal alcohol; prostate cancers
13.  Dyspnea in Chronic Fatigue Syndrome (CFS): Comparison of Two Prospective Cross-Sectional Studies 
Chronic Fatigue Syndrome (CFS) subjects have many systemic complaints including shortness of breath. Dyspnea was compared in two CFS and control cohorts to characterize pathophysiology. Cohort 1 of 257 CFS and 456 control subjects were compared using the Medical Research Council chronic Dyspnea Scale (MRC Score; range 0–5). Cohort 2 of 106 CFS and 90 controls answered a Dyspnea Severity Score (range 0–20) adapted from the MRC Score. Subsets of both cohorts completed CFS Severity Scores, fatigue, and other questionnaires. A subset had pulmonary function and total lung capacity measurements. Results show MRC Scores were equivalent between sexes in Cohort 1 CFS (1.92 [1.72–2.16]; mean [95% C.I.]) and controls (0.31 [0.23–0.39]; p<0.0001). Receiver-operator curves identified 2 as the threshold for positive MRC Scores in Cohort 1. This indicated 54% of CFS, but only 3% of controls, had significant dyspnea. In Cohort 2, Dyspnea Score threshold of 4 indicated shortness of breath in 67% of CFS and 23% of controls. Cohort 2 Dyspnea Scores were higher for CFS (7.80 [6.60–9.00]) than controls (2.40 [1.60–3.20]; p<0.0001). CFS had significantly worse fatigue and other complaints compared to controls. Pulmonary function was normal in CFS, but Borg scores and sensations of chest pain and dizziness were significantly greater during testing than controls. General linear model of Cohort 2 CFS responses linked Dyspnea with rapid heart rate, chest pain and dizziness. In conclusion, sensory hypersensitivity without airflow limitation contributed to dyspnea in CFS. Correlates of dyspnea in controls were distinct from CFS suggesting different mechanisms.
doi:10.5539/gjhs.v5n2p94
PMCID: PMC4209305  PMID: 23445698
Medical Research Council Dyspnea Score; fatigue; CFS Severity Score; fibromyalgia; pulmonary function tests; total lung capacity; central sensitization
14.  Blood Purification and Mortality in Sepsis: A Meta-analysis of Randomized Trials 
Critical care medicine  2013;41(9):2209-2220.
Objectives
Although blood purification improves outcomes in animal studies of sepsis, results of clinical trials have been mixed. We conducted a systematic review and meta-analysis of randomized trials to determine the association between various blood purification techniques and all-cause mortality in humans with sepsis.
Data Sources
We searched for relevant studies in MEDLINE, EMBASE, and the Cochrane Library database from January 1966 until May 2012.
Study Selection
Inclusion required a diagnosis of sepsis and comparison of blood purification techniques including hemofiltration, hemoperfusion, plasma exchange, or hemodialysis with no blood purification (control group).
Data Extraction
Two authors independently selected studies and extracted data. Summary statistics, risk ratios (RRs), and CIs were calculated using random-effects modeling. Study quality was assessed using Jadad score, and publication bias using funnel plots and Egger’s statistic.
Data Synthesis
Overall, blood purification decreased mortality compared to no blood purification (35.7% versus 50.1%; RR, 0.69; 95% CI, 0.56–0.84; p < 0.001; 16 trials, n=827). However, these results were driven mainly by hemoperfusion (RR, 0.63; 95% CI, 0.50–0.80; p < 0.001; 10 trials, n=557), and plasma exchange (RR, 0.63; 95% CI, 0.42–0.96; p = 0.03; 2 trials, n=128). Pooling of all trials of blood purification for treatment of sepsis was no longer associated with lower mortality (RR, 0.89; 95% CI, 0.71–1.13; p = 0.36; 8 trials, n=457) after excluding trials using polymyxin B hemoperfusion.
Conclusions
Blood purification techniques including hemoperfusion, plasma exchange, and hemofiltration with hemoperfusion were associated with lower mortality in patients with sepsis. These results were mainly influenced by studies using polymyxin B hemoperfusion from Japan.
doi:10.1097/CCM.0b013e31828cf412
PMCID: PMC3758418  PMID: 23860248
sepsis; blood purification; cytokines; inflammation; mortality; meta-analysis
15.  Selective targeting of KRAS-Mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-Mutant cells 
Oncotarget  2014;5(17):7635-7650.
MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors.
PMCID: PMC4202150  PMID: 25245095
Colorectal Cancer; miRNA; miR-126; KRAS; KRAS mutant
16.  Theory on the Dynamics of Oscillatory Loops in the Transcription Factor Networks 
PLoS ONE  2014;9(8):e104328.
We develop a detailed theoretical framework for various types of transcription factor gene oscillators. We further demonstrate that one can build genetic-oscillators which are tunable and robust against perturbations in the critical control parameters by coupling two or more independent Goodwin-Griffith oscillators through either -OR- or -AND- type logic. Most of the coupled oscillators constructed in the literature so far seem to be of -OR- type. When there are transient perturbations in one of the -OR- type coupled-oscillators, then the overall period of the system remains constant (period-buffering) whereas in case of -AND- type coupling the overall period of the system moves towards the perturbed oscillator. Though there is a period-buffering, the amplitudes of oscillators coupled through -OR- type logic are more sensitive to perturbations in the parameters associated with the promoter state dynamics than -AND- type. Further analysis shows that the period of -AND- type coupled dual-feedback oscillators can be tuned without conceding on the amplitudes. Using these results we derive the basic design principles governing the robust and tunable synthetic gene oscillators without compromising on their amplitudes.
doi:10.1371/journal.pone.0104328
PMCID: PMC4128676  PMID: 25111803
17.  A p21-ZEB1 Complex Inhibits Epithelial-Mesenchymal Transition through the MicroRNA 183-96-182 Cluster 
Molecular and Cellular Biology  2014;34(3):533-550.
The tumor suppressor p21 acts as a cell cycle inhibitor and has also been shown to regulate gene expression by functioning as a transcription corepressor. Here, we identified p21-regulated microRNAs (miRNAs) by sequencing small RNAs from isogenic p21+/+ and p21−/− cells. Three abundant miRNA clusters, miR-200b-200a-429, miR-200c-141, and miR-183-96-182, were downregulated in p21-deficient cells. Consistent with the known function of the miR-200 family and p21 in inhibition of the epithelial-mesenchymal transition (EMT), we observed EMT upon loss of p21 in multiple model systems. To explore a role of the miR-183-96-182 cluster in EMT, we identified its genome-wide targets and found that miR-183 and miR-96 repressed common targets, including SLUG, ZEB1, ITGB1, and KLF4. Reintroduction of miR-200, miR-183, or miR-96 in p21−/− cells inhibited EMT, cell migration, and invasion. Conversely, antagonizing miR-200 and miR-183-96-182 cluster miRNAs in p21+/+ cells increased invasion and elevated the levels of VIM, ZEB1, and SLUG mRNAs. Furthermore, we found that p21 forms a complex with ZEB1 at the miR-183-96-182 cluster promoter to inhibit transcriptional repression of this cluster by ZEB1, suggesting a reciprocal feedback loop.
doi:10.1128/MCB.01043-13
PMCID: PMC3911499  PMID: 24277930
18.  Angiomotins link F-actin architecture to Hippo pathway signaling 
Molecular Biology of the Cell  2014;25(10):1676-1685.
Angiomotin proteins, together with LATS kinase, regulate the Hippo pathway transcriptional coactivator YAP in response to changes in the F-actin cytoskeleton. Competition between F-actin and YAP for binding to angiomotins makes YAP regulation responsive to F-actin levels. Phosphorylation by LATS can switch angiomotins from F-actin to YAP binding.
The Hippo pathway regulates the transcriptional coactivator YAP to control cell proliferation, organ size, and stem cell maintenance. Multiple factors, such as substrate stiffness, cell density, and G protein–coupled receptor signaling, regulate YAP through their effects on the F-actin cytoskeleton, although the mechanism is not known. Here we show that angiomotin proteins (AMOT130, AMOTL1, and AMOTL2) connect F-actin architecture to YAP regulation. First, we show that angiomotins are required to relocalize YAP to the cytoplasm in response to various manipulations that perturb the actin cytoskeleton. Second, angiomotins associate with F-actin through a conserved F-actin–binding domain, and mutants defective for F-actin binding show enhanced ability to retain YAP in the cytoplasm. Third, F-actin and YAP compete for binding to AMOT130, explaining how F-actin inhibits AMOT130-mediated cytoplasmic retention of YAP. Furthermore, we find that LATS can synergize with F-actin perturbations by phosphorylating free AMOT130 to keep it from associating with F-actin. Together these results uncover a mechanism for how F-actin levels modulate YAP localization, allowing cells to make developmental and proliferative decisions based on diverse inputs that regulate actin architecture.
doi:10.1091/mbc.E13-11-0701
PMCID: PMC4019498  PMID: 24648494
20.  AXL is a key regulator of inherent and chemotherapy-induced invasion and predicts a poor clinical outcome in early stage colon cancer 
Purpose
Despite the use of 5-FU-based adjuvant treatments, a large proportion of high risk stage II/III colorectal cancer (CRC) patients will relapse. Thus, novel therapeutic strategies are needed for early stage CRC. Residual micrometastatic disease from the primary tumour is a major cause of patient relapse.
Experimental Design
In order to model CRC tumour cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) CRC cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins which underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumours from patients with early stage CRC (N=336) were examined for AXL expression.
Results
Invasive CRC cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability and an attenuation of EGFR/HER2 autocrine signalling. Receptor tyrosine kinase arrays showed significant increases in AXL levels in all invasive sub-lines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion and targeting AXL using pharmacologic inhibition or RNAi approaches, suppressed basal and 5-FU-induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early stage CRC tissues.
Conclusions
We have identified AXL as poor prognostic marker and important mediator of cell migration/invasiveness in CRC. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in CRC, in particular in the adjuvant disease where EGFR/VEGF-targeted therapies have failed.
doi:10.1158/1078-0432.CCR-13-1354
PMCID: PMC3885388  PMID: 24170546
21.  Optimization of Pyrrolamides as Mycobacterial GyrB ATPase Inhibitors: Structure-Activity Relationship and In Vivo Efficacy in a Mouse Model of Tuberculosis 
Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 μg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 μg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10−6 to 10−8, and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.
doi:10.1128/AAC.01751-13
PMCID: PMC3910769  PMID: 24126580
22.  Hydatid cyst of lung: An uncommon cause of chest pain in young 
Echinococcosis can involve any organ. The liver is the most common organ involved, followed by the lungs. Depending on the location of involvement it can have varied presentation. We describe a young adult presenting with chest pain secondary to a pulmonary giant hydatid cyst. A pulmonary hydatid cyst should be considered in the differential diagnosis of patients presenting with chest pain without conventional risk factors of coronary artery disease, especially in a tropical region.
doi:10.4103/0970-2113.135770
PMCID: PMC4129599  PMID: 25125814
Chest pain; chest radiograph; echinococcosis
23.  Effect of Noni (Morinda citrifolia Linn.) Fruit and Its Bioactive Principles Scopoletin and Rutin on Rat Vas Deferens Contractility: An Ex Vivo Study 
The Scientific World Journal  2014;2014:909586.
This study examined the effect of methanolic extract of Morinda citrifolia Linn. (MMC) and its bioactive principles, scopoletin and rutin, on dopamine- and noradrenaline-evoked contractility in isolated rat vas deferens preparations. MMC (1–40 mg/mL), scopoletin (1–200 μg/mL), and rutin hydrate (0.6–312.6 μg/mL) dose-dependently inhibited the contractility evoked by submaximal concentrations of both dopamine and noradrenaline, respectively. Haloperidol and prazosin, reference dopamine D2, and α1-adrenoceptors antagonists significantly reversed the dopamine- and noradrenaline-induced contractions, respectively, in a dose-dependent manner. Interestingly, MMC per se at higher doses (60–100 mg/mL) showed dose-dependent contractile response in rat vas deferens which was partially inhibited by high doses of haloperidol but not by prazosin. These results demonstrated the biphasic effects of MMC on dopaminergic system; that is, antidopaminergic effect at lower concentrations (<40 mg/mL) and dopaminergic agonistic effect at higher concentrations (>60 mg/mL). However, similar contractile response at high doses of scopoletin (0.5–5 mg/mL) and rutin hydrate (0.5–5 mg/mL) per se was not observed. Therefore, it can be concluded that the bioactive principles of MMC, scopoletin, and rutin might be responsible for the antidopaminergic and antiadrenergic activities of MMC.
doi:10.1155/2014/909586
PMCID: PMC4090441  PMID: 25045753
24.  Monitoring Organ Donors to Improve Transplantation Results (MOnIToR) trial methodology 
Background
Despite efforts to increase organ donation, there remain critical shortages in organ donors and organs procured per donor. Our trial is a large-scale, multicentre, randomised controlled trial in brain-dead donors, to compare protocolised care (using minimally invasive haemodynamic monitoring) with usual care. We describe the study design and discuss unique aspects of doing research in this population.
Methods
Our study will randomise brain-dead patients to protocolised or usual care. The primary end point is the number of organs transplanted per donor. Secondary end points include number of transplantable organs per donor, recipient 6-month hospital-free survival time, and the relationship between the level of interleukin-6 and the number and usability of organs transplanted. The primary analysis will be an intention-to-treat analysis; secondary analyses include modified intention-to-treat and as-treated analyses. The study will also compare the ratio of observed to expected number of organs transplanted per donor, by treatment arm, as a secondary end point. Preplanned subgroup analyses include restriction to extended criteria donors, and donors older or younger than 65 years.
Results and conclusions
Several unique challenges for study design and execution can be seen in our trial, and it should generate results that will inform and influence the fields of organ donation and transplantation.
PMCID: PMC4058763  PMID: 23944211
25.  Dexmedetomidine use in the ICU: Are we there yet? 
Critical Care  2013;17(3):320.
Expanded abstract
Citation
Jakob SM, Ruokonen E, Grounds RM, Sarapohja T, Garratt C, Pocock SJ, Bratty JR, Takala J; Dexmedeto midine for Long-Term Sedation Investigators: Dexmedetomidine vesus midazolam or propofol for sedation during prolonged mechanical ventilation: two randomized controlled trials. JAMA 2012, 307:1151-1160.
Background
Long-term sedation with midazolam or propofol in intensive care units (ICUs) has serious adverse effects. Dexmedetomidine, an alpha-2 agonist available for ICU sedation, may reduce the duration of mechanical ventilation and enhance patient comfort.
Methods
Objective
The objective was to determine the efficacy of dexmedetomidine versus midazolam or propofol (preferred usual care) in maintaining sedation, reducing duration of mechanical ventilation, and improving patients' interaction with nursing care.
Design
Two phase 3 multicenter, randomized, double-blind trials were conducted.
Setting
The MIDEX (Midazolam vs. Dexmedetomidine) trial compared midazolam with dexmedetomidine in ICUs of 44 centers in nine European countries. The PRODEX (Propofol vs. Dexmedetomidine) trial compared propofol with dexmedetomidine in 31 centers in six European countries and two centers in Russia.
Subjects
The subjects were adult ICU patients who were receiving mechanical ventilation and who needed light to moderate sedation for more than 24 hours.
Intervention
After enrollment, 251 and 249 subjects were randomly assigned midazolam and dexmedetomidine, respectively, in the MIDEX trial, and 247 and 251 subjects were randomly assigned propofol and dexmedetomidine, respectively, in the PRODEX trial. Sedation with dexmedetomidine, midazolam, or propofol; daily sedation stops; and spontaneous breathing trials were employed.
Outcomes
For each trial, investigators tested whether dexmedetomidine was noninferior to control with respect to proportion of time at target sedation level (measured by Richmond Agitation Sedation Scale) and superior to control with respect to duration of mechanical ventilation. Secondary end points were the ability of the patient to communicate pain (measured by using a visual analogue scale [VAS]) and length of ICU stay. Time at target sedation was analyzed in per-protocol (midazolam, n = 233, versus dexmedetomidine, n = 227; propofol, n = 214, versus dexmedetomidine, n = 223) population.
Results
Dexmedetomidine/midazolam ratio in time at target sedation was 1.07 (95% confidence interval (CI) 0.97 to 1.18), and dexmedetomidine/propofol ratio in time at target sedation was 1.00 (95% CI 0.92 to 1.08). Median duration of mechanical ventilation appeared shorter with dexmedetomidine (123 hours, interquartile range (IQR) 67 to 337) versus midazolam (164 hours, IQR 92 to 380; P = 0.03) but not with dexmedetomidine (97 hours, IQR 45 to 257) versus propofol (118 hours, IQR 48 to 327; P = 0.24). Patient interaction (measured by using VAS) was improved with dexmedetomidine (estimated score difference versus midazolam 19.7, 95% CI 15.2 to 24.2; P <0.001; and versus propofol 11.2, 95% CI 6.4 to 15.9; P <0.001). Lengths of ICU and hospital stays and mortality rates were similar. Dexmedetomidine versus midazolam patients had more hypotension (51/247 [20.6%] versus 29/250 [11.6%]; P = 0.007) and bradycardia (35/247 [14.2%] versus 13/250 [5.2%]; P <0.001).
Conclusions
Among ICU patients receiving prolonged mechanical ventilation, dexmedetomidine was not inferior to midazolam and propofol in maintaining light to moderate sedation. Dexmedetomidine reduced duration of mechanical ventilation compared with midazolam and improved the ability of patients to communicate pain compared with midazolam and propofol. Greater numbers of adverse effects were associated with dexmedetomidine.
doi:10.1186/cc12707
PMCID: PMC3706806  PMID: 23731973

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