Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs).
The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified.
This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.
To evaluate the cytotoxicity of dimeric naphthoquinones (BiQs) in prostate cancer cells.To assess the interaction of dimeric naphthoquinones with common therapies including radiation and docetaxel.
MATERIALS AND METHODS
The cytotoxicity of 12 different dimeric naphthoquinones was assessed in androgen-independent (PC-3, DU-145) and androgen-responsive (LNCaP, 22RV1) prostate cancer cell lines and in prostate epithelial cells (PrECs).BiQ2 and BiQ11 were selected for determination of dose response, effects on colony formation and initial exploration into mechanism of action.Synergistic effects with radiation and docetaxel were explored using colony-forming and MTT assays.
At concentrations of 15μM, BiQ2, BiQ3, BiQ11, BiQ12, and BiQ15 demonstrated cytotoxicity in all prostate cancer cell lines.Treatment with BiQs limited the ability of prostate cancer cells to form colonies in clonogenic assays.Exposure of prostate cancer to BiQs increased cellular reactive oxygen species (ROS), decreased ATP production, and promoted apoptosis.BiQ cytotoxicity was independent of NADP(H):quinone oxidoreductase 1 (NQO1) activity in PrECs, PC-3 and 22RV1, but not DU-145 cells.Exposure of prostate cancer cells to radiation before treatment with BiQs increased their activity allowing for inhibitory effects well below the IC50s of these compounds in PrECs.Co-administration of BiQs with docetaxel had minimal additive effects.
Dimeric naphthoquinones represent a new class of compounds with prostate cancer cytotoxicity and synergistic effects with radiation. The cytotoxic effect of these agents is probably contributed to by the accumulation of ROS and mitochondrial dysfunction.Further studies are warranted to better characterize this class of potential chemotherapeutics.
dimeric naphthoquinones; oxidative stress; cytotoxicity; prostate cancer; radiation synergy
During slow-wave sleep, general anesthesia, and generalized seizures, there is an absence of consciousness. These states are characterized by low-frequency large-amplitude traveling waves in scalp electroencephalogram. Therefore the oscillatory state might be an indication of failure to form coherent neuronal assemblies necessary for consciousness. A generalized seizure event is a pathological brain state that is the clearest manifestation of waves of synchronized neuronal activity. Since gap junctions provide a direct electrical connection between adjoining neurons, thus enhancing synchronous behavior, reducing gap-junction conductance should suppress seizures; however there is no clear experimental evidence for this. Here we report theoretical predictions for a physiologically-based cortical model that describes the general anesthetic phase transition from consciousness to coma, and includes both chemical synaptic and direct electrotonic synapses. The model dynamics exhibits both Hopf (temporal) and Turing (spatial) instabilities; the Hopf instability corresponds to the slow (≲8 Hz) oscillatory states similar to those seen in slow-wave sleep, general anesthesia, and seizures. We argue that a delicately balanced interplay between Hopf and Turing modes provides a canonical mechanism for the default non-cognitive rest state of the brain. We show that the Turing mode, set by gap-junction diffusion, is generally protective against entering oscillatory modes; and that weakening the Turing mode by reducing gap conduction can release an uncontrolled Hopf oscillation and hence an increased propensity for seizure and simultaneously an increased sensitivity to GABAergic anesthesia.
Seizure; Gap junctions; Mean-field cortical model; Hopf oscillations; Turing patterns; Nonlinear interactions; Phase coherence
Eleven genetic loci have reached genome-wide significance in a recent meta-analysis of genome-wide association studies in Parkinson disease (PD) based on populations of Caucasian descent. The extent to which these genetic effects are consistent across different populations is unknown.
Investigators from the Genetic Epidemiology of Parkinson's Disease Consortium were invited to participate in the study. A total of 11 SNPs were genotyped in 8,750 cases and 8,955 controls. Fixed as well as random effects models were used to provide the summary risk estimates for these variants. We evaluated between-study heterogeneity and heterogeneity between populations of different ancestry.
In the overall analysis, single nucleotide polymorphisms (SNPs) in 9 loci showed significant associations with protective per-allele odds ratios of 0.78–0.87 (LAMP3, BST1, and MAPT) and susceptibility per-allele odds ratios of 1.14–1.43 (STK39, GAK, SNCA, LRRK2, SYT11, and HIP1R). For 5 of the 9 replicated SNPs there was nominally significant between-site heterogeneity in the effect sizes (I2 estimates ranged from 39% to 48%). Subgroup analysis by ethnicity showed significantly stronger effects for the BST1 (rs11724635) in Asian vs Caucasian populations and similar effects for SNCA, LRRK2, LAMP3, HIP1R, and STK39 in Asian and Caucasian populations, while MAPT rs2942168 and SYT11 rs34372695 were monomorphic in the Asian population, highlighting the role of population-specific heterogeneity in PD.
Our study allows insight to understand the distribution of newly identified genetic factors contributing to PD and shows that large-scale evaluation in diverse populations is important to understand the role of population-specific heterogeneity. Neurology® 2012;79:659–667
To examine the efficacy of adjuvant chemoradiotherapy after pancreaticoduodenectomy (PD) for pancreatic adenocarcinoma (PC) in patients undergoing resection at Johns Hopkins Hospital (JHH; Baltimore, MD).
Patients and Methods
Between August 30, 1993, and February 28, 2005, a total of 908 patients underwent PD for PC at JHH. A prospective database was reviewed to determine which patients received fluorouracil (FU) -based CRT. Excluded patients had metastatic disease, died 60 or fewer days after PD, received preoperative therapy, an experimental vaccine, adjuvant chemotherapy or radiation alone. The final cohort includes 616 patients.
The median follow-up was 17.8 months (interquartile range, 9.7 to 33.5 months). Overall median survival was 17.9 months (95% CI, 16.3 to 19.5 months). Groups were similar with respect to tumor size, nodal status, and margin status, but the CRT group was younger (P < .001), and less likely to present with a severe comorbid disease (P = .001). Patients with carcinomas larger than 3 cm (P = .001), grade 3 and 4 (P < .001), margin-positive resection (P = .001), and complications after surgery (P = .017) had poor long-term survival. Patients receiving CRT experienced an improved median (21.2 v 14.4 months; P < .001), 2-year (43.9% v 31.9%), and 5-year (20.1% v 15.4%) survival compared with no CRT. After controlling for high-risk features, CRT was still associated with improved survival (relative risk = 0.74; 95% CI, 0.62 to 0.89).
These data suggest that adjuvant concurrent FU-based CRT significantly improves survival after PD for PC when compared with patients not receiving CRT. These data support the use of combined adjuvant CRT for PC.
Cancer cells reprogram their metabolism due to genetic alteration to compensate for increased energy demand and enhanced anabolism, cell proliferation, and protection from oxidative damage. Here, we assessed the cytotoxicity of three dimeric naphthoquinones against the glycolytic MCF-7 versus the oxidative MDA-453 breast carcinoma cell lines. Dimeric naphthoquinones 1 and 2 impaired MDA-453, but not MCF-7, cell growth at IC50 = 15 μM. Significant increase in reactive oxygen species, decrease in oxygen consumption and ATP production were observed in MDA-453 cells but not in MCF-7 cell. These findings suggest that oxidative stress and mitochondrial dysfunction are mechanisms by which these agents exert their cytotoxic effects. Cyclic voltammetry and semi-empirical molecular orbital calculations further characterized the electrochemical behavior of these compounds. These results also suggest that dimeric naphthoquinones may be used to selectively target cancer cells that depend on oxidative phosphorylation for energy production and macromolecular synthesis.
Dimeric Naphthoquinones; Anticancer Agents; Cytotoxicity; Oxidative Stress; Tumor Metabolism
Numerous studies have now demonstrated that many older women retain an interest in their sexual lives. Yet, how many old age psychiatrists commonly check with older women about whether the depression they are treating, or the SSRIs (Selective Serotonin Re-uptake Inhibitors) they have prescribed, have adversely affected their patient’s sexual lives? We consider the latest evidence regarding cultural, social and medical influences on older women’s sexual lives and some specific issues which affect lesbian and transsexual people. We examine how mental illness and psychotropic medication in particular can adversely affect older women’s sexual functioning and at how difficult it often proves to be for women to seek help. We also focus on why doctors and in particular psychiatrists may not take a sexual history, look for sexual side effects or refer for appropriate treatment, especially when interviewing older women patients. Most published information about psychiatric training and sexual issues focuses on the younger male patient. We therefore aimed to provide a broad-ranging review of the literature regarding female sexual functioning in old age, the difficulties that can arise and the role that old age psychiatrists have an opportunity to fulfil, in this often neglected aspect of their patients’ treatment. From our review it was clear that, in the light of the increasing cultural acceptability of discussions regarding sexuality and older women, the training of student doctors and trainee psychiatrists needs to reflect this change so that old age psychiatrists can enhance the quality of their patient care.
Female; old age; sexuality; chronic disease; help-seeking; psychiatry
Here we describe the Parametric Response Map (PRM), a voxel-wise approach for image analysis and quantification of hemodynamic alterations during treatment for 44 patients with high-grade glioma. Relative cerebral blood volume (rCBV) and flow (rCBF) maps were acquired before treatment and after 1 and 3 weeks of therapy. We compared the standard approach using region-of-interest analysis for change in rCBV or rCBF to the change in perfusion parameters on the basis of PRM (PRMrCBV and PRMrCBF) for their accuracy in predicting overall survival. Neither the percentage change of rCBV or rCBF predicted survival, whereas the regional response evaluations based upon PRM were highly predictive of survival. Even when accounting for baseline rCBV, which is prognostic, PRMrCBV proved more predictive of overall survival.
Mathematical models exist that quantify the effect of temperature on poikilotherm growth rate. One family of such models assumes a single rate-limiting ‘master reaction’ using terms describing the temperature-dependent denaturation of the reaction's enzyme. We consider whether such a model can describe growth in each domain of life.
A new model based on this assumption and using a hierarchical Bayesian approach fits simultaneously 95 data sets for temperature-related growth rates of diverse microorganisms from all three domains of life, Bacteria, Archaea and Eukarya. Remarkably, the model produces credible estimates of fundamental thermodynamic parameters describing protein thermal stability predicted over 20 years ago.
The analysis lends support to the concept of universal thermodynamic limits to microbial growth rate dictated by protein thermal stability that in turn govern biological rates. This suggests that the thermal stability of proteins is a unifying property in the evolution and adaptation of life on earth. The fundamental nature of this conclusion has importance for many fields of study including microbiology, protein chemistry, thermal biology, and ecological theory including, for example, the influence of the vast microbial biomass and activity in the biosphere that is poorly described in current climate models.
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α–specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML.
intestinal botulism; Crohn disease; Clostridium botulinum; bacteria; adult intestinal toxemia botulism; Canada
American Indian/Alaska Native youth represent the strength and continued survival of many Nations and Tribes. However, they currently experience numerous health disparities and challenges, including the highest rate of suicide among 15 to 24 year-olds in the United States. Our comprehensive review of the literature on the mental health of AI/AN youth highlighted seven focal causes of behavioral health disparities: 1) high levels of violence and trauma exposure and traumatic loss, 2) past and current oppression, racism, and discrimination, 3) underfunded systems of care, 4) disregard for effective indigenous practices in service provision, policy, and funding, 5) overreliance on evidence-based practices, 6) lack of cultural competence among systems of care and providers, and 7) barriers to care. Seven policy recommendations that recognize the importance of moving beyond exclusive reliance on western models of care and that seek to foster transformation of individuals, families, communities, behavioral health service systems of care, and social structures are presented, supported, and discussed.
Adolescent; Alaska Native; American Indian; Behavioral Health; Evidence-Based Intervention; Historical Trauma; Mental Health Services; Native American; Policy
Palerasnitsynus ohlhoffi gen. et sp. n. is described fromBurmese amber of late Albian (Lower Cretaceous) age. This is the first record of the family Psychomyiidae from Burmese amber, and the earliest fossil record of the family. The genus Palerasnitsynus gen. n. differs from all other known psychomyiid genera by the absence of fork III in the forewings.
Fossil Trichoptera; fossil insects; aquatic insects; fossil taxonomy; palaeoenvironment
According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1–mediated survival was as efficient as that induced by the cytokine IFN-β and provided an additive, increased delay in apoptosis when given in combination with IFN-β. VCAM-1 delivered its antiapoptotic effect through binding the integrin α9β1. The α9β1 signaling pathway shares significant features with the IFN-β survival signaling pathway, requiring PI3 kinase, NF-κB activation, as well as de novo protein synthesis, but the kinetics of NF-κB activation by VCAM-1 were slower and more sustained compared with IFN-β. This study demonstrates a novel functional role for α9β1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues.
The six-layered mammalian neocortex evolved from the three-layered paleocortex, which is retained in present-day reptiles such as the turtle. Thus the turtle offers an opportunity to examine which cellular and circuit properties are fundamental to cortical function. We characterized the dendritic properties of pyramidal neurons in different cortical regions of mature turtles, Pseudemys scripta elegans, using whole cell recordings and calcium imaging from the axon, soma, and dendrites in a slice preparation. The firing properties, in response to intrasomatic depolarization, resembled those previously recorded with sharp electrodes in this preparation. Somatic spikes led to active backpropagating high-amplitude dendritic action potentials and intracellular calcium ion concentration ([Ca2+]i) changes at all dendritic locations, suggesting that both backpropagation and dendritic voltage-gated Ca2+ channels are primitive traits. We found no indication that Ca2+ spikes could be evoked in the dendrites, but fast Na+ spikes could be initiated there following intradendritic stimulation. Several lines of evidence indicate that fast, smaller-amplitude somatic spikes (“prepotentials”) that are easily recorded in this preparation are generated in the axon. Most synaptically activated [Ca2+]i changes resulted from Ca2+ entry through voltage-gated channels. In some cells synaptic stimulation evoked a delayed Ca2+ wave due to release from internal stores following activation of metabotropic glutamate receptors. With some small differences these properties resemble those of pyramidal neurons in mammalian species. We conclude that spike backpropagation, dendritic Ca2+ channels, and synaptically activated Ca2+ release are primitive and conserved features of cortical pyramidal cells, and therefore likely fundamental to cortical function.
We study the dynamics of the transition between the low- and high-firing states of the cortical slow oscillation by using intracellular recordings of the membrane potential from cortical neurons of rats. We investigate the evidence for a bistability in assemblies of cortical neurons playing a major role in the maintenance of this oscillation. We show that the trajectory of a typical transition takes an approximately exponential form, equivalent to the response of a resistor–capacitor circuit to a step-change in input. The time constant for the transition is negatively correlated with the membrane potential of the low-firing state, and values are broadly equivalent to neural time constants measured elsewhere. Overall, the results do not strongly support the hypothesis of a bistability in cortical neurons; rather, they suggest the cortical manifestation of the oscillation is a result of a step-change in input to the cortical neurons. Since there is evidence from previous work that a phase transition exists, we speculate that the step-change may be a result of a bistability within other brain areas, such as the thalamus, or a bistability among only a small subset of cortical neurons, or as a result of more complicated brain dynamics.
Cortex; Neurons; Slow wave sleep; Phase transition
In cortical pyramidal neurons, the axon initial segment (AIS) is pivotal in synaptic integration. It has been asserted that this is because there is a high density of Na+ channels in the AIS. However, we found that action potential–associated Na+ flux, as measured by high-speed fluorescence Na+ imaging, was about threefold larger in the rat AIS than in the soma. Spike-evoked Na+ flux in the AIS and the first node of Ranvier was similar and was eightfold lower in basal dendrites. At near-threshold voltages, persistent Na+ conductance was almost entirely axonal. On a time scale of seconds, passive diffusion, and not pumping, was responsible for maintaining transmembrane Na+ gradients in thin axons during high-frequency action potential firing. In computer simulations, these data were consistent with the known features of action potential generation in these neurons.
Natural health products (NHP) use may have implications with respect to adverse effects, drug interactions and adherence yet the prevalence of NHP use by patients with acute cardiovascular disease and the best method to ascertain this information is unknown.
To identify the best method to ascertain information on NHP, and the prevalence of use in a population with acute cardiovascular disease.
Structured interviews were conducted with a convenience sample of consecutive patients admitted with acute cardiovascular disease to the University of Alberta Hospital during January 2009. NHP use was explored using structured and open-ended questions based on Health Canada's definition of NHP. The medical record was reviewed, and documentation of NHP use by physicians, nurses, and pharmacists, compared against the gold-standard structured interview.
88 patients were interviewed (mean age 62 years, standard deviation [SD 14]; 80% male; 41% admitted for acute coronary syndromes). Common co-morbidities included hypertension (59%), diabetes (26%) and renal impairment (19%). NHP use was common (78% of patients) and 75% of NHP users reported daily use. The category of NHP most commonly used was vitamins and minerals (73%) followed by herbal products (20%), traditional medicines including Chinese medicines (9%), homeopathic preparations (1%) and other products including amino acids, essential fatty acids and probiotics (35%). In a multivariable model, only older age was associated with increased NHP use (OR 1.5 per age decile [95%CI 1.03 to 2.2]). When compared to the interview, the highest rate of NHP documentation was the pharmacist history (41%). NHP were documented in 22% of patients by the physician and 19% by the nurse.
NHP use is common in patients admitted with acute cardiovascular disease. However, health professionals do not commonly identify NHP as part of the medication profile despite its potential importance. Structured interview appears to be the best method to accurately identify patient use of NHP.
High concentrations of enflurane will induce a characteristic electroencephalogram pattern consisting of periods of suppression alternating with large short paroxysmal epileptiform discharges (PEDs). In this study, we compared a theoretical computer model of this activity with real local field potential data obtained from anesthetized rats.
After implantation of a high-density 8x8 electrode array in the visual cortex, the patterns of local field potential and multiunit spike activity were recorded in rats during 0.5, 1.0, 1.5 and 2.0 minimum alveolar anesthetic concentration (MAC) enflurane anesthesia. These recordings were compared with computer simulations from a mean field model of neocortical dynamics. The neuronal effect of increasing enflurane concentration was simulated by prolonging the decay time constant of the inhibitory postsynaptic potential (IPSP). The amplitude of the excitatory postsynaptic potential (EPSP) was modulated, inverse to the neocortical firing rate.
In the anesthetized rats, increasing enflurane concentrations consistently caused the appearance of suppression pattern (>1.5MAC) in the local field potential recordings. The mean rate of multiunit spike activity decreased from 2.54/s (0.5MAC) to 0.19/s (2.0MAC). At high MAC the majority of the multiunit action potential events became synchronous with the PED. In the theoretical model, prolongation of the IPSP decay time and activity-dependent EPSP modulation resulted in output that was similar in morphology to that obtained from the experimental data. The propensity for rhythmic seizure-like activity in the model could be determined by analysis of the eigenvalues of the equations.
It is possible to use a mean field theory of neocortical dynamics to replicate the PED pattern observed in local field potentials in rats under enflurane anesthesia. This pattern requires a combination of a moderately increased total area under the IPSP, prolonged IPSP decay time, and also activity-dependent modulation of EPSP amplitude.
JAK2 kinase inhibitors were developed for the treatment of myeloproliferative
neoplasms (MPNs), following the discovery of activating JAK2 mutations in the
majority of patients with MPN. However, to date JAK2 inhibitor treatment has shown
limited efficacy and apparent toxicities in clinical trials. We report here that an
HSP90 inhibitor, PU-H71, demonstrated efficacy in cell line and mouse models of the
MPN polycythemia vera (PV) and essential thrombocytosis (ET) by disrupting JAK2
protein stability. JAK2 physically associated with both HSP90 and PU-H71 and was
degraded by PU-H71 treatment in vitro and in vivo, demonstrating that JAK2 is an
HSP90 chaperone client. PU-H71 treatment caused potent, dose-dependent inhibition of
cell growth and signaling in JAK2 mutant cell lines and in primary MPN patient
samples. PU-H71 treatment of mice resulted in JAK2 degradation, inhibition of
JAK-STAT signaling, normalization of peripheral blood counts, and improved survival
in MPN models at doses that did not degrade JAK2 in normal tissues or cause
substantial toxicity. Importantly, PU-H71 treatment also reduced the mutant allele
burden in mice. These data establish what we believe to be a novel therapeutic
rationale for HSP90 inhibition in the treatment of JAK2-dependent MPN.
Fruit and vegetable consumption has been hypothesized to reduce the risk of renal cell cancer. We conducted a pooled analysis of 13 prospective studies, including 1,478 incident cases of renal cell cancer (709 women and 769 men) among 530,469 women and 244,483 men followed for up to 7 to 20 years. Participants completed a validated food-frequency questionnaire at baseline. Using the primary data from each study, the study-specific relative risks (RRs) were calculated using the Cox proportional hazards model and then pooled using a random effects model. We found that fruit and vegetable consumption was associated with a reduced risk of renal cell cancer. Compared with <200 g/d of fruit and vegetable intake, the pooled multivariate RR for ≥600 g/d was 0.68 (95% CI = 0.54–0.87; P value, test for between-studies heterogeneity = 0.86; P value, test for trend = 0.001). Compared with <100 g/d, the pooled multivariate RRs (95% CIs) for ≥400 g/d were 0.79 (0.63–0.99; P value, test for trend = 0.03) for total fruit, and 0.72 (0.48–1.08; P value, test for trend = 0.07) for total vegetables. For specific carotenoids, the pooled multivariate RRs (95% CIs) comparing the highest and lowest quintiles were 0.87 (0.73–1.03) for α-carotene, 0.82 (0.69–0.98) for β-carotene, 0.86 (0.73–1.01) for β-cryptoxanthin, 0.82 (0.64–1.06) for lutein/zeaxanthin, and 1.13 (0.95–1.34) for lycopene. In conclusion, increasing fruit and vegetable consumption is associated with decreasing risk of renal cell cancer; carotenoids present in fruit and vegetables may partly contribute to this protection.
Dimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones.
Exposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC50s in the µM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC50. Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS). Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(P)H dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ.
In yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(P)H:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells.
Botulinum neurotoxin elicits its paralytic activity through a zinc-dependant metalloprotease that cleaves proteins involved in neurotransmitter release. Currently, no drugs are available to reverse the effects of botulinum intoxication. Herein we report the design of a novel series of mercaptoacetamide small-molecule inhibitors active against botulinum neurotoxin serotype A. These analogs show low micromolar inhibitory activity against the isolated enzyme. Structure-activity relationship studies for a series of mercaptoacetamide analogs of 5-amino-3-phenylpyrazole reveal components essential for potent inhibitory activity.
Botulism; Botulinum; BoNT/A; Metalloprotease Inhibitors; Mercaptoacetamides
Cell-based screening can facilitate rapid identification of compounds inducing complex cellular phenotypes. Advancing a compound toward the clinic, however, generally requires identification of precise mechanisms of action. We previously found that epidermal growth factor receptor (EGFR) inhibitors induce acute myeloid leukemia (AML) differentiation via a non-EGFR mechanism. In this report, we integrated proteomic and RNAi-based strategies to identify their off-target anti-AML mechanism. These orthogonal approaches identified Syk as a target in AML. Genetic and pharmacological inactivation of Syk with a drug in clinical trial for other indications promoted differentiation of AML cells and attenuated leukemia growth in vivo. These results demonstrate the power of integrating diverse chemical, proteomic, and genomic screening approaches to identify therapeutic strategies for cancer.