Gatekeeper T790M mutation in EGFR is the most prevalent factor underlying acquired resistance. Acrylamide-bearing quinazoline derivatives are powerful irreversible inhibitors for overcoming resistance. Nevertheless, concerns about the risk of non-specific covalent modification have motivated the development of novel cysteine-targeting inhibitors. In this paper, we demonstrate that fluoro-substituted olefins can be tuned to alter Michael addition reactivity. Incorporation of these olefins into the quinazoline templates produced potent EGFR inhibitors with improved safety and pharmacokinetic properties. A lead compound 5a was validated against EGFRWT, EGFR T790M as well as A431 and H1975 cancer cell lines. Additionally, compound 5a displayed a weaker inhibition against the EGFR-independent cancer cell line SW620 when compared withafatinib. Oral administration of 5a at a dose of 30mg/kg induced tumor regression in a murine-EGFRL858R/T790M driven H1975 xenograft model. Also, 5a exhibited improved oral bioavailability and safety, as well as favorable tissue distribution properties and enhanced brain uptake. These findings provide the basis of a promising strategy toward the treatment of NSCLC patients with drug resistance.
We conducted a meta-analysis of positron emission tomography (PET) findings in Alzheimer's disease (AD) and mild cognitive impairment (MCI) to clarify the changes underpinning these conditions. All studies that utilised the PET tracers Pittsburgh Compound-B (PIB) or 2-[18F]fluoro-2-deoxy-D-glucose (FDG) to investigate patients with MCI or AD, were considered for the meta-analysis. Meta-analyses of PIB-PET and FDG-PET changes between patients and controls were undertaken with the effect-size signed differential mapping (ES-SDM) voxel-based meta-analytic method. A total of 24 studies were included involving 728 AD patients, 211 MCI patients and 658 healthy controls. Individuals with AD showed a significant PIB retention in bilateral precuneus and temporal, supramarginal, cingulate and fusiform gyri, as well as right insula and putamen. In addition, AD patients showed significant glucose hypometabolism in bilateral precuneus and temporal, supramarginal, cingulate, fusiform, angular, inferior parietal and middle frontal gyri, as well as left precentral and parahippocampal gyri and right superior frontal gyrus and thalamus. An exploratory meta-analysis of the few studies on MCI showed mildly decreased glucose metabolism with a similar regional distribution than in patients with AD. We suggest that our results can be used for further region-of-interest studies of AD and MCI patients.
Long intergenic non-protein-coding RNA 152 (LINC00152) is one of the long noncoding RNAs (lncRNAs) abnormally expressed in gastric cancer tissues. However, its value in the diagnosis of gastric cancer is unclear. The aim of this study is to evaluate the clinical significance of plasma LINC00152 as a biomarker in the screening of gastric cancer and to explore the possible mechanism underling its stable existence in blood. We analyzed the levels of plasma LINC00152 in patients with gastric cancer and gastric epithelial dysplasia and healthy controls using quantitative reverse transcription polymerase chain reaction and then confirmed by sequencing. We also compared its levels in paired preoperative and postoperative plasma samples. In addition, we compared the levels of LINC00152 in plasma and in exosomes, which were extracted from the same plasma and confirmed by transmission electron microscopy. The levels of plasma LINC00152 were significantly elevated in gastric cancer patients compared with healthy controls. The sensitivity and specificity of plasma LINC00152 in the diagnosis of gastric cancer were 48.1 and 85.2 %, respectively. There were no significant differences of LINC00152 levels between gastric epithelial dysplasia patients and healthy controls. LINC00152 levels in preoperative plasma samples were lower than those in postoperative ones. There were also no differences between LINC00152 levels in plasma and in exosomes. All these results suggested that LINC00152 can be detected in plasma, and one of the possible mechanisms of its stable existence in blood was protected by exosomes. It has the possibility to be applied in gastric cancer diagnosis as a novel blood-based biomarker.
Serotonin 5-hydroxytryptamine 4(5-HT4) receptor agonists have been widely prescribed as a prokinetics drug for patients with gastro-esophageal reflux disease and functional dyspepsia. QX100626, one of the 5-HT4 receptor agonists, has been studied as a promising agent for this clinical use. The objective of the present study was to identify possible target organs of toxicity and propose a non-toxic dose of QX100626 for clinical usage. After single lethal dose oral and intravenous testing in rodents, some signs indicative of adverse CNS effects were observed. The minimum toxic dose of QX100626 for a single oral administration for dogs was 90.0mg/kgb.w., and the severe toxic dose was more than 300mg/kgb.w. The No Observed Adverse Effect Level (NOAEL) of QX100626 by daily oral administration for rats and dogs was 20mg/kg and 10mg/kg, respectively, whereas the minimum toxic dosages were 67 and 30mg/kg, respectively. All of the adverse effects suggested that kidney, digestive tract, as well as nervous, hematological, and respiratory systems might be the target organs of toxicity for humans induced by QX100626. The compound could be a safe alternative to other existing prokinetic agents for the treatment of functional bowel disorders.
Allogeneic peripheral blood stem cell transplantation from unrelated donors (UR-PBSCT) is an alternative treatment for many hematologic diseases due to lack of human leukocyte antigen (HLA)-identical sibling donors. This study aimed to evaluate the impact of the degree of the HLA match on the clinical efficacy of UR-PBSCT.
Heritable variations in natural populations, including genetic variation and epigenetic variation, are the motivation for adaptation and evolution of plant species. Germplasm can be transferred stably from generation to generation by mitosis and/or meiosis without alteration in DNA sequence and protein expression. Here, two important sources of epigenetic variation, distant hybridization and polyploidy, and various biotic and abiotic stresses, are introduced. The application of epigenetic variation in crop improvement and its limitation, as well as optional solutions, are also summarized.
Slo2 channels are prominent K(+) channels in mammalian neurons but their physiological functions are not well understood. Here we investigate physiological functions and regulation of the Caenorhabditis elegans homologue SLO-2 in motor neurons through electrophysiological analyses of wild-type and mutant worms. We find that SLO-2 is the primary K(+) channel conducting delayed outward current in cholinergic motor neurons, and one of two K(+) channels with this function in GABAergic motor neurons. Loss-of-function mutation of slo-2 increases the duration and charge transfer rate of spontaneous postsynaptic current bursts at the neuromuscular junction, which are physiological signals used by motor neurons to control muscle cells, without altering postsynaptic receptor sensitivity. SLO-2 activity in motor neurons depends on Ca(2+) entry through EGL-19, an L-type voltage-gated Ca(2+) channel (CaV1), but not on other proteins implicated in either Ca(2+) entry or intracellular Ca(2+) release. Thus, SLO-2 is functionally coupled with CaV1 and regulates neurotransmitter release.
Although inter-laboratory validation efforts of the in-vivo micronucleus (MN) assay based on flow cytometry (FCM) have taken place in the EU and US, none have been organized in China. Therefore, an inter-laboratory study that included eight laboratories in China and one experienced reference laboratory in the US was coordinated to validate the in-vivo FCM MicroFlow(®) method to determine the frequency of micro-nucleated reticulocytes (MN-RETs) in rat blood. Assay reliability and reproducibility were evaluated with four known genotoxicants, and the results obtained with the FCM method were compared with the outcome of the traditional evaluation of bone-marrow micronuclei by use of microscopy. Each of the four chemicals was tested at three sites (two in China and the one US reference laboratory). After three consecutive daily exposures to a genotoxicant, blood and bone-marrow samples were obtained from rats 24h after the third dose. MN-RET frequencies were measured in 20,000 RET in blood by FCM, and micro-nucleated polychromatic erythrocyte (MN-PCE) frequencies were measured in 2,000 PCEs in bone marrow by microscopy. For both methods, each genotoxicant was shown to induce a statistically significant increase in the frequency of MN after treatment with at least one dose. Where more doses than one caused an increase, responses occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM-based MN-RET vs microscopy-based MN-PCE measurements (eight experiments, 200 paired measurements) was 0.723, indicating a high degree of correspondence between methods and compartments. The rs value for replicate FCM MN-RET measurements performed at the eight collaborative laboratories was 0.940 (n=200), and between the eight FCM laboratories with the reference laboratory was 0.933 (n=200), suggesting that the automated method is very well transferable between laboratories. The FCM micronucleus analysis method is currently used in many countries worldwide, and these data support its use for evaluating the in-vivo genotoxic potential of test chemicals in China.
To compare visual outcomes and spectral-domain optical coherence tomography results following treatment with intravitreal ranibizumab (IVR) or IVR combined with intravitreal triamcinolone acetonide (IVTA) for macular edema (ME) secondary to central retinal vein occlusion (CRVO).
Host-plant resistance is the most practical and economical approach to control the rice planthoppers. However, up to date, few rice germplasm accessions that are resistant to the all three kinds of planthoppers (1) brown planthopper (BPH; Nilaparvata lugens Stål), (2) the small brown planthopper (SBPH; Laodelphax striatellus Fallen), and (3) the whitebacked planthopper (WBPH, Sogatella furcifera Horvath) have been identified; consequently, the genetic basis for host-plant broad spectrum resistance to rice planthoppers in a single variety has been seldom studied. Here, one wild species, Oryza officinalis (Acc. HY018, 2n = 24, CC), was detected showing resistance to the all three kinds of planthoppers. Because resistance to WBPH and BPH in O. officinalis has previously been reported, the study mainly focused on its SBPH resistance. The SBPH resistance gene(s) was (were) introduced into cultivated rice via asymmetric somatic hybridization. Three QTLs for SBPH resistance detected by the SSST method were mapped and confirmed on chromosomes 3, 7, and 12, respectively. The allelic/non-allelic relationship and relative map positions of the three kinds of planthopper resistance genes in O. officinalis show that the SBPH, WBPH, and BPH resistance genes in O. officinalis were governed by multiple genes, but not by any major gene. The data on the genetics of host-plant broad spectrum resistance to planthoppers in a single accession suggested that the most ideally practical and economical approach for rice breeders is to screen the sources of broad spectrum resistance to planthoppers, but not to employ broad spectrum resistance gene for the management of planthoppers. Pyramiding these genes in a variety can be an effective way for the management of planthoppers.
GRD081, a newly developed dual PI3K/mTOR inhibitor, is now being considered for evaluation in phase I clinical trial. In this work, the subchronic toxicity of GRD081 in Sprague-Dawley (SD) rats and beagle dogs has been characterized. Rats and dogs received GRD081 orally (2, 5, 10 and 1, 2, 4mg/kg/day, respectively) on a consecutive daily dosing schedule for 28days following a 14days of recovery period. The treatment resulted in unscheduled mortality in rats receiving 5mg/kg/day and 10mg/kg/day. The adverse effects of GRD081 on rats and dogs mainly included myelosuppression, immunosuppression, hematological toxicity, and moderate liver, pancreas and kidney toxicity. These observations are consistent with pharmacologic perturbations of physiologic processes associated with the intended molecular targets for this class of PI3K/mTOR signaling inhibitors. Most of the treatment-induced effects were reversible upon discontinuation of treatment. The no-observed-adverse-effect level (NOAEL) of GRD081 was 1mg/kg/day for beagle dogs and less than 2mg/kg/day for SD rats.
This study investigated the relationship between N5,N10-methylene tetrahydrofolic acid reductase (MTHFR) polymorphisms, smoking, and vascular dementia (VD). Polymerase chain reaction-restriction fragment length polymorphism analysis was used to analyze the frequency of the C/T polymorphism at position 677 of the MTHFR gene in 304 VD patients and 300 control patients with nondementia cerebral infarction. The CC, CT, and TT genotype frequencies of the MTHFR gene were 43.42%, 32.57%, and 24.01%, respectively, in the VD group, and 50.67%, 32.00%, and 17.33%, respectively, in the control group. The T allele frequency was significantly higher in the VD group than in the control group (P < .05). Among patients who smoked, the relative risk of VD in patients with the TT genotype and T allele was higher than in the control group (P < .05). Therefore, the smoking group with the T allele has the highest risk of VD, and synergy appears to exist between the MTHFR gene polymorphisms and smoking in susceptibility to VD.
While stem cells can sense and respond to physical properties of their environment, the molecular aspects how physical information is translated into biochemical signals remain unknown. Here we show that human mesenchymal stem cells (hMSCs) harvest and assemble plasma fibronectin into their extracellular matrix (ECM) fibrils within 24 hours. hMSCs pro-actively pull on newly assembled fibronectin ECM fibrils, and the fibers are more stretched on rigid than on soft fibronectin-coated polyacrylamide gels. Culturing hMSCs on single stretched fibronectin fibers upregulates hMSC osteogenesis. Osteogenesis was increased when ?v?3 integrins were blocked on relaxed fibronectin fibers, and decreased when ?5?1 integrins were blocked or when epidermal growth factor (EGF) receptor signaling was inhibited on stretched fibronectin fibers. This suggests that hMSCs utilize their own contractile forces to translate environmental cues into differential biochemical signals by stretching fibronectin fibrils. Mechanoregulation of fibronectin fibrils may thus serve as check point to regulate hMSC osteogenesis.
Nematode neurons generally produce graded potentials instead of action potentials. It is unclear how the graded potentials control postsynaptic cells under physiological conditions. Here we show that postsynaptic currents frequently occur in bursts at the neuromuscular junction of Caenorhabditis elegans. Cholinergic bursts concur with facilitated action potential firing, elevated cytosolic [Ca(2+)] and contraction of the muscle whereas GABAergic bursts suppress action potential firing. The bursts, distinct from artificially evoked responses, are characterized by a persistent current (the primary component of burst-associated charge transfer) and increased frequency and mean amplitude of postsynaptic current events. The persistent current of cholinergic postsynaptic current bursts is mostly mediated by levamisole-sensitive acetylcholine receptors, which correlates well with locomotory phenotypes of receptor mutants. Eliminating command interneurons abolishes the bursts whereas mutating SLO-1 K(+) channel, a potent presynaptic inhibitor of exocytosis, greatly increases the mean burst duration. These observations suggest that motoneurons control muscle by producing postsynaptic current bursts.
Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.
Lead is a heavy metal and important environmental toxicant and nerve poison that can destruction many functions of the nervous system. Lead poisoning is a medical condition caused by increased levels of lead in the body. Lead interferes with a variety of body processes and is toxic to many organs and issues, including the central nervous system. It interferes with the development of the nervous system, and is therefore particularly toxic to children, causing potentially permanent neural and cognitive impairments. In this study, we investigated the relationship between lead poisoning and the intellectual and neurobehavioral capabilities of children.
A lesion-mimic mutant in rice (Oryza sativa L.), spotted leaf 5 (spl5), displays a disease-resistance-enhanced phenotype, indicating that SPL5 negatively regulates cell death and resistance responses. To understand the molecular mechanisms of SPL5 mutation-induced cell death and resistance responses, a proteomics-based approach was used to identify differentially accumulated proteins between the spl5 mutant and wild type (WT).
The aim of this study was to investigate the role of connexin 43 (Cx43) and its hemichannel (HC1) in the death of astrocytes following ischemia/reperfusion (IR) or oxygen-glucose deprivation/reoxygenation (OGDR) insult. Wistar rats had their bilateral common carotid artery clamped for 1.5 h followed by 0, 4, and 24 h of reperfusion (n = 8 for each time point), respectively. All rats were sacrificed and Cx43, HC1, and caspase 3 (Casp3) in cerebral ischemic tissues were examined by immunohistochemistry and western blotting. Astrocytes cell line, astrocytes transduced with a retroviral empty vector (Psup astrocyte), or a Cx43-specific shRNA construct (shRNA astrocytes) were treated with OGDR insult for various periods. The viability of astrocytes was assessed by MTT assay. The expression of Cx43, HC1, and Casp3 was detected with western blotting. The results showed that the expression of Cx43, HC1, and Casp3 in rats brain, astrocytes, and Psup astrocytes was significantly increased after 4 h of IR/OGDR and recovered on 24 h of the insult. Cell viability decreased after 4 h of the insult whereas the cell viability increased on 24 h after the insult. In contrast, the expression of Cx43, HC1, Casp3, and cell viability had no statistical differences in the null Cx43 gene-shRNA transfected astrocytes after the treatment of OGDR. The results suggest that Cx43 and HC1 are likely to play the pivotal roles in the mediation of the astrocytic death.
C. elegans body-wall muscle cells are electrically coupled through gap junctions. Previous studies suggest that UNC-9 is an important, but not the only, innexin mediating the electrical coupling. Here we analyzed junctional current (I j ) for mutants of additional innexins to identify the remaining innexin(s) important to the coupling. The results suggest that a total of six innexins contribute to the coupling, including UNC-9, INX-1, INX-10, INX-11, INX-16, and INX-18. The I j deficiency in each mutant was rescued completely by expressing the corresponding wild-type innexin specifically in muscle, suggesting that the innexins function cell-autonomously. Comparisons of I j between various single, double, and triple mutants suggest that the six innexins probably form two distinct populations of gap junctions with one population consisting of UNC-9 and INX-18 and the other consisting of the remaining four innexins. Consistent with their roles in muscle electrical coupling, five of the six innexins showed punctate localization at muscle intercellular junctions when expressed as GFP- or epitope-tagged proteins, and muscle expression was detected for four of them when assessed by expressing GFP under the control of innexin promoters. The results may serve as a solid foundation for further explorations of structural and functional properties of gap junctions in C. elegans body-wall muscle.
Rapid atrial pacing (RAP) can induce electrical and autonomic remodeling and facilitate atrial fibrillation (AF). Recent reports showed that low-level vagosympathetic nerve stimulation (LLVNS) can suppress AF, as an antiarrhythmic effect. We hypothesized that LLVNS can reverse substrate heterogeneity induced by RAP.
BACKGROUND: We continue our exploration of the large polysaccharide polymer Chitosan as an acute therapy for severe damage to the nervous system. We tested the action of subcutaneously injected nanoparticles (~ 100 -- 200 nanometers in diameter; 1 mg per ml) against control injections (silica particle of the same size and concentration) in a standardized in vivo spinal cord injury model. These functional tests used standardized physiological measurements of evoked potentials arriving at the sensorimotor cortex subsequent to stimulation of the tibial nerve of the contralateral hindlimb. We further explored the degree of acetylation and molecular weight of chitosan on the success of sealing cell damage using specific probes of membrane integrity. RESULTS: Not one of the control group showed restored conduction of evoked potentials stimulated from the tibial nerve of the hindleg -- through the lesion -- and recorded at the sensorimotor cortex of the brain. Investigation if the degree of acetylation and molecular weight impacted "membrane sealing" properties of Chitosan were unsuccessful. Dye - exchange membrane probes failed to show a difference between the comparators in the function of Chitosan in ex vivo injured spinal cord tests. CONCLUSIONS: We found that Chitosan nanoparticles effectively restore nerve impulse transmission through the crushed adult guinea pig spinal cord in vivo after severe crush/compression injury. The tests of the molecular weight (MW) and degree of acetylation did not produce any improvement in Chitosans membrane sealing properties.
Recent studies suggested quantitative analysis of diffusion-weighted magnetic resonance imaging as a promising tool for early prognostication of cardiac arrest patients. However, most of their methods involve significant manual image handling often subjective and difficult to reproduce. Therefore developing a computerized analysis method using easy-to-define characteristics would be useful.
Dystrobrevin is a major component of a dystrophin-associated protein complex. It is widely expressed in mammalian tissues, including the nervous system, in which it is localized to the presynaptic nerve terminal with unknown function. In a genetic screen for suppressors of a lethargic phenotype caused by a gain-of-function isoform of SLO-1 in Caenorhabditis elegans, we isolated multiple loss-of-function (lf) mutants of the dystrobrevin gene dyb-1.dyb-1(lf) phenocopied slo-1(lf), causing increased neurotransmitter release at the neuromuscular junction, increased frequency of Ca(2+) transients in body-wall muscle, and abnormal locomotion behavior. Neuron- and muscle-specific rescue experiments suggest that DYB-1 is required for SLO-1 function in both neurons and muscle cells. DYB-1 colocalized with SLO-1 at presynaptic sites in neurons and dense body regions in muscle cells, and dyb-1(lf) caused SLO-1 mislocalization in both types of cells without altering SLO-1 protein level. The neuronal phenotypes of dyb-1(lf) were partially rescued by mouse ?-dystrobrevin-1. These observations revealed novel functions of the BK channel in regulating muscle Ca(2+) transients and of dystrobrevin in controlling neurotransmitter release and muscle Ca(2+) transients by localizing the BK channel.
The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.
The recombinant soluble human TRAIL mutant (DATR), derived from tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), is a promising agent for cancer therapy. The present study evaluated the toxicity of DATR in rats and monkeys. Based on the results, the safety and toxic doses of DATR intravenously injected to rats for 50 days were 60 and 180 mg/kg, respectively, and when delivered intravenously guttae to monkeys for 50 days, these levels were 10 and 30 mg/kg, respectively. The main toxic effects in rats were red blood cell count and haemoglobin decreases; blood urea nitrogen and creatinine increases. The main toxic effects in monkeys included red blood cell count and haemoglobin decreases; alanine aminotransferase and aspartate aminotransferase increases; high proliferation of karyocytes of the erythrocyte series; and regional hydropic degeneration of hepatic parenchymal cells. The TUNEL assay showed both 90 mg/kg DATR- and TRAIL-induced apoptosis of the liver in monkeys, which confirmed the hepatotoxicity of DATR. These findings indicated that the target toxic organs of DATR might be the haematological system. Furthermore, kidney in rats and liver in monkeys are also likely target toxic organs. The toxicity was reversible and did not differ from that associated with TRAIL administered at the same dosage.
Calcium (Ca) plays a crucial role as a second messenger in intracellular signaling elicited by developmental and environmental cues. Calcineurin B-like proteins (CBLs) and their target proteins, CBL-interacting protein kinases (CIPKs) have emerged as a key Ca(2+)-mediated signaling network in response to stresses in plants. Bioinformatic analysis was used to identify 43 putative ZmCIPK (Zea mays CIPK) genes in the genome of maize inbred line B73. Based on gene structures, these ZmCIPKs were divided into intron-rich and intron-poor groups. Phylogenetic analysis indicated that the ZmCIPK family had a high evolutionary relationship with the rice CIPK family of 30 members. Microarray data and RT-PCR assay showed that ZmCIPK genes transcriptionally responded to abiotic stresses, and that 24, 31, 20 and 19 ZmCIPK genes were up-regulated by salt, drought, heat and cold stresses, respectively. There were different expression patterns of ZmCIPKs between cold-tolerant inbred line B73 and cold-sensitive inbred line Mo17 under cold stress. Our findings will aid further molecular dissection of biological functions of the CIPKs in maize, and provide new insight into the CBL-CIPK signaling network in plants.
Aortic pseudoaneurysms are rare but life-threatening complications of aortic procedures. Operation on the femorofemoral bypass with hypothermic circulatory arrest has been the method of choice. Iatrogenic ascending aorta pseudoaneurysm combined with infective endocarditis of the aortic valve has never been reported.
The objective was to investigate corneal re-epithelialisation of surface-modified polymethacrylate hydrogel implants in order to evaluate them as potential materials for an artificial cornea. Polymethacrylate hydrogels were modified with amines and then coated with different extracellular matrix proteins (collagen I, IV, laminin and fibronectin). The modified hydrogels were surgically implanted into bovine corneas maintained in a 3-D culture system for 5 days. The epithelial growth across the implant surface was evaluated using fluorescent, light and electron microscopy. Full epithelialisation was achieved on 1,4-diaminobutane-modified hydrogels after coating with collagen IV. Hydrogels modified with 1,4-diaminobutane but without further coating only showed partial re-epithelialisation. Hydrogels modified with other amines (1,2-diaminoethane or 1,3-diaminopropane) showed only partial re-epithelialisation; further coating with extracellular matrix proteins improved epithelialisation of these surfaces but did not result in complete re-epithelialisation. Evaluation of the corneas implanted with the 1,4-diaminobutane-modified hydrogels coated with collagen IV showed that the artificial corneas remain clear, integrate well and become covered by a healthy stratified epithelium. In conclusion the 1,4-diaminobutane surface-modified hydrogel coated with collagen IV supported the growth of a stable stratified epithelium. With further refinement this hydrogel has the potential to be used clinically for an artificial cornea.
The BK channel is a Ca²+- and voltage-gated potassium channel with many important physiological functions. To identify proteins important to its function in vivo, we screened for Caenorhabditis elegans mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of the BK channel ?-subunit SLO-1. BKIP-1 (for BK channel interacting protein), a small peptide with no significant homology to any previously characterized molecules, was thus identified. BKIP-1 and SLO-1 showed similar expression and subcellular localization patterns and appeared to interact physically through discrete domains. bkip-1 loss-of-function (lf) mutants phenocopied slo-1(lf) mutants in behavior and synaptic transmission and suppressed the lethargy, egg-laying defect, and deficient neurotransmitter release caused by SLO-1(gf). In heterologous expression systems, BKIP-1 decreased the activation rate and shifted the conductance-voltage relationship of SLO-1 in a Ca²+-dependent manner and increased SLO-1 surface expression. Thus, BKIP-1 is a novel auxiliary subunit critical to SLO-1 function in vivo.
To identify the role of the Notch signaling pathway in corneal wound healing, rat corneas receiving either epithelial or stromal wounds were placed in organ culture for up to 3 and 14 days, respectively. Localization of Notch receptors--Notch1, Notch2, and their ligands--Delta1, Jagged1 was determined by immunofluorescence. Wounds were treated with a ?-secretase inhibitor to suppress Notch signaling or recombinant Jagged1 to enhance Notch signaling and morphological changes in the epithelium and stroma were recorded. The expressions of markers of cell proliferation (Ki67) and epithelial differentiation (cytokeratin 3) were assessed by immunohistology. Notch1 and Notch2 were localized to suprabasal epithelial cells in normal corneas. During corneal wound healing, both Notch receptors were detected in suprabasal and superficial epithelial layers. Delta1 and Jagged1 were observed throughout all corneal epithelial cell layers and occasional keratocytes of the stroma in normal and wounded corneas. ?-secretase inhibition of Notch resulted in increased epithelial cell layers, with recombinant Jagged1 activation of Notch leading to a reduction in epithelial cell layers during corneal wound healing. Correspondingly, the activation of Notch resulted in a decreased cytokeratin 3 expression in the corneal epithelium, with no effect on cellular expression of Ki67. Notch signaling pathway suppressed corneal epithelial differentiation during corneal wound healing, but had no effect on epithelial cell proliferation.
Although the neuromuscular system of C. elegans has been studied intensively, little is known about the properties of muscle action potentials (APs). By combining mutant analyses with in vivo electrophysiological recording techniques and Ca2+ imaging, we have established the fundamental properties and molecular determinants of body-wall muscle APs. We show that, unlike mammalian skeletal muscle APs, C. elegans muscle APs occur in spontaneous trains, do not require the function of postsynaptic receptors, and are all-or-none overshooting events, rather than graded potentials as has been previously reported. Furthermore, we show that muscle APs depend on Ca2+ entry through the L-type Ca2+ channel EGL-19 with a contribution from the T-type Ca2+ channel CCA-1. Both the Shaker K+ channel SHK-1 and the Ca2+/Cl?-gated K+ channel SLO-2 play important roles in controlling the speed of membrane repolarization, the amplitude of afterhyperpolarization (AHP) and the pattern of AP firing; SLO-2 is also important in setting the resting membrane potential. Finally, AP-elicited elevations of [Ca2+]i require both EGL-19 and the ryanodine receptor UNC-68. Thus, like mammalian skeletal muscle, C. elegans body-wall myocytes generate all-or-none APs, which evoke Ca2+ release from the sarcoplasmic reticulum (SR), although the specific ion channels used for AP upstroke and repolarization differ.
Cerebral ischemia is a major cause of adult disability and death worldwide. Evidence suggests that Bax-dependent initiation and activation of intrinsic apoptotic pathways contribute to ischemic brain injury. We investigated the Bax-inhibiting peptide VPALR, designed from the rat Ku70-Bax inhibiting domain, on the apoptotic neuronal cell death and behavioral deficits following global cerebral ischemia. The pentapeptide was infused into the left lateral ventricle of the rat brain by intracerebroventricular (i.c.v.) injection 1 h after cerebral ischemia, and results showed that it highly permeated hippocampal neurons and bound to Bax protein in vivo. Post-treatment with VPALR reduced the delayed neuronal damage by approximately 78% compared to the non-treated ischemic control and scrambled peptide-treated rats. TUNEL analysis revealed that VPALR markedly reduced the ischemia-induced increase in apoptotic neuronal death in rat hippocampal CA1 region. VPALR post-treatment also significantly attenuated Bax activation and its mitochondrial translocation as compared with scrambled peptide-treated animals. Concomitantly, Bax-inhibiting peptide-treated rats showed reduced cytochrome c release from mitochondria to cytosol and reduced caspase-3 activation in response to cerebral ischemia, indicating that activation of the intrinsic apoptotic pathway was reduced. Furthermore, Bax-inhibiting peptide improved spatial learning and memory performance in the Morris water maze, which was seriously affected by global cerebral ischemia. In conclusion, Bax inhibition by cell-permeable pentapeptides reduced apoptotic neuronal injury in the hippocampal CA1 region and behavioral deficits following global ischemia. These results suggest that Bax is a potential target for pharmacological neuroprotection and that Bax-inhibiting peptide may be a promising neuroprotective strategy for cerebral ischemia.
The impact of the grafting techniques (individual or sequential grafts) on the graft patency of off-pump coronary artery bypass grafting (OPCAB) has not been reported. The mid-term patency rates for individual and sequential saphenous vein grafts (SVGs) as coronary bypass conduits of OPCAB were compared.
The BK channel, a voltage- and Ca(2+)-gated large-conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN-1, a homologue of mammalian ?-catulin, is required for subcellular localization of SLO-1, the Caenorhabditis elegans BK channel ?-subunit, in body-wall muscle cells. CTN-1 was identified in a genetic screen for mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of SLO-1. In body-wall muscle cells, CTN-1 coclusters with SLO-1 at regions of dense bodies, which are Z-disk analogs of mammalian skeletal muscle. In ctn-1 loss-of-function (lf) mutants, SLO-1 was mislocalized in body-wall muscle but its transcription and protein level were unchanged. Targeted rescue of ctn-1(lf) in muscle was sufficient to reinstate the lethargic phenotype in slo-1(gf);ctn-1(lf). These results suggest that CTN-1 plays an important role in BK channel function by mediating channel subcellular localization.
The innexin family of gap junction proteins has 25 members in Caenorhabditis elegans. Here, we describe the first high-resolution expression map of all members through analysis of live worms transformed with green fluorescent protein under the control of entire promoter regions. Our analyses show that innexins have dynamic expression patterns throughout development and are found in virtually all cell types and tissues. Complex tissues, such as the pharynx, intestine, gonad, as well as scaffolding tissues and guidepost cells express a variety of innexins in overlapping or complementary patterns, suggesting they may form heteromeric and heterotypic channels. Innexin expression occurs in several types of cells that are not known to form gap junctions as well as in a pair of migrating cells, suggesting they may have hemichannel function. Therefore, innexins likely play roles in almost all body functions, including embryonic development, cell fate determination, oogenesis, egg laying, pharyngeal pumping, excretion, and locomotion.
A bioabsorbable nanofibrous scaffold was developed for early adhesion of mesenchymal stem cells (MSCs). Collagen nanofibers with diameters of 430 +/- 170 nm were fabricated by electrospinning. Over 45% of the MSC population adhered to this collagen nanofiber after 30 min at room temperature. Remarkably, collagen-coated P(LLA-CL) electrospun nanofibers were almost as efficient as collagen nanofibers whereas collagen cast film did not enhance early capture when it was applied on cover slips. The adhesive efficiency could be further increased to over 20% at 20 min and over 55% at 30 min when collagen nanofibers were grafted with monoclonal antibodies recognizing CD29 or CD49a. These data demonstrate that the early adhesive behavior is highly dependent on both the surface texture and the surface chemistry of the substrate. These findings have potential applications for early capture of MSCs in an ex vivo setting under time constraints such as in a surgical setting.
Off-pump coronary artery bypass graft surgery (OPCAB) has been performed for many years and its use is increasing frequently, but it remains an open question whether OPCAB provides similar patency to conventional coronary artery bypass graft (CCABG) surgery with cardiopulmonary bypass. The present study assessed the graft patency in patients that had coronary arterial bypass grafting (CABG) performed on-pump and off-pump. A total of 237 patients with CABG performed by a single surgeon were retrospectively studied, in which 100 patients underwent CCABG and 137 patients underwent OPCAB; the two groups were well matched according to relative factors and no significant differences were found in both groups. Postoperatively, systematic assessment on the graft patency of all the patients was conducted with 64-slice multidetector spiral computed tomography angiography (MSCTA) at one month, 1 year, 2 years, 3 years and 4 years, respectively, to provide 641 grafts for analysis. Patency of left internal mammary artery (LIMA) was higher than that of saphenous vein (SVG) in both groups; no significant difference was seen in LIMA patency and SVG patency in both groups. Results of 64-slice MSCTA indicate that OPCAB provides similar patency to CCABG surgery with CPB.
Safety evaluation of synthetic human brain natriuretic peptide (shBNP) was carried out in cynomolgus monkeys (Macaca fascicularis) by 2-week intravenous toxicity studies. System exposure was also assessed throughout the whole administration. Three test groups received doses of 432, 1440 and 4320 microg/kg/day of shBNP, with a high infusion rate of 36 mL/kg/hr for 30 min compared to the clinical protocol of continuous infusion over 24h. Commercially available recombinant human brain natriuretic peptide (rhBNP) of 1440 microg/kg/day was used as positive control. The 2-week repeated intravenous doses of shBNP resulted in reversible increased serum LDH and CPK in monkeys receiving 1440 and 4320 microg/kg/day dose with no pertinent histopathological changes. Some changes related to the pharmacologic effects of BNP including hypotension was observed after administration. No treatment-related mortalities or renal dysfunction were found. Controversy about the safety issue of BNP as an exogenous hormone concerning ventricular remodeling and myocardial cell apoptosis, coupled with our results, were also discussed. The no-observed-adverse-effect level (NOAEL) was considered to be 432 microg/kg /day, which is about 20 times higher than the commonly used clinical dose. The results of the present study advocate the safety of shBNP in cynomolgus monkeys at levels used in the study.
To compare the blood flow in sequential and individual saphenous vein grafts (SVGs) and to analyze the influence of the location of the target vessel in off-pump coronary artery bypass grafting (OPCAB).
The recombinant soluble human TRAIL mutant (DATR), derived from tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), is a promising agent for cancer therapy. The present study evaluated the synergetic toxicity of DATR in combination with traditional chemotherapeutics, including irinotecan, polyene paclitaxel and oxaliplatin in rats. Rats treated with polyene paclitaxel alone or in combination with DATR showed severe diarrhea, appetite inhibition, increasing sodium (Na(+)), potassium (Cl(-)) and glucose (GLU) and serious disorders in the haematological system. Increasing total bilirubin (TBIL) and blood urea nitrogen (BUN) were detected in the rats treated with oxaliplatin alone or in combination with DATR. Furthermore, except that the BUN and Crea of male rats treated with irinotecan in combination with DATR were higher than those of treated with irinotecan, the addition of DATR does not increase the toxicity induced by irinotecan, polyene paclitaxel and oxaliplatin. In conclusion, DATR probably increases kidney lesions of rat with irinotecan, but does not increase the toxicity induced by polyene paclitaxel and oxaliplatin. This indicated that DATR has promising potential in clinical combination therapies. Furthermore, the toxicity induced by DATR on the liver, kidneys and haematological system should be considered carefully if DATR is used in combination with traditional chemotherapeutics.
The pannexin-1 (Panx1) channel (often referred to as the Panx1 hemichannel) is a large-conductance channel in the plasma membrane of many mammalian cells. While opening of the channel is potentially detrimental to the cell, little is known about how it is regulated under physiological conditions. Here we show that stomatin inhibited Panx1 channel activity. In transfected HEK-293 cells, stomatin reduced Panx1-mediated whole-cell currents without altering either the total or membrane surface Panx1 protein expression. Stomatin coimmunoprecipitated with full-length Panx1 as well as a Panx1 fragment containing the fourth membrane-spanning domain and the cytosolic carboxyl terminal. The inhibitory effect of stomatin on Panx1-mediated whole-cell currents was abolished by truncating Panx1 at a site in the cytosolic carboxyl terminal. In primary culture of mouse astrocytes, inhibition of endogenous stomatin expression by small interfering RNA enhanced Panx1-mediated outward whole-cell currents. These observations suggest that stomatin may play important roles in astrocytes and other cells by interacting with Panx1 carboxyl terminal to limit channel opening.
To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1?kPa-2.3?MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5?kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.
MicroRNAs (miRNAs) exhibit tumor-specific expression signatures and play crucial roles in tumorigenesis by targeting oncogenes. Here, through analyzing the miRNA-array profiles of human glioblastoma tissues and the adjacent normal brain tissues, we found miR-483-5p was significantly down-regulated in gliomas, which was confirmed in both human glioma specimens and cell lines. The overexpression of miR-483-5p suppressed glioma cell proliferation and induced a G0/G1 arrest. In contrast, miR-483-5p inhibition promoted cell proliferation. Furthermore, by a dual-luciferase reporter assay and expression analysis, we identified extracellular signal-regulated kinase 1 (ERK1) as a direct target of miR-483-5p. ERK1 knockdown can block cell proliferation induced by miR-483-5p inhibition. Thus, our findings provide the first evidence that miR-483-5p can serve as a tumor suppressor in gliomas.
SIM010603, a promising multi-targeted receptor tyrosine kinase (RTK) inhibitor, is now being considered for evaluation in phase clinical trial. In this work, the subchronic toxicity of SIM010603 in SD rats and beagle dogs have been characterized. Rats and dogs received SIM010603 orally (0-20 and 0-10mg/kg/day, respectively) on a consecutive daily dosing schedule for 28 days following a 14 days recovery period. Sunitinib was used as a positive control. The No Observed Adverse Effect Level (NOAEL) of SIM010603 was 5mg/kg/day for rats, and undefined for dogs. The treatment resulted in unscheduled mortality in dogs receiving 10mg/kg of SIM010603 or Sunitinib. The adverse effects of SIM010603 on rats and dogs mainly included gastrointestinal toxicity, skeletal toxicity, myelosuppression, thymus atrophy, bronchopneumonia, cardiovascular dysfunction, and pancreatic toxicity. Similar observations have also been noted with this class of RTK signaling inhibitors and are consistent with pharmacologic perturbations of physiologic/angiogenic processes associated with the intended molecular targets. Most treatment-induced effects were reversible or showed ongoing recovery upon discontinuation of treatment. SIM010603 has shown comparable toxicity effect on beagle dogs, while better tolerability on SD rats when compared to Sunitinib.
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