To investigate the impact of establishing regional collaborative network on reperfusion time and prognosis of patients with ST-segment elevated myocardial infarction (STEMI) admitting to community hospitals without percutaneous coronary intervention (PCI) capacity (Non-PCI hospital).
Imperatorin (IMP) is the major active ingredient in many common medicinal herbs. We examined the irreversible inhibitory effect of IMP on cytochrome P450 2B6 (CYP2B6). IMP produced a time- and concentration-dependent inactivation of CYP2B6. About 70% of activity of CYP2B6 was suppressed after its incubation with 1.5 ?M IMP for 9 min. KI and kinact were found to be 0.498 ?M and 0.079 min(-1), respectively. The loss of CYP2B6 activity required the presence of NADPH. Glutathione (GSH) and catalase/superoxide dismutase showed little protection against the IMP-induced enzyme inactivation. Ticlopidine, a substrate of CYP2B6, showed protection of the enzyme against the inactivation induced by IMP. The estimated partition ratio of the inactivation was approximately 4. Additionally, a ?-ketoenal intermediate was identified in microsomal incubations with IMP. CYPs 1A2, 2A6, 2B6, 2D6, 2E1, 3A4, and 3A5 were found to be involved in bioactivation of IMP. In conclusion, IMP is a mechanism-based inactivator of CYP2B6. The formation of ?-ketoenal intermediate may account for the enzyme inactivation.
Abstract Context: AXIN1 is a central component of Wnt signalling pathway which is essential for embryonic development. Objective: To investigate whether polymorphisms of AXIN1 contribute to ASD susceptibility. Materials and methods: Three tag SNPs (rs12921862, rs370681 and rs1805105) in AXIN1 were genotyped in 208 ASD patients and 302 healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a Chinese population. Results: Significantly increased ASD risk was observed to be associated with the A allele of rs12921862 (p?0.0001, OR?=?3.096, 95% CI?=?2.037-4.717). Increased ASD risk was observed to be associated with rs370681 in a codominant (p?=?0.043, OR?=?1.52, 95% CI?=?1.04-2.22) and overdominant model (p?=?0.016, OR?=?1.57, 95% CI?=?1.08-2.27). Conclusion: rs12921862 and rs370681 may contribute to ASD susceptibility.
The tumor suppressor menin is recognized as a key regulator of ? cell proliferation. To induce tumorigenesis within pancreatic ? cells, floxed alleles of Men1 were selectively ablated using Cre-recombinase driven by the insulin promoter. Despite the ? cell specificity of the RipCre, glucagon-expressing tumors as well as insulinomas developed in old mutant mice. These glucagon-expressing tumor cells were menin-deficient and expressed the mature ? cell-specific transcription factors Brn4 and MafB. Moreover, inactivation of ? cell-specific transcription factors was observed in mutant ? cells. Our work shows that Men1 ablation in the pancreatic ? cells leads to the inactivation of specific transcription factors resulting in glucagon-expressing tumor development, which sheds light on the mechanisms of islet tumorigenesis.
Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. In this Review, we summarize the common approaches and applications of these still-evolving tools as they are being used in the most popular model developmental systems. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms.
Many furan-containing compounds have been reported to be toxic and/or carcinogenic. Furanoids have been found in a wide range of fruits, herbs, foods, and beverages. The risks for intake of toxic furans have been rising, due to the rapid growth of globe-wide consumption of natural products. The objective of the study was to develop an analytical platform to screen cis-enediones (cis-enedials or ?-ketoenals) resulting from metabolic activation of potentially harmful furans. 2,5-Dimethylfuran (DMF), a model furan compound, was incubated with rat liver microsomes supplemented with glutathione (GSH) and 4-bromobenzylamine (BBA) as trapping agents, to produce a GSH/BBA-derived pyrrole. The incubation mixture was monitored by acquiring neutral loss scan of 129 Da and precursor ion scans of m/z 272, 169, and 171 in polarity switch mode. Four individual chromatograms showed the respective peak with the same retention time. An additional six furan-containing compounds were tested by the same approach, and similar observation was obtained. The system also showed its extremely high sensitivity, and an estimate of the limit of detection for DMF bioactivated in rat liver microsomes was <100 fmol. We also applied inductively coupled plasma mass spectrometry (ICP-MS) to monitor the formation of the bromine-tagged pyrrole derivatives. Crude extracts obtained from traditional Chinese medicine Dioscorea bulbifera L., known to contain furanoditerpenoids, were analyzed by the approach. In conclusion, the platform has been proven selective, sensitive, effective, and reliable, and ICP MS allows us to estimate the resulting bromine-labeled pyrroles without authentic standards.
Abstract The innate and adaptive immune response could be initiated by toll like receptors (TLRs) by recognizing the conserved components of microbes. Among human TLR family, TLR9 was critical in sensing DNA viruses and endogenous DNA. Previous researches confirmed that activation of TLR9 could initiate many important cytokines such as IL-6, IL-8, IL-10 and IFN-?. The aim of this study was to analyze expression of more molecules upon TLR9 agonist stimulation, including tumor-related factors, kinase signal molecules, adhesion molecules and co-stimulators. Peripheral blood mononuclear cells (PBMCs) were isolated from health volunteer and stimulated by CpG. RNA extraction and supernatant collection were conducted four hours post CpG treatment. Reatl-time PCR and antibody chip were introduced to detect the expression of immune-related molecules in RNA and protein secretion in supernatant, respectively. The results indicated that activation of TLR9 pathway greatly influenced the expression and secretion of many interleukins, cytokine, chemokines, tumor-related genes, adhesion molecules, kinase signal molecules and co-stimulators. This is the first systematical analysis of immune-related molecules in PBMSCs upon TLR9 activation. Future study should focus on the role of the candidate molecules in TLR9-mediating biological functions.
A high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry-UV-irradiation (HPLC-QTOF-MS-UV) method was established for rapid separation and structural identification of the constituents in Re Du Ning Injections (RDNI). A total of 20 potentially bioactive compounds including 10 caffeoylquinic acids and 10 iridoid glycosides were identified or tentatively characterized in RDNI by comparing their retention times and MS spectra with those of authentic standards or literature data. In particular, UV-irradiation was employed in the identification of the cis/trans isomers of caffeoylquinic acids. Furthermore, each compound was assigned to the individual raw materials (Artemisia annua L., Lonicera japonica Thunb. or Gardenia jasminoides Ellis) present in RDNI. This is the first time that an HPLC-QTOF-MS-UV analytical method has been used for the identification of caffeoylquinic acids in RDNI.
A method was developed for the simultaneous determination of congmunoside II, congmunoside IV, congmunoside V, congmunoside X and congmuyenoside II in different parts of Aralia elata by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The chromatographic separation was performed on an Alltima C18 analytical column (250 mm x 4.6 mm, 5 microm) using the mobile phases of acetonitrile and 0.1% (v/v) formic acid aqueous solution with gradient elution. The separated compounds were detected in multiple reaction monitoring (MRM)) mode via positive electrospray ionization (ESI+). The linear ranges of congmunoside II, congmunoside IV, congmunoside V, congmunoside X and congmuyenoside II were 0.17-108 microg/L, 0.53-329 microg/L, 0.77-480 microg/L, 0.77-480 microg/L and 0.82-510 microg/L, respectively. The extraction recoveries varied from 99.0% to 100.2%. The developed method is rapid, accurate and reproducible, and is successfully utilized as a quality control method for Aralia elata. The results indicate that the order of contents of these five saponins in different parts of Aralia elata is root bark > leaves > seeds > buds.
Emerging evidence clearly indicates that EZH2 plays a crucial role in tumor angiogenesis. However, the role of EZH2 in angiogenesis is still unknown in nasopharyngeal carcinoma (NPC). We here showed that the elevated EZH2 level was closely associated with an aggressive and poor prognostic phenotype, and was positively correlated with microvessel density (MVD) in NPC tissues. Functional studies showed that EZH2 upregulation promoted cell proliferation, migration and tubule formation of endothelial cells, and knockdown of EZH2 suppressed tumor growth, metastasis and angiogenesis in vivo. Mechanistic investigations revealed that EZH2 inhibited miR-1 transcription via promoter binding activity, leading to enhanced expression of Endothelin-1 (ET-1) which is suppressed by miR-1 targeting of ET-1 3'UTR. Furthermore, knockdown of EZH2 or overexpression of miR-1 exerted anti-angiogenic effect on NPC cells. More importantly, the neutralizing antibody against ET-1 significantly abrogated the pro-angiogenic effect of EZH2, and forced expression of ET-1 rescued the anti-angiogenic effect induced by EZH2 knockdown. In clinical specimens, ET-1 was widely overexpressed and associated with clinical stage and MVD. Taken together, our results identify a novel signaling pathway involved in NPC angiogenesis, and also suggest that EZH2-miR-1-ET-1 axis represents multiple potential therapeutic targets for NPC.
A comprehensive computational simulation model of sound transmission through the porcine lung is introduced and experimentally evaluated. This "subject-specific" model utilizes parenchymal and major airway geometry derived from x-ray CT images. The lung parenchyma is modeled as a poroviscoelastic material using Biot theory. A finite element (FE) mesh of the lung that includes airway detail is created and used in comsol FE software to simulate the vibroacoustic response of the lung to sound input at the trachea. The FE simulation model is validated by comparing simulation results to experimental measurements using scanning laser Doppler vibrometry on the surface of an excised, preserved lung. The FE model can also be used to calculate and visualize vibroacoustic pressure and motion inside the lung and its airways caused by the acoustic input. The effect of diffuse lung fibrosis and of a local tumor on the lung acoustic response is simulated and visualized using the FE model. In the future, this type of visualization can be compared and matched with experimentally obtained elastographic images to better quantify regional lung material properties to noninvasively diagnose and stage disease and response to treatment.
Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid-? protein (A?), the hallmark of AD, invokes a cascade of mitochondrial dysfunction and eventually leads to neuronal death. l-3-n-Butylphthalide (l-NBP) has shown the potent neuroprotective effects in stroke and AD animal models. The present study is to evaluate the neuroprotective effect of l-NBP on A?25-35-induced neuronal injury and the possible mechanism in the human neuroblastoma SH-SY5Y cells. Our results showed that l-NBP significantly attenuated A?25-35-induced cell death and reduced neuronal apoptosis. l-NBP significantly inhibited A?25-35-induced mitochondrial dysfunction, including mitochondrial membrane potential reduction, and reactive oxygen species production. Furthermore, l-NBP could partially reverse the elevations of A?25-35-induced active caspase-3, caspase-9, and cytochrome c expressions, and the downregulation of anti-apoptosis protein Bcl-2. Moreover, l-NBP markedly inhibited the activations of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway. These results demonstrated that l-NBP was capable of protecting neuronal cells from A?25-35-induced toxicity through a mitochondrial-dependent apoptotic pathway. Thus, l-NBP shows promising candidate of multi-target neuronal protective agent for the treatment of AD.
Seven new aromatic acid derivatives (1-7), together with five known analogs, were isolated from the lateral roots of Aconitum carmichaelii. Structures of the new compounds were determined by spectroscopic and chemical methods as 4-methyl ( - )-(R)-hydroxyeucomate (1), 4-butyl ( - )-(R)-hydroxyeucomate (2), 4-butyl-1-methyl (+)-(R)-2-O-(4'-hydroxy-3'-methoxybenzoyl)malate (3), 1-butyl-4-methyl (+)-(R)-2-O-(4'-hydroxy-3'-methoxybenzoyl)malate (4), dimethyl (+)-(R)-2-O-(4'-hydroxy-3'-methoxybenzoyl)malate (5), dimethyl (+)-(R)-2-O-(4'-hydroxybenzoyl)malate (6), and methyl ( ± )-3-(4'-hydroxy-3'-methoxyphenyl)-3-sulfopropionate (7), respectively. Compounds 1 and 2 are 2-benzylmalates (eucomate derivatives), 3-6 belong to 2-O-benzoylmalates, and 7 is a rare phenylpropionate containing a sulfonic acid group. The absolute configurations of eucomate derivatives were confirmed by X-ray crystallographic analysis of 4-methyl eucomate (11).
The successful gene delivery into the brain is a major challenge due to the presence of the blood-brain barrier (BBB). In order to transport plasmid DNA across the BBB and target the brain glioma, the PEGylated liposomes (PLs) modified with OX26 and chlorotoxin (CTX) were developed as a dual-targeting gene delivery system, and the therapeutic efficacy of OX26/CTX-PL/pC27 against glioma was evaluated using in vitro and in vivo experimental models.
Our previous study demonstrated that treatment of Coxiella burnetii with the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive transfer of 1E4 would protect SCID mice against C. burnetii aerosol infection. The results indicated that 1E4 conferred significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preventing C. burnetii natural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibit C. burnetii infection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4. In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages. Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis against C. burnetii exposure.
Whether clopidogrel should be added to aspirin for stroke prevention remained controversial for the risk of hemorrhagic complications. This meta-analysis was aimed to assess the efficacy and safety of adding clopidogrel to aspirin on stroke prevention in high vascular risk patients, and to provide evidence for a suitable duration of dual antiplatelet therapy.
Noninvasive measurement of mechanical wave motion (sound and vibration) in the lungs may be of diagnostic value, as it can provide information about the mechanical properties of the lungs, which in turn are affected by disease and injury. In this study, two previously derived theoretical models of the vibroacoustic behavior of the lung parenchyma are compared: (1) a Biot theory of poroviscoelasticity and (2) an effective medium theory for compression wave behavior (also known as a "bubble swarm" model). A fractional derivative formulation of shear viscoelasticity is integrated into both models. A measurable "fast" compression wave speed predicted by the Biot theory formulation has a significant frequency dependence that is not predicted by the effective medium theory. Biot theory also predicts a slow compression wave. The experimentally measured fast compression wave speed and attenuation in a pig lung ex vivo model agreed well with the Biot theory. To obtain the parameters for the Biot theory prediction, the following experiments were undertaken: quasistatic mechanical indentation measurements were performed to estimate the lung static shear modulus; surface wave measurements were performed to estimate lung tissue shear viscoelasticity; and flow permeability was measured on dried lung specimens. This study suggests that the Biot theory may provide a more robust and accurate model than the effective medium theory for wave propagation in the lungs over a wider frequency range.
A facile method was found to incorporate a mussel-inspired adhesive moiety into synthetic polymers, and mussel mimetic polyurethanes were developed as adhesive hydrogels. In these polymers, a urethane backbone was substituted for the polyamide chain of mussel adhesive proteins, and dopamine was appended to mimic the adhesive moiety of adhesive proteins. A series of mussel mimetic polyurethanes were created through a step-growth polymerization based on hexamethylene diisocyanate as a hard segment, PEG having different molecular weights as a soft segment, and lysine-dopamine as a chain extender. Upon a treatment with Fe(3+), the aqueous mussel mimetic polyurethane solutions can be triggered by pH adjustment to form adhesive hydrogels instantaneously; these materials can be used as injectable adhesive hydrogels. Upon a treatment with NaIO4, the mussel mimetic polyurethane solutions can be cured in a controllable period of time. The successful combination of the unique mussel-inspired adhesive moiety with a tunable polyurethane structure can result in a new kind of mussel-inspired adhesive polymers.
Prunus tomentosa seeds were researched for antioxidant and anti-inflammatory constituents. By activity-guided fractionation of P. tomentosa seed extract, six new dihydrobenzofuran neolignans, prunustosanans AI-IV (1-4) and prunustosanansides AI and AII (5 and 6), together with 10 known compounds (7-16) were isolated from bioactive fraction. The structures were determined by spectroscopic analyses, especially NMR, HRESIMS, and CD spectra. The antioxidant activity was greatest for 5, 10, and 12 against DPPH radical and for 8, 9, and 13 against ABTS radical. Moreover, compounds 7 and 11 exhibited much stronger inhibitory activity on nitric oxide (NO) production in murine microglia BV-2 compared with positive control minocycline (IC50 = 19.7 ± 1.5 ?M). The results show that P. tomentosa seeds can be regarded as a potential source of antioxidants and inflammation inhibitors.
Alcohol consumption induces inflammatory damage in vessels, and the underlying mechanism is unclear. Valsartan, as one of the angiotensin receptor blockers (ARBs), plays a role in the inhibition of inflammatory reactions in vascular dysfunction. This study is to investigate the role of Toll-like receptor 2 (TLR2) in alcohol-induced inflammatory damage in vascular endothelial cells in vitro and to explore the protective effect of valsartan on alcohol-induced and TLR2-mediated inflammatory damage.
Rhubarb is often used to establish chronic diarrhea and spleen (Pi)-deficiency syndrome animal models in China. In this study, we utilized the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) method to detect changes in bacterial diversity in feces and the bowel mucosa associated with this model. Total microbial genomic DNA from the small bowel (duodenum, jejunum, and ileum), large bowel (proximal colon, distal colon, and rectum), cecum, and feces of normal and rhubarb-exposed rats were used as templates for the ERIC-PCR analysis. We found that the fecal microbial composition did not correspond to the bowel bacteria mix. More bacterial diversity was observed in the ileum of rhubarb-exposed rats (P<0.05). Furthermore, a 380 bp product was found to be increased in rhubarb-exposed rats both in faces and the bowel mucosa. The product was cloned and sequenced and showed high similarity with regions of the Bacteroides genome. AS a result of discriminant analysis with the SPSS software, the Canonical Discriminant Function Formulae for model rats was established.
The present study aimed to investigate the metabolism of Forsythoside A (FTA) by human fecal bacteria to clarify the relationship between its intestinal metabolism and its pharmacological activities. FTA was incubated with human fecal microflora in vitro to investigate its metabolic process, and highly sensitive and specific ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed using MetaboLynx™ software for metabolite analysis. Caffeic acid (CA) and hydroxytyrosol (HT) were obtained by hydrolysis of FTA, and CA was further hydrogenated to form 3,4-dihydroxybenzenepropionic acid (DCA). The anticomplementary, antimicrobial and antiendotoxin activities of FTA and its metabolites by human fecal microflora were evaluated in vitro with a hemolysis assay, the agar disc-diffusion method, the MIC value and the gel clot LAL assay, respectively. The metabolites showed higher biological activity than FTA, especially HT and DCA. Orally administered FTA may be metabolized to HT and DCA, and the pharmacological effects of FTA may be dependent on intestinal bacterial metabolism.
Diosbulbin B (DIOB), a furan-containing diterpenoid lactone, is the most abundant component of Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Administration of purified DIOB or DB extracts has been reported to cause liver injury in animals. The mechanisms of DIOB-induced hepatotoxicity remain unknown. The major objective of this study was to identify reactive metabolites of DIOB. A DIOB-derived cis-enedial was trapped by N-acetyl lysine (NAL) and glutathione (GSH) or N-acetyl cysteine (NAC) in rat and human liver microsomal incubation systems after exposure to DIOB. Four metabolites (M1-M4) associated with GSH were detected by liquid chromatography coupled to tandem mass spectrometry. Apparently, M1 was derived from both NAL and GSH. M2 and M3 resulted from the reaction of GSH without the involvement of NAL. Two molecules of GSH participated in the formation of M4. M2 and M3 were also detected in bile and urine of rats given DIOB. M5, a DIOB-derived NAC/NAL conjugate, was detected in microsomal incubations with DIOB fortified with NAC and NAL as trapping agents. Biomimetic M1-M5 were prepared by oxidation of DIOB with Oxone for metabolite identification. Microsomal incubation study demonstrated that ketoconazole inhibited the production of the enedial in a concentration-dependent manner, and CYP3A4 was found to be the enzyme responsible for the metabolic activation of DIOB. The metabolism study facilitates the understanding of the role of bioactivation of DIOB in its hepatotoxicity.
Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography-mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.
The interferon-induced helicase C domain-containing protein 1 (IFIH1) is a cytosolic RNA sensor belonging to the pattern-recognition receptor (PPR) family. Activation of PPRs on innate immune cells is widely believed to control the development of virus-induced autoimmunity in myocarditis and subsequent dilated cardiomyopathy (DCM). We conducted a pilot study to test whether single nucleotide polymorphisms (SNPs) in IFIH1 were associated with the risk and prognosis of DCM. The TaqMan SNP Genotyping Assay was used to genotype rs1990760 and rs3747517 in 351 DCM patients and 359 controls. The frequency of T allele and CT/TT genotypes at rs1990760 were significantly increased in DCM patients compared to control subjects (p?=?0.046 and p?=?0.027, respectively). The CC homozygosity was associated with worse prognosis expressed by the endpoint of cardiac death compared with allele T carriers of rs3747517 in both univariable (p?=?0.04) and multivariable survival analysis after adjusting for age, sex, left ventricular end-diastolic diameter and ejection fraction (p?=?0.01). The results revealed that rs1990760 was associated with susceptibility to DCM and rs3747517 played a role in the prognostic assessment of DCM, reflecting the distinct genetic contributions of innate IFIH1 polymorphisms in controlling the onset and outcome of DCM.
Chest physical examination often includes performing chest percussion, which involves introducing sound stimulus to the chest wall and detecting an audible change. This approach relies on observations that underlying acoustic transmission, coupling, and resonance patterns can be altered by chest structure changes due to pathologies. More accurate detection and quantification of these acoustic alterations may provide further useful diagnostic information. To elucidate the physical processes involved, a realistic computer model of sound transmission in the chest is helpful. In the present study, a computational model was developed and validated by comparing its predictions with results from animal and human experiments which involved applying acoustic excitation to the anterior chest, while detecting skin vibrations at the posterior chest. To investigate the effect of pathology on sound transmission, the computational model was used to simulate the effects of pneumothorax on sounds introduced at the anterior chest and detected at the posterior. Model predictions and experimental results showed similar trends. The model also predicted wave patterns inside the chest, which may be used to assess results of elastography measurements. Future animal and human tests may expand the predictive power of the model to include acoustic behavior for a wider range of pulmonary conditions.
Abstract Spinocerebellar ataxia type 3 (SCA3) is the most common type of spinocerebellar ataxia, which are inherited neurodegenerative diseases. CAG repeat expansions that translate into an abnormal length of glutamine residues are considered to be the disease-causing mutation. The pathological mechanisms of SCA3 are not fully elucidated but may include aggregate or inclusion formation, imbalance of cellular protein homeostasis, axonal transportation dysfunction, translation dysregulation, mitochondrial damage and oxidative stress, abnormal neural signaling pathways, etc. Currently, symptom relief is the only available therapeutic route; however, promising therapeutic targets have been discovered, such as decreasing the mutant protein through RNA interference (RNAi) and antisense oligonucleotides (AONs) and replacement therapy using stem cell transplantation. Other potential targets can inhibit the previously mentioned pathological mechanisms. However, additional efforts are necessary before these strategies can be used clinically.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-? (A?) deposition and neurofibrillary tangles. Dl-PHPB [potassium 2-(1-hydroxypentyl)-benzoate], has been shown to have neuroprotective effects on cerebral ischemic, vascular dementia, and A?-induced animal models by inhibiting oxidative injury, neuronal apoptosis, and glial activation. The aim of the present study was to examine the effect of dl-PHPB on learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic AD mouse models (APP/PS1) and the mechanisms of dl-PHPB in reducing A? accumulation and ? phosphorylation. Twelve-month-old APP/PS1 mice were given 30 mg/kg dl-PHPB by oral gavage for 3 months. Dl-PHPB treatment significantly improved the spatial learning and memory deficits compared with the vehicle-treated APP/PS1 mice. In the meantime, dl-PHPB obviously reduced ? hyperphosphorylation at Ser199, Thr205, and Ser396 sites in APP/PS1 mice. This reduction was accompanied by APP phosphorylation reduction and protein kinase C activation. In addition, expression of cyclin-dependent kinase and glycogen synthase kinase 3?, the most important kinases involved in ? phosphorylation, was markedly decreased by dl-PHPB treatment. Phosphorylated protein kinase B and phosphoinositide 3-kinase levels of APP/PS1 mice were significantly reduced compared with levels in wild-type mice, and dl-PHPB reversed the reduction. The effects of dl-PHPB effecting a decrease in ? phosphorylation and kinase activation were further confirmed in neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. These data raised the possibility that dl-PHPB might be a promising multitarget neuronal protective agent for the treatment of AD.
Two new lignan glycosides forsythiayanoside A (1) and forsythiayanoside B (2), one new stereoisomer of lignan glycoside (+)-8-hydroxyepipinoresinol-4-O-?-D-glucopyranoside (3), together with seven known compounds (4-10), were isolated from the fruits of Forsythia suspense (Thunb.) Vahl. Compounds 4 and 10 were isolated from this species for the first time. The structures of 1-3 were elucidated on the basis of chemical and spectral analyses, including 1D and 2D NMR data and HR-ESI-MS. The absolute configurations were determined by the circular dichroism method. All isolates were tested for their cytotoxicities against five human cancer cell lines (A549, Colo-205, Hep-3B, HL60, and KB). In particular, compound 3 showed significant cytotoxicity with IC50 values of 9.48, 7.75, 0.59, 4.06, and 38.38 ?M, respectively.
The present study aimed to investigate the metabolism of Entadae Semen by human fecal bacteria to clarify the relationship between its pharmacological activities and intestinal metabolism. Three major components (phaseoloidin, entadamide A-?-D-glucopyranoside and entadamide A) were isolated and identified from Entadae Semen and then incubated with human fecal microflora in vitro to investigate the metabolic processes. The metabolites were analyzed with high-performance liquid chromatography (HPLC). The anti-complement activities of the three components and their metabolites produced by human fecal microflora were evaluated in vitro using a hemolysis assay. Phaseoloidin and entadamide A-?-D-glucopyranoside were metabolized into their respective aglycones during the incubation process, which enhanced their anti-complement effects. These results indicated that the presence of intestinal bacteria likely plays an important role and that the pharmacological effects of Entadae Semen may be dependent on intestinal bacterial metabolism.
Human brain natriuretic peptide (BNP) is utilized in the treatment of acute decompensated congestive heart failure. However, BNP has limited clinical use owing to its rapid clearance and the need for continuous intravenous infusion. Thus, we generated human serum albumin (HSA)-BNP fusion constructs to produce long-acting fusion proteins in Pichia pastoris. Four fusion proteins, BNP-HSA, (BNP)2-HSA, (BNP)4-HSA, and HSA-(BNP)2, were constructed, with different numbers of BNP molecules and fusion orientations. BNP-HSA was most abundantly expressed in Pichia pastoris and (BNP)4-HSA had the lowest yield, probably because of the high copy number of BNP. Western blot analysis confirmed the immunogenicity of both BNP and HSA for the four fusion proteins. A cGMP activity assay was used to measure the ability of fusion proteins to stimulate intracellular cGMP expression. Results showed that the fusion protein HSA-(BNP)2 activated human natriuretic peptide receptor A (hNPR-A) with potency similar to that of BNP, despite using a 10-fold higher dosage than BNP. The other three fusion proteins (BNP-HSA, (BNP)2-HSA, and (BNP)4-HSA), only slightly increased NPR-A activity. In addition, fusion with HSA successfully prolonged BNP bioactivity, stimulating intracellular cGMP expression over 24 h. In conclusion, HSA-(BNP)2, with two BNP molecules fused at the C-terminus of HSA, has the highest and most prolonged BNP bioactivity in activating human NPR-A.
The present study aimed to investigate the impact of hypothyroidism on left ventricular systolic function using real-time three-dimensional speckle tracking imaging (RT3D-STI). Thirty hypothyroidism patients and forty healthy volunteers were recruited and received RT3DSTI measurement of global longitudinal strain (GLS), global circumferential strain (GCS), global radial strain (GRS), and global area strain (GAS). A comparison of differences between the hypothyroidism patients and those in the healthy group was carried out and we obtained the results as followings. The values of GLS were (-18.93 +/- 3.89) vs. (-21.44 +/- 1.99), with P < 0.01, GRS were (51.13 +/- 11.95) vs. (56.10 +/- 5.76), with P < 0.0; and GAS were (-31.63 +/- 5.38) vs. (-34.40 +/- 2.32), with P < 0.01, i.e. they were lower in hypothyroidism group than those in the health group. While GCS were (-17.75 +/- 1.92) vs. 17.03 +/- 3.45), with P > 0.05, which were not significantly different between the two groups. In linear regres sion, GLS showed significant correlation with both TSH (b = -0.69, P < 0.01) and FT3 (b = 0.71, P < 0.01). Meanwhile, the GRS (b = 2.98, P < 0.05) and GAS (b = 3.11, P < 0.05) linearly correlated with FT3 level. In conclusion, the present study shows that the global longitudinal and radial moves of left ventricular are weaker in patients with hypothyroidism than healthy controls. And the impairment of left ventricular function would aggravate as FSH rises or FT3 declines.
The effect of changing temperature on an individual's cerebrovascular risk is both biologically plausible and supported by epidemiologic evidence. We used a global proteomic-based approach to analyze the expression alterations of proteins in artificial cold exposure (ACE)-induced hypertensive stroke in renovascular hypertensive rats (RHR) and to identify the biomarker of ACE-induced hypertensive stroke.
In past 2 decades, nonmedical consumption of cough mixture has become a serious social problem in certain regions of China. Cough mixture abuse causes psychiatric symptoms. Moreover, there has been an increasing concern about the physical disorders associated with cough mixture abuse.
Myofilaments are composed of thin and thick filaments that coordinate with each other to regulate muscle contraction and relaxation. PTMs together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down MS has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to the study of cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed.
?-tocotrienol (GT3), an analogue of vitamin E, has gained increasing scientific interest recently as it provides significant health benefits. It has been shown that emulsified GT3, after subcutaneous administration, has long-term biological effects. However, whether the effects are due to the increase of GT3 level in the early phase following administration or the persistent functions after accumulation in tissues is unknown. This study was conducted to determine the levels of GT3 in different tissues by high performance liquid chromatography (HPLC) with a fluorescence detector after a single-dose of GT3 with polyethylene glycol (PEG-400) emulsion via subcutaneous injection. Previous studies have explored that GT3 has favorable effects on bone and can inhibit osteoclast formation. To confirm the persistent biological activity of accumulated GT3 in tissues, receptor activator of NF-?B ligand (RANKL) and osteoprotegerin (OPG) gene expressions, which have an important role in regulating osteoclast formation, were also evaluated in bone tissue on day 1, 3, 7 and 14 after a signal subcutaneous injection of GT3.
Adrenal Cushing's syndrome is caused by excess production of glucocorticoid from adrenocortical tumors and hyperplasias, which leads to metabolic disorders. We performed whole-exome sequencing of 49 blood-tumor pairs and RNA sequencing of 44 tumors from cortisol-producing adrenocortical adenomas (ACAs), adrenocorticotropic hormone-independent macronodular adrenocortical hyperplasias (AIMAHs), and adrenocortical oncocytomas (ADOs). We identified a hotspot in the PRKACA gene with a L205R mutation in 69.2% (27 out of 39) of ACAs and validated in 65.5% of a total of 87 ACAs. Our data revealed that the activating L205R mutation, which locates in the P+1 loop of the protein kinase A (PKA) catalytic subunit, promoted PKA substrate phosphorylation and target gene expression. Moreover, we discovered the recurrently mutated gene DOT1L in AIMAHs and CLASP2 in ADOs. Collectively, these data highlight potentially functional mutated genes in adrenal Cushing's syndrome.
It was previously reported that cytokines and neurotoxins released from activated inflammatory cells induced the loss of projecting dopaminergic neurons in the substantia nigra, which triggered the pathogenesis of PD. The present study investigated the effect of treatment with tetramethylpyrazine (TMP) on the central cytokine synthesis, striatal dopamine content and glutamatergic transmission, and behavioral performance in the rotarod task in mice injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Treatment with TMP significantly improved the behavioral performance in the rotarod task in mice injected with MPTP. It also decreased the upregulation of cytokines (tumor necrosis factor-? and interleukin-1?) in the substantia nigra and striatum in these modeled mice. Furthermore, treatment with TMP significantly improved the dopamine deficits and attenuated the upregulation of striatal basal glutamatergic strength in the striatum of mice injected with MPTP. These results indicated that TMP might serve as a novel approach for the treatment of patients with PD.
The partial trisomy 9q syndrome is a well-defined chromosomal disorder with over 40 reported cases in the literature. However, 9q duplications derived from an insertional translocation have rarely been reported.
Dissolved organic nitrogen (DON) from atmospheric deposition has been a growing concern in the world and atmospheric nitrogen (N) deposition is increasing quickly in China especially Southeastern China. In our study, DON wet deposition was estimated by collecting and analyzing rainwater samples continuously over eight years (2005-2012) in a typical red-soil farmland ecosystem, Southeast China. Results showed that the volume-weighted-average DON concentration varied from 0.2 to 3.3 mg N L(-1) with an average of 1.2 mg N L(-1). DON flux ranged from 5.7 to 71.6 kg N ha(-1) year(-1) and averaged 19.7 kg N ha(-1) year(-1) which accounted for 34.6% of the total dissolved nitrogen (TDN) in wet deposition during the eight-year period. Analysis of DON concentration and flux, contribution of DON to TDN, rainfall, rain frequency, air temperature and wind frequency and the application of pig manure revealed possible pollution sources. Significant positive linear relation of annual DON flux and usage of pig manure (P<0.0001) suggested that agricultural activities, especially application of pig manure, were the main source of DON in the study area. In conclusion, DON wet deposition was an important part of TDN and would have a possible effect on N cycle in the red-soil agro-ecosystem in the future.
Astaxanthin is a strong antioxidant with the ability of reducing the markers of inflammation. To explore the protective effect of astaxanthin on maternal ethanol induced embryonic deficiency, and to investigate the underlying mechanisms, we detected the morphology, expression of neural marker genes, oxidative stress indexes, and inflammatory factors in mice model of fetal alcohol spectrum disorder with or without astaxanthin pretreatment. Our results showed that astaxanthin blocked maternal ethanol induced retardation of embryonic growth, and the down-regulation of neural marker genes, Otx1 and Sox2. Moreover, astaxanthin also reversed the increases of malondialdehyde (MDA), hydrogen peroxide (H2O2), and the decrease of glutathione peroxidase (GPx) in fetal alcohol spectrum disorder. In addition, maternal ethanol induced up-regulation of toll-like receptor 4 (TLR4), and the down-streaming myeloid differentiation factor 88 (MyD88), NF-?B, TNF-?, and IL-1? in embryos, and this was inhibited by astaxanthin pretreatment. These results demonstrated a protective effect of astaxanthin on fetal alcohol spectrum disorder, and suggested that oxidative stress and TLR4 signaling associated inflammatory reaction are involved in this process.
Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)(2)-HSA, (SS28)(3)-HSA, and HSA-(SS28)(2), were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)(3)-HSA was much lower than (SS28)(2)-HSA and HSA-(SS28)(2) due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)(2)-HSA being the most effective one. A pharmacokinetics study showed that (SS28)(2)-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. Cholinesterase inhibitors are widely used for the symptomatic treatment of Alzheimer's disease to enhance central cholinergic transmission. In this study, a bioactivity-oriented screening platform based on a modified Ellman's method and HPLC-QTOF MS technique was developed to rapidly screen active agents of Anemarrhena asphodeloides Bge. The 60% ethanol fraction from an ethyl acetate extract exhibited the most potential anticholinesterase activity. Fifteen steroid saponins were identified by the mass spectrum, standards and literature reports. Twenty-five compounds were isolated from the active fraction. The results showed that compounds with the C6-C3-C6 skeleton probably had both AChE and BuChE inhibitory activities. Xanthone and benzene derivatives exhibited no or little activity. Lignans showed weak BuChE inhibitory activity. The steroidal saponins demonstrated moderate or weak AChE inhibitory activity.
Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.
Detection of chromosome copy number variation (CNV) plays an important role in the diagnosis of patients with unexplained clinical symptoms and for the identification of chromosome disease syndromes in the established fetus. In current clinical practice, karyotyping, in conjunction with array-based methods, is the gold standard for detection of CNV. To increase accessibility and reduce patient costs for diagnostic CNV tests, we speculated that next-generation sequencing methods could provide a similar degree of sensitivity and specificity as commercial arrays. CNV in patient samples was assessed on a medium-density single nucleotide polymorphism array and by low-coverage massively parallel CNV sequencing (CNV-seq), with mate pair sequencing used to confirm selected CNV deletion breakpoints. A total of 10 ng of input DNA was sufficient for accurate CNV-seq diagnosis, although 50 ng was optimal. Validation studies of samples with small CNVs showed that CNV-seq was specific and reproducible, suggesting that CNV-seq may have a potential genome resolution of approximately 0.1 Mb. In a blinded study of 72 samples with known gross and submicroscopic CNVs originally detected by single nucleotide polymorphism array, there was high diagnostic concordance with CNV-seq. We conclude that CNV-seq is a viable alternative to arrays for the diagnosis of chromosome disease syndromes.
Resistance to chemotherapy and the side effects of anticancer drugs are the major obstacles for glioma treatment. The aim of the present study was to develop a novel approach for the treatment of gliomas that improved the therapeutic effect; the anticancer drug, doxorubicin (DOX), was combined with short interfering (si)RNA and monomethoxy polyethylene glycol polyethylenimine superparamagnetic iron oxide nanoparticle (mPEG-PEI-SPION), a magnetic resonance imaging (MRI)-visible nanoparticle. Specific siRNA molecules, delivered by mPEG-PEI-SPION, were employed to knockdown the PIN2-interacting protein 1 (PinX1) gene in C6 glioma cells. PinX1 is a nucleolar protein associated with telomere and telomerase. C6 cells were treated with DOX and/or PinX1-siRNA. The results of the transfection experiments revealed that siRNA/mPEG-PEI-SPION was transfected into C6 cells with high efficiency. PinX1-siRNA was unable to inhibit C6 cells, while in the PinX1-siRNA + DOX group, the same dose of DOX caused an increased loss of cell viability. Therefore, mPEG-PEI-SPION was shown to be viable for siRNA delivery into C6 cells and coadministration of DOX with PinX1-siRNA may be a potential therapeutic method for inhibiting gliomas.
A major challenge for further promotion of lipase productivity in Penicillium expansum PE-12 is to find a suitable promoter that can function efficiently in this industrial strain. In this study, the 5' flanking region of P. expansum lipase (Ppel) containing a putative novel promoter sequence was characterized by fusing to ?-glucuronidase (GUS) and subsequently introducing into P. expansum. As a result, all the transformants showed blue color quickly after incubation in GUS detection buffer, suggesting a strong promoter activity of this fragment. Glucose repression was identified for the promoter, whereas olive oil acted as a positive regulator. Facilitated by this novel promoter, P. expansum PE-12 was genetically modified, with an improved lipase yield, via a recombinant plasmid with P. expansum lipase gene (PEL) under the control of Ppel promoter and TtrpC terminator. The highest lipase yield among the modified strains could attain 2,100 U/mL, which is more than twofold of the previous industrial strain (900 U/mL). The engineered strain through molecular breeding method as well as this new promoter has great value in lipase industry.
Oral delivery is the preferred route of administration and therefore good absorption after oral dosing is a prerequisite for a compound to be successful in the clinic. The prediction of oral bioavailability from in vitro permeability assays is thus a valuable tool during drug discovery and development. Caco-2 cell monolayers mimic the human intestinal epithelium in many aspects. These monolayers form tight junctions between cells and have been widely used as a model of human intestinal absorption. Caco-2 cells also express a variety of transporter proteins although the transformed nature of the cells results in unpredictable differentiation markers, transport properties and enzyme expression. Thus various modifications of the Caco-2 assay are used in laboratories across the globe. The purpose of this paper is to provide an overview of a time and resource saving 7-day Caco-2 assay protocol. We also discuss the impact of various experimental conditions on permeability measurements and its applications during lead optimization in early discovery and for clinical candidate characterization, specifically for prediction of absorption in human, at a later stage in drug development.
The role of interleukin-22 (IL-22) in intracellular bacterial infections is a controversial issue, although the contribution of this cytokine to host defense against extracellular bacterial pathogens has been well established. In this study, we focused on an intra-cellular bacterium, Chlamydia, and evaluated the production and function of IL-22 in host defense against chlamydial lung infection using a mouse model. We found that Chlamydia muridarum infection elicited quick IL-22 responses in the lung, which increased during infection and were reduced when bacterial loads decreased. More importantly, blockade of endogenous IL-22 using neutralizing anti-IL-22 monoclonal antibodies (mAb) resulted in more severe disease in the mice, leading to significantly higher weight loss and bacterial growth and much more severe pathological changes than treatment with isotype control antibody. Immunological analyses identified significantly lower T helper 1 (Th1) and Th17 responses in the IL-22-neutralized mice. In contrast, intranasal administration of exogenous IL-22 significantly enhanced protection following chlamydial lung infection, which was associated with a significant increase of Th17 response. The data demonstrate that IL-22 is a critical cytokine, mediating host defense against chlamydial lung infection and coordinating the function of distinct Th-cell subsets, particularly Th1 and Th17, in the process.
The Internet is increasingly being applied in health education worldwide; however there is little knowledge of its use in Chinese higher education institutions. The present study provides the first review and highlights the deficiencies and required future advances in Chinese Internet-based health education.
A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the first time for the identification and quantification of curdione in rabbit plasma after vaginal drug administration and intravenous administration of zedoary turmeric oil (ZTO) solution (10 mg/kg). The analysis was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive ionization mode. After mixing with internal standard diazepam, plasma samples were extracted with ethyl ether-acetic ether (1:1, v/v). Chromatographic separation was carried out on a C18 column with gradient elution using a mixture of water and acetonitrile (both containing 0.1% formic acid) as mobile phases. Linearity ranged over 1.06-106 and 10.6-530 ng/mL (r ? 0.995) with the lower limit of quantfication 1.06 ng/mL. The intra- and inter-day precision relative standard deviation values were <12% and the accuracy relative error was from -10.6 to -6.1% at all quality control sample levels. The method was applied to a study of the pharmacokinetics of curdione after vaginal drug administration and intravenous administration of ZTO.
?-Tocotrienol (GT3), an analogue of vitamin E, has gained increasing scientific interest recently as it provides significant health benefits. GT3 exerts its biological effects not only by virtue of antioxidant properties but also by inhibiting hydroxy-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. Studies have reported that the mevalonate pathway is relevant for bone metabolism and HMG-CoA reductase inhibitors can increase bone mass and are useful in osteoporosis therapy. However, whether it is involved in the bone anabolic activity of GT3 is not clear. This study was conducted to investigate the ability of GT3 to protect against ovariectomy-induced bone loss, as well as the correlation between the protections and mevalonate pathway. Results showed that mice supplemented with 100mg/kg emulsified GT3 via subcutaneous injection once per month for three months were significantly protected from ovariectomy-induced bone loss as evaluated by various bone structural parameters, bone metabolic gene expression levels and serum levels of biochemical markers for bone resorption and bone formation. Importantly, the effect of GT3 on preventing against ovariectomy-induced bone loss could be reversed by daily supplementation with mevalonate, indicating that GT3 may via an HMG-CoA reductase-dependent mechanism to protect against ovariectomy-induced bone loss. Our results suggest that GT3 is suitable as dietary supplement and has potential as an alternative drug to treat or prevent osteoporosis.
In the present study, the functional properties and pharmacology of two-pore domain potassium channel (K2P) TREK-1 in primary cultured rat brain astrocytes were investigated. Western blot, patch clamping techniques, and ELISA were used to detect the distribution and function of TREK-1 as well as the expression of brain-derived neurotrophic factor (BDNF) on the primary cultured astrocytes. It was shown that TREK-1 protein expressed in astrocytes was 2.4-fold higher than it was expressed in microglia. Single channel recording via patch clamping showed that the TREK-1 outward currents in astrocytes could be activated by arachidonic acid (AA) or chloroform with the conductance of 113?±?14 and 120?±?13 pS, respectively. The current was also sensitive to mechanical stretch and intracellular acidification. Negative pressure (-30 cm H2O) and acidification of intracellular solution (pH 6.8 or 6.3) both enhanced TREK-1 channel open probability significantly. Further pharmacological studies showed that TREK-1 antagonist penfluridol inhibited AA-induced currents, and both penfluridol and methionine (TREK-1 blockers) significantly increased BDNF level in astrocytes by 50 %. These results indicated that TREK-1 channel current was a major component of K2P currents in astrocytes. TREK-1 channels might play important roles in regulating the function of astrocytes and might be used as a drug target for neuroprotection.
ETHNOPHARMACOLOGICAL RELEVANCE SCUTELLARIA BAICALENSIS: Georgi (Labiatae) is a well-known traditional Chinese medicine to treat inflammation, cardiovascular diseases, respiratory and gastrointestinal infections, etc. The present study was to understand the metabolism of the root of Scutellaria baicalensis (a.k.a. Huangqin in Chinese) in the gastrointestinal tract and the correlation between the metabolites and their respective pharmacological activities.
Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been used to trace the transmission of M. tuberculosis, the causative agent of tuberculosis (TB). Previously published studies using WGS were conducted in developed countries with a low TB burden. We sought to evaluate the relative usefulness of traditional VNTR and SNP typing methods, WGS and epidemiological investigations to study the recent transmission of M. tuberculosis in a high TB burden country. We conducted epidemiological investigations of 42 TB patients whose M. tuberculosis isolates were classified into three clusters based on variable-number tandem repeat (VNTR) typing. We applied WGS to 32 (76.2%) of the 42 strains and calculated the pairwise genomic distances between strains within each cluster. Eighteen (56.3%) of the 32 strains had genomic differences ?100 SNPs with every other strain, suggesting that direct transmission did not likely occurred. Ten strains were grouped into four WGS-based clusters with genomic distances ?5 SNPs within each cluster, and confirmed epidemiological links were identified in two of these clusters. Our results indicate that WGS provides reliable resolution for tracing the transmission of M. tuberculosis in high TB burden settings. The high resolution of WGS is particularly useful to confirm or exclude the possibility of direct transmission events defined by traditional typing methods.
The rapid advancements in mass spectrometry (MS) instrumentation, particularly in Fourier transform (FT) MS, have made the acquisition of high-resolution and high-accuracy mass measurements routine. However, the software tools for the interpretation of high-resolution MS data are underdeveloped. Although several algorithms for the automatic processing of high-resolution MS data are available, there is still an urgent need for a user-friendly interface with functions that allow users to visualize and validate the computational output. Therefore, we have developed MASH Suite, a user-friendly and versatile software interface for processing high-resolution MS data. MASH Suite contains a wide range of features that allow users to easily navigate through data analysis, visualize complex high-resolution MS data, and manually validate automatically processed results. Furthermore, it provides easy, fast, and reliable interpretation of top-down, middle-down, and bottom-up MS data. MASH Suite is convenient, easily operated, and freely available. It can greatly facilitate the comprehensive interpretation and validation of high-resolution MS data with high accuracy and reliability.
Toll-like receptors (TLRs) play an essential role in the activation and regulation of the innate and adaptive immune responses through the recognition of specific components of pathogens. TLR1/2 on the cell surface plays an important role in defending against Gram-positive bacteria. The aim of the present study was to examine the expressional variation of immunomodulatory molecules in peripheral blood leukocytes (PBLs) treated with the TLR1/2 agonist, Pam3Cys. The quantitative polymerase chain reaction result showed dramatically increased expression of immune-related factors treated with Pam3Cys. Antibody-chip assays confirmed that activation of TLR1/2 could induce secretion of four important immune factors [interleukin (IL)-6, IL-8, macrophage inflammatory protein-1? and interferon-?). Western-blot analysis indicated the upregulation of three significant signal kinase proteins (phosphorylated signal transducer and activator of transcription 3, extracellular signal-related kinase and c-Jun N-terminal kinase 2). The study demonstrated that there were numerous molecules involved in the immune response of PBLs stimulated by the TLR1/2 ligand. Our future studies will focus on the mechanisms of these molecules in the TLR1/2 agonist-mediated immune response.
Hypertension is common after acute stroke onset. Previous studies showed controversial effects of early blood pressure (BP) lowering on stroke outcomes. The aim of this study is to assess the effects of early BP lowering on early and long-term outcomes after acute stroke.
Albumin fusion technology, the combination of small molecular proteins or peptides with human serum albumin (HSA), is an effective method for improving the medicinal values of natural small molecular proteins or peptides. However, comparative studies between HSA-fusion proteins or peptides and the parent small molecules in biological and molecular mechanisms are less reported. In this study, we examined the binding property of two novel somatostatin-HSA fusion proteins, (SST14)2-HSA and (SST28)2-HSA, to human SSTRs in stably expressing SSTR1-5 HEK 293 cells; observed the regulation of receptor internalization and internalized receptor recycling; and detected the receptors activation of HSA fusion proteins in stably expressing SSTR2- and SSTR3-EGFP cells. We showed that both somatostatin-HSA fusion proteins had high affinity to all five SSTRs, stimulated the ERK1/2 phosphorylation and persistently inhibited the accumulation of forskolin-stimulated cAMP in SSTR2- and SSTR3-expressing cells; but were less potent than the synthetic somatostatin-14 (SST-14). Our experiments also showed that somatostatin-HSA fusion proteins did not induce the receptors internalization; rather, they accelerated the recycling of the internalized receptors induced by SST-14 to the plasma membrane. Our results indicated that somatostatin-HSA fusion proteins, different from SST-14, exhibit some particular properties in binding, regulating, and activating somatostatin receptors.
Radiation-induced reactive oxygen species (ROS) can damage DNA and most other biological macromolecules in skin and radiation-induced skin injury is a serious concern for radiation therapy. Skin possesses an extremely efficient antioxidant system, which is conferred by two systems: antioxidant enzymes and small molecules that can scavenge ROS by donating electrons. Amphibian skin is a multifunctional organ, which protects against dangers of various oxidative stresses. Recently, a small peptide called RP-1 was isolated from the skin secretions of Rana pleurade, which shows strong antioxidant activity. However, this RP-1 peptide is limited because its inability to across the cell membrane. Protein transduction domains (PTDs) have demonstrated high efficiency for facilitating the internalization of both homologous and heterogeneous proteins into cells. This study aims to elucidate the protective effects of a HIV-TAT (TAT) PTD-coupled RP-1 fusion protein (TAT-RP1) on radiation-induced skin injury in vitro and in vivo. The synthesized fusion TAT-RP1 peptide can be incorporated into human keratinocyte HaCaT cells in a dose- and time-dependent manner without cytotoxicity. We then evaluated the protective role of TAT-RP1 against ionizing radiation. TAT-RP1 supplementation increased anti-superoxide anion ability of HaCaT cells and decreased HaCaT cell radiosensitivity to irradiation. Moreover, TAT-RP1 was able to penetrate the skin of rats, entering epidermis as well as the dermis of the subcutaneous layer in skin tissue. Topical spread of TAT-RP1 promoted the amelioration of radiation-induced skin damage in rats. These results suggest that TAT-RP1 has potential as a protein therapy for radiation-induced skin injury.
Setting: Multidrug-resistant tuberculosis (MDR-TB) has emerged as a serious global public health problem. In China, the risk factors for MDR-TB have not been systematically evaluated. Objective: To identify risk factors associated with MDR-TB among previously treated patients in China. Design: A case-control study was carried out. Cases were selected from previously treated MDR-TB patients who were resistant to both isoniazid and rifampin, and controls were selected from previously treated TB patients who were sensitive to isoniazid and rifampin (non-MDR-TB). Information was collected from the registration database and a structured questionnaire. Results: A total of 61 cases and 50 controls were recruited. A multivariate analysis showed that the family annual per-capita income ?7,000 Yuan (odds ratio [OR]=3.238; 95% confidence interval [CI]: 1.270-8.252), no history of fixed dose combinations (FDCs) in anti-TB treatment (OR=4.027; 95% CI: 1.457-11.129), and adverse reactions in the course of TB treatment (OR=3.568; 95% CI: 1.402-9.085) were independent predictors of MDR-TB. Moreover, among the TB patients who had adverse reactions, quitting the treatment was shown as a risk factor for MDR-TB (p=0.009). Conclusion: In the control of MDR-TB among previously treated patients, lower socioeconomic groups, the expanding use of FDCs, and improving adherence to treatment by implementing Directly Observed Therapy Short Course-Plus (DOTS-Plus), strictly should become a priority that requires strong commitment and collaboration among health organizations.
BackgroundTumors affecting the head, neck, and brain account for significant morbidity and mortality. The curative efficacy of radiotherapy for these tumors is well established, but radiation carries a significant risk of neurologic injury. So far, neuroprotective therapies for radiation-induced brain injury are still limited. In this study we demonstrate that Stichodactyla helianthus (ShK)-170, a specific inhibitor of the voltage-gated potassium (Kv)1.3 channel, protected mice from radiation-induced brain injury.MethodsMice were treated with ShK-170 for 3 days immediately after brain irradiation. Radiation-induced brain injury was assessed by MRI scans and a Morris water maze. Pathophysiological change of the brain was measured by immunofluorescence. Gene and protein expressions of Kv1.3 and inflammatory factors were measured by quantitative real-time PCR, reverse transcription PCR, ELISA assay, and western blot analyses. Kv currents were recorded in the whole-cell configuration of the patch-clamp technique.ResultsRadiation increased Kv1.3 mRNA and protein expression in microglia. Genetic silencing of Kv1.3 by specific short interference RNAs or pharmacological blockade with ShK-170 suppressed radiation-induced production of the proinflammatory factors interleukin-6, cyclooxygenase-2, and tumor necrosis factor-? by microglia. ShK-170 also inhibited neurotoxicity mediated by radiation-activated microglia and promoted neurogenesis by increasing the proliferation of neural progenitor cells.ConclusionsThe therapeutic effect of ShK-170 is mediated by suppression of microglial activation and microglia-mediated neurotoxicity and enhanced neurorestoration by promoting proliferation of neural progenitor cells.
MicroRNA (miRNA) deregulation and pathway alterations have been implicated in nasopharyngeal carcinoma (NPC), a highly invasive and metastatic cancer widely prevalent in southern China. In this study, we report that miR-9 is commonly downregulated in NPC specimens and NPC cell lines with important functional consequences. The reduced expression of miR-9 was inversely correlated with clinical stages and marked the progression from locoregional to metastatic tumors. The CpG island hypermethylation contributed to miR-9 silencing in NPC cell lines and tissues. Ectopic expression of miR-9 dramatically inhibited the proliferative, migratory and invasive capacities of NPC cells in vitro and in vivo. We found that miR-9 strongly reduced the expression of CXCR4 in NPC cells. Luciferase assay demonstrated that miR-9 could directly bind to the 3()-untranslated region of CXCR4. Similar to the restoring miR-9 expression, CXCR4 downregulation inhibited cell growth, migration and invasion, whereas CXCR4 overexpression rescued the suppressive effect of miR-9. Mechanistic investigations revealed that CXCR4 functionally mediated the SDF-1-stimulated activation of p38 MAPK pathway in NPC cells with miR-9 downregulation or CXCR4 overexpression. In clinical specimens, CXCR4 and phospho-p38 were widely overexpressed, and the levels increased with the progression from locoregional to metastatic tumors in NPC tissues. The levels of CXCR4 were inversely correlated with miR-9 or phospho-p38 expression. Taken together, our results indicate that miR-9 functions as a tumor suppressive miRNA in NPC, and that its suppressive effects are mediated chiefly by repressing CXCR4 expression.
Drug metabolism studies, including in vivo and in vitro metabolism studies, are significant in the design of candidate compounds and screening of lead compounds at drug discovery/development stages. Compared with in vivo metabolism studies, in vitro metabolism studies have the advantages of rapidity, simplicity, without consumption of large amounts of samples and animals. Moreover, it is convenient for researchers to observe the selective interaction between compound and target. Therefore, in vitro metabolism studies are appropriate for high throughput screening of compounds which are lack of metabolism information and have been widely used during drug discovery stages. This article briefly introduced the application of in vitro drug metabolism studies based on the metabolic stability, reaction phenotyping and metabolic drug-drug interactions, aiming to raise valuable evaluation strategies for innovative drug discovery in China.
The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.
Three novel xanthonolignoid C-glycosides, glomexanthones A-C, with a trans-dihydrobenzofuran on B ring and a 2-hydroxymethyl-5-hydroxyl-2-pentenoic acid moiety in the sugar chain were isolated from an ethanol extract of Polygala glomerata. Their structures and absolute configurations were characterized by extensive NMR, MS, and CD spectroscopic studies. Screening results indicated that compounds 1-3 showed moderate neuroprotective effects on l-Glutamic acid-induced cellular damage in human neuroblastoma SK-N-SH cells.
The lipid droplet (LD) is a unique cellular organelle containing a neutral-lipid core enclosed by a phospholipid monolayer and associated proteins. Despite the important function of LDs at the hub of cellular energy homeostasis regulation, major questions in the field of LD biology are still unanswered. Drosophila melanogaster has been used as a model organism to make fundamental discoveries in biology for over a century. In recent years, genome-wide unbiased reverse genetic screens using Drosophila cells or transgenic lines have been proven to provide valuable knowledge to the field of LD biology. Here we summarize the methods we use for functional genomic screens in Drosophila S2 cells to identify genes involved in LD biology, and the methods used for studying LD function in vivo using Drosophila as a model to combat metabolic diseases.
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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.