JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Effects of gabexate mesilate on coagulopathy and organ dysfunction in rats with endotoxemia: a potential use of thrombelastography in endotoxin-induced sepsis.
Blood Coagul. Fibrinolysis
PUBLISHED: 11-15-2014
Show Abstract
Hide Abstract
Sepsis and its associated multiple organ failure are related to high mortality in critical patients. Several studies have reported that gabexate mesilate, a synthetic inhibitor of trypsin-like serine protease, protects tissues/organs against injury in the models of endotoxemia. The aim of this study was to examine whether gabexate mesilate could attenuate coagulopathy and organ dysfunction in lipopolysaccharide (LPS)-induced sepsis model by using thrombelastography (TEG). LPS (7.5?mg/kg/h, intravenouly for 4?h) was administered to male adult Wistar rats. Some of the LPS rats received a continuous infusion of gabexate mesilate (10?mg/kg/h, intravenously for 8.5?h) for 30?min before the LPS administration. Variable parameters of hemodynamics, biochemistry, hemostasis and inflammatory response were measured for 6?h after the LPS infusion. TEG variables (R-time, K-time, ?-angle, and maximal amplitude) were also measured. The pretreatment of LPS rats with gabexate mesilate significantly attenuated the lung, liver and kidney dysfunction, consumptive coagulopathy, the increases in serum tumor necrosis factor-?, interleukin-6, plasma thrombin-antithrombin complex and plasminogen activator inhibitor-1, and neutrophils infiltration score in lung, liver and kidney, compared with the LPS alone group. In addition, TEG parameters correlated with tissue and liver injury in the late phase of endotoxemia. In particular, a strong negative correlation between maximal amplitude at 4?h and Ln (lactate dehydrogenase) at 6?h after LPS infusion was noted (r?=?-0.752, P?
Related JoVE Video
Sensitization of Intracellular Salmonella enterica Serovar Typhimurium to Aminoglycosides In Vitro and In Vivo by a Host-Targeted Antimicrobial Agent.
Antimicrob. Agents Chemother.
PUBLISHED: 09-29-2014
Show Abstract
Hide Abstract
Aminoglycosides exhibit relatively poor activity against intracellular Salmonella enterica serovar Typhimurium due to their low permeativity across eukaryotic cell membranes. Previously, we identified the unique ability of AR-12, a celecoxib-derived small-molecule agent, to eradicate intracellular Salmonella Typhimurium in macrophages by facilitating autophagosome formation and suppressing Akt kinase signaling. In light of this unique mode of antibacterial action, we investigated the ability of AR-12 to sensitize intracellular Salmonella to aminoglycosides in macrophages and in an animal model. The antibacterial activities of AR-12 combined with various aminoglycosides, including streptomycin, kanamycin, gentamicin, and amikacin, against intracellular S. Typhimurium in murine RAW264.7 macrophages were assessed. Cells were infected with S. Typhimurium followed by treatment with AR-12 or individual aminoglycosides or with combinations for 24 h. The in vivo efficacies of AR-12, alone or in combination with gentamicin or amikacin, were also assessed by treating S. Typhimurium-infected BALB/c mice daily for 14 consecutive days. Exposure of S. Typhimurium-infected RAW264.7 cells to a combination of AR-12 with individual aminoglycosides led to a reduction in bacterial survival (P < 0.05), both intracellular and extracellular, that was greater than that seen with the aminoglycosides alone. This sensitizing effect, however, was not associated with increased aminoglycoside penetration into bacteria or macrophages. Moreover, daily intraperitoneal injection of AR-12 at 0.1 mg/kg of body weight significantly increased the in vivo efficacy of gentamicin and amikacin in prolonging the survival of S. Typhimurium-infected mice. These findings indicate that the unique ability of AR-12 to enhance the in vivo efficacy of aminoglycosides might have translational potential for efforts to develop novel strategies for the treatment of salmonellosis.
Related JoVE Video
Large-scale filament formation inhibits the activity of CTP synthetase.
Elife
PUBLISHED: 07-18-2014
Show Abstract
Hide Abstract
CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.
Related JoVE Video
Assessment of myocardial reactivity to controlled hypercapnia with free-breathing T2-prepared cardiac blood oxygen level-dependent MR imaging.
Radiology
PUBLISHED: 04-17-2014
Show Abstract
Hide Abstract
To examine whether controlled and tolerable levels of hypercapnia may be an alternative to adenosine, a routinely used coronary vasodilator, in healthy human subjects and animals.
Related JoVE Video
Determination of location, size, and transmurality of chronic myocardial infarction without exogenous contrast media by using cardiac magnetic resonance imaging at 3 T.
Circ Cardiovasc Imaging
PUBLISHED: 03-28-2014
Show Abstract
Hide Abstract
Late-gadolinium-enhanced (LGE) cardiac MRI (CMR) is a powerful method for characterizing myocardial infarction (MI), but the requisite gadolinium infusion is estimated to be contraindicated in ?20% of patients with MI because of end-stage chronic kidney disease. The purpose of this study is to investigate whether T1 CMR obtained without contrast agents at 3 T could be an alternative to LGE CMR for characterizing chronic MIs using a canine model of MI.
Related JoVE Video
Drug-induced conformational population shifts in topoisomerase-DNA ternary complexes.
Molecules
PUBLISHED: 03-07-2014
Show Abstract
Hide Abstract
Type II topoisomerases (TOP2) are enzymes that resolve the topological problems during DNA replication and transcription by transiently cleaving both strands and forming a cleavage complex with the DNA. Several prominent anti-cancer agents inhibit TOP2 by stabilizing the cleavage complex and engendering permanent DNA breakage. To discriminate drug binding modes in TOP2-? and TOP2-?, we applied our newly developed scoring function, dubbed AutoDock4RAP, to evaluate the binding modes of VP-16, m-AMSA, and mitoxantrone to the cleavage complexes. Docking reproduced crystallographic binding mode of VP-16 in a ternary complex of TOP2-? with root-mean-square deviation of 0.65 Å. Molecular dynamics simulation of the complex confirmed the crystallographic binding mode of VP-16 and the conformation of the residue R503. Drug-related conformational changes in R503 have been observed in ternary complexes with m-AMSA and mitoxantrone. However, the R503 rotamers in these two simulations deviate from their crystallographic conformations, indicating a relaxation dynamics from the conformations determined with the drug replacement procedure. The binding mode of VP-16 in the cleavage complex of TOP2-? was determined by the conjoint use of docking and molecular dynamics simulations, which fell within a similar binding pocket of TOP2-? cleavage complex. Our findings may facilitate more efficient design efforts targeting TOP2-? specific drugs.
Related JoVE Video
TERT promoter mutation in resectable hepatocellular carcinomas: a strong association with hepatitis C infection and absence of hepatitis B infection.
Int J Surg
PUBLISHED: 02-07-2014
Show Abstract
Hide Abstract
Mutation in the core promoter of the telomerase reverse transcriptase (TERT) gene was determined to be a frequent event in malignant melanoma and other cancers. However, the role of TERT promoter mutation in hepatocellular carcinomas (HCCs) remains largely unknown.
Related JoVE Video
AID downregulation is a novel function of the DNMT inhibitor 5-aza-deoxycytidine.
Oncotarget
PUBLISHED: 01-25-2014
Show Abstract
Hide Abstract
Activation-induced cytidine deaminase (AID) was originally identified as an inducer of somatic hypermutation (SHM) and class switch recombination (CSR) in immunoglobulin genes. However, AID can also cause mutations in host genes and contribute to cancer progression and drug resistance. In this study, molecular docking showed the interaction of free 5-aza-CdR and Zebularine (Zeb) with AID. However, only 5-aza-CdR-incorporated ssDNA bound to the active site of AID and inhibited AID expression through proteasomal degradation. 5-aza-CdR demonstrated cytotoxicity against AID-positive and -negative hematopoietic cancer cells. In contrast, Zeb exhibited a cytotoxic effect only in AID-negative cells due to its inability to inhibit AID expression. This differential effect might be due to the DNMT1 stabilization induced by AID, thus restricting the ability of Zeb to deplete DNMT1 and induce tumor suppressor genes (TSGs), such as p21, in AID-positive cells. Moreover, the in vivo anticancer effect of 5-aza-CdR but not Zeb in AID-positive hematopoietic cancer cells was demonstrated. The study not only displays the association of AID and DNMT1 and identifies a novel biological function of AID, but also provides novel information regarding the use of DNMT inhibitors to treat AID-positive hematopoietic cancers.
Related JoVE Video
Morphological subclassification of intrahepatic cholangiocarcinoma: etiological, clinicopathological, and molecular features.
Mod. Pathol.
PUBLISHED: 01-10-2014
Show Abstract
Hide Abstract
On the basis of morphological features, we subclassified 189 intrahepatic cholangiocarcinomas into two subtypes: bile duct and cholangiolar. The cholangiolar type is composed of cuboidal to low columnar tumor cells that contain scanty cytoplasm. The bile duct type is composed of tall columnar tumor cells arranged in a large glandular pattern. In this study, 77 (41%) tumors were classified as the cholangiolar type and 112 (59%) tumors were classified as the bile duct type. The cholangiolar-type intrahepatic cholangiocarcinoma was more frequently associated with viral hepatitis, whereas all but one intrahepatic cholangiocarcinoma associated with intrahepatic lithiasis were classified as the bile duct type. Biliary intraepithelial neoplasm or intraductal papillary neoplasm of the bile duct could be identified in 50 bile duct-type intrahepatic cholangiocarcinomas (45%), but in only 3 cholangiolar-type intrahepatic cholangiocarcinomas (4%). Cholangiolar-type intrahepatic cholangiocarcinomas frequently expressed N-cadherin, whereas bile duct intrahepatic cholangiocarcinomas were more likely to express S100P, Trefoil factor 1, and anterior gradient 2. KRAS is mutated in 23 of 98 (23%) bile duct-type intrahepatic cholangiocarcinomas and in only 1 of 76 (1%) cholangiolar-type intrahepatic cholangiocarcinomas. Cholangiolar-type intrahepatic cholangiocarcinomas had a higher frequency of IDH1 or 2 mutations than did the bile duct-type intrahepatic cholangiocarcinomas. The molecular features of the bile duct-type intrahepatic cholangiocarcinoma were similar to those of hilar cholangiocarcinoma. Patients with the cholangiolar-type intrahepatic cholangiocarcinoma had higher 5-year survival rates than those of patients with the bile duct-type intrahepatic cholangiocarcinoma. Our results indicated that intrahepatic cholangiocarcinoma was a heterogeneous tumor. Subclassification of intrahepatic cholangiocarcinomas based on cholangiocytic differentiation divides them into two groups with different etiologies, clinical manifestations, and molecular pathogeneses.
Related JoVE Video
Homozygous truncating PTPRF mutation causes athelia.
Hum. Genet.
PUBLISHED: 01-08-2014
Show Abstract
Hide Abstract
Athelia is a very rare entity that is defined by the absence of the nipple-areola complex. It can affect either sex and is mostly part of syndromes including other congenital or ectodermal anomalies, such as limb-mammary syndrome, scalp-ear-nipple syndrome, or ectodermal dysplasias. Here, we report on three children from two branches of an extended consanguineous Israeli Arab family, a girl and two boys, who presented with a spectrum of nipple anomalies ranging from unilateral hypothelia to bilateral athelia but no other consistently associated anomalies except a characteristic eyebrow shape. Using homozygosity mapping after single nucleotide polymorphism (SNP) array genotyping and candidate gene sequencing we identified a homozygous frameshift mutation in PTPRF as the likely cause of nipple anomalies in this family. PTPRF encodes a receptor-type protein phosphatase that localizes to adherens junctions and may be involved in the regulation of epithelial cell-cell contacts, peptide growth factor signaling, and the canonical Wnt pathway. Together with previous reports on female mutant Ptprf mice, which have a lactation defect, and disruption of one allele of PTPRF by a balanced translocation in a woman with amastia, our results indicate a key role for PTPRF in the development of the nipple-areola region.
Related JoVE Video
Gas6/Axl pathway promotes tumor invasion through the transcriptional activation of Slug in hepatocellular carcinoma.
Carcinogenesis
PUBLISHED: 11-14-2013
Show Abstract
Hide Abstract
Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. Other than the sorafenib treatment, no effective systemic therapy has been available thus far. Most targets in molecularly targeted therapy for cancer are receptor tyrosine kinases (RTKs). Therefore, identifying activated RTKs in HCC is critical for developing new molecularly targeted therapies. Using a phospho-RTK array, we found that Axl is one of the most frequently activated RTKs in liver cancer cell lines. The knockdown of Axl by RNA interference significantly reduced cell migration and invasion in the HCC cell lines HA22T and Mahlavu. Stimulation of HCC cell lines by Axl ligand growth arrest-specific 6 (Gas6) enhanced cell migration and invasion. The Gas6/Axl pathway enhanced the expression of the epithelial-mesenchymal transition-inducing transcription factor Slug, which is essential for the invasion-promoting activity of Axl. Treating HCC cells with the Axl inhibitor bosutinib suppressed Slug expression and decreased the invasiveness of HCC cell lines. These findings indicate that Gas6/Axl regulates tumor invasion through the transcriptional activation of Slug.
Related JoVE Video
Inhibition of aldolase A blocks biogenesis of ATP and attenuates Japanese encephalitis virus production.
Biochem. Biophys. Res. Commun.
PUBLISHED: 11-12-2013
Show Abstract
Hide Abstract
Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication.
Related JoVE Video
Deletion of CPEB3 enhances hippocampus-dependent memory via increasing expressions of PSD95 and NMDA receptors.
J. Neurosci.
PUBLISHED: 10-25-2013
Show Abstract
Hide Abstract
Long-term memory requires activity-dependent synthesis of plasticity-related proteins (PRPs) to strengthen synaptic efficacy and consequently consolidate memory. Cytoplasmic polyadenylation element binding protein (CPEB)3 is a sequence-specific RNA-binding protein that regulates translation of several PRP RNAs in neurons. To understand whether CPEB3 plays a part in learning and memory, we generated CPEB3 knock-out (KO) mice and found that the null mice exhibited enhanced hippocampus-dependent, short-term fear memory in the contextual fear conditioning test and long-term spatial memory in the Morris water maze. The basal synaptic transmission of Schaffer collateral-CA1 neurons was normal but long-term depression evoked by paired-pulse low-frequency stimulation was modestly facilitated in the juvenile KO mice. Molecular and cellular characterizations revealed several molecules in regulating plasticity of glutamatergic synapses are translationally elevated in the CPEB3 KO neurons, including the scaffolding protein PSD95 and the NMDA receptors along with the known CPEB3 target, GluA1. Together, CPEB3 functions as a negative regulator to confine the strength of glutamatergic synapses by downregulating the expression of multiple PRPs and plays a role underlying certain forms of hippocampus-dependent memories.
Related JoVE Video
Carbon nanotube alignment driven rapid actuations.
Nanotechnology
PUBLISHED: 10-24-2013
Show Abstract
Hide Abstract
Suspended micro-beams made from aligned carbon nanotubes and parylene deflect reversibly in an ac field and the deflection rate is three orders of magnitude greater than those for existing devices. The direction of beam deflection is determined by the area moment of inertia and the actuation mechanism involves rapid accumulation of charges at tube surfaces, the creation of Coulomb repulsive forces between tubes, beam dilation and the formation of compressive stresses at beam ends. Tube alignment plays a crucial role in the first step as is verified by experimental data and calculation.
Related JoVE Video
Effect of intragastric balloon on gastric emptying time in humans for weight control.
Clin Nucl Med
PUBLISHED: 10-04-2013
Show Abstract
Hide Abstract
This study evaluated the effect of fluid-filled intragastric balloon treatment on the scintigraphic gastric emptying times in humans for weight control.
Related JoVE Video
Scoring functions for prediction of protein-ligand interactions.
Curr. Pharm. Des.
PUBLISHED: 09-06-2013
Show Abstract
Hide Abstract
The scoring functions for protein-ligand interactions plays central roles in computational drug design, virtual screening of chemical libraries for new lead identification, and prediction of possible binding targets of small chemical molecules. An ideal scoring function for protein-ligand interactions is expected to be able to recognize the native binding pose of a ligand on the protein surface among decoy poses, and to accurately predict the binding affinity (or binding free energy) so that the active molecules can be discriminated from the non-active ones. Due to the empirical nature of most, if not all, scoring functions for protein-ligand interactions, the general applicability of empirical scoring functions, especially to domains far outside training sets, is a major concern. In this review article, we will explore the foundations of different classes of scoring functions, their possible limitations, and their suitable application domains. We also provide assessments of several scoring functions on weakly-interacting protein-ligand complexes, which will be useful information in computational fragment-based drug design or virtual screening.
Related JoVE Video
Autocrine-derived epidermal growth factor receptor ligands contribute to recruitment of tumor-associated macrophage and growth of basal breast cancer cells in vivo.
Oncol. Res.
PUBLISHED: 07-25-2013
Show Abstract
Hide Abstract
Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-alpha, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-alpha in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-alpha increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-alpha increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-alpha on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-alpha were somewhat interchangeable in their effects on EGFR signaling; however, TGF-alpha had a greater effect in vitro and AREG had a greater effect in vivo.
Related JoVE Video
Design and synthesis of dual-action inhibitors targeting histone deacetylases and 3-hydroxy-3-methylglutaryl coenzyme A reductase for cancer treatment.
J. Med. Chem.
PUBLISHED: 04-22-2013
Show Abstract
Hide Abstract
A series of dual-action compounds were designed to target histone deacetylase (HDAC) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) by having a hydroxamate group essential for chelation with the zinc ion in the active site of HDAC and the key structural elements of statin for binding with both proteins. In our study, the statin hydroxamic acids prepared by a fused strategy are most promising in cancer treatments. These compounds showed potent inhibitory activities against HDACs and HMGR with IC50 values in the nanomolar range. These compounds also effectively reduced the HMGR activity as well as promoted the acetylations of histone and tubulin in cancer cells, but were not toxic to normal cells.
Related JoVE Video
The role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer cells.
Biochim. Biophys. Acta
PUBLISHED: 04-02-2013
Show Abstract
Hide Abstract
We have reported previously that LIM homeobox transcription factor 1? (LMX1A) is hypermethylated and functions as a metastasis suppressor in cervical cancer cells. However, the regulation of LMX1A in carcinogenesis has not been reported. We aim to clarify whether specificity protein 1 (Sp1) and enhancer of zeste homolog 2 (EZH2) are involved in the regulation of LMX1A in cervical cancer. First we characterized the LMX1A promoter and used overexpression, knockdown, and reporter assays to show that Sp1 increased LMX1A promoter activity. Next, we used site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Sp1-binding sites were important for Sp1-mediated activation of the LMX1A promoter. Chromatin immunoprecipitation data demonstrated that Sp1 could bind directly to the LMX1A promoter and activate endogenous LMX1A expression in cells pretreated with 5-aza-2-deoxycytidine (5-aza-dC). Knockdown of EZH2 decreased H3K27me3 histone modification but was insufficient to restore LMX1A expression. To explore the effect of EZH2 on the endogenous LMX1A promoter, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A (TSA) and then depleted the cells of drugs for 3days. H3K14ac was enriched at the LMX1A promoter in EZH2-knockdown cells and LMX1A mRNA was still expressed. Taken together, these data imply that Sp1 may activate LMX1A expression upon oncogenic stress during cervical cancer development. Moreover, suppression of EZH2 may delay resilencing of LMX1A after the removal of 5-aza-dC and TSA.
Related JoVE Video
Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons.
Acta Neuropathol.
PUBLISHED: 04-01-2013
Show Abstract
Hide Abstract
The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had >1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization, two iPSC lines from each subject were selected, differentiated into postmitotic neurons, and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs, iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover, repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism.
Related JoVE Video
The nipple: a simple intersection of mammary gland and integument, but focal point of organ function.
J Mammary Gland Biol Neoplasia
PUBLISHED: 03-09-2013
Show Abstract
Hide Abstract
Having glands that secrete milk to nourish neonatal offspring characterizes all mammals. We provide a brief overview of the development and anatomy of nipples and mammary glands in monotremes, marsupials, and marine mammals, and focus on the nipples and mammary glands in terrestrial eutherian species. We first classify eutherians into three groups: the altricial, precocial, and arboreal types based on their rearing system. We then summarize the physiology of lactation and the cell biology of nipples with specific focus on comparing these in the mouse, cow, and human, which represent the three different groups. Finally we propose that the nipple is an example of specialized epidermis. As specialized epidermis, it is dependent the underlying stroma for development and maintenance in adult life. The development of the nipple and signaling pathways that regulate its formation are described.
Related JoVE Video
Analysis of androgen receptor and anti-Müllerian hormone pathways in human granulosa cells under luteinizing hormone treatment.
Reprod. Biol. Endocrinol.
PUBLISHED: 02-13-2013
Show Abstract
Hide Abstract
The objective of this study was to determine the gene expression profiles of the androgen/androgen receptor (AR) and anti-Müllerian hormone (AMH)/ Sry-related high-mobility group box 9 (SOX9) pathways in granulosa-luteal cells from patients undergoing standard in vitro fertilization (IVF) with or without recombinant luteinizing hormone (rLH) therapy.
Related JoVE Video
The use of oral rehydration salt in managing children under 5 y old with diarrhea in the Gambia: knowledge, attitude, and practice.
Nutrition
PUBLISHED: 01-15-2013
Show Abstract
Hide Abstract
Diarrhea is a leading cause of mortality in children under the age of 5 y in developing countries. To our knowledge, no other studies have investigated the management of diarrhea in the Gambia. The aim of this study was to assess maternal knowledge, attitude, and practice in the causes, prevention, and management of diarrhea in children under the age of 5 y in the Gambia.
Related JoVE Video
All-trans retinoic acid in combination with primaquine clears pneumocystis infection.
PLoS ONE
PUBLISHED: 01-04-2013
Show Abstract
Hide Abstract
Pneumocystis pneumonia (PcP) develops in immunocompromised patients. Alveolar macrophages play a key role in the recognition, phagocytosis, and degradation of Pneumocystis, but their number is decreased in PcP. Our study of various inflammatory components during PcP found that myeloid-derived suppressor cells (MDSCs) accumulate in the lungs of mice and rats with Pneumocystis pneumonia (PcP). We hypothesized that treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, may effectively control Pneumocystis (Pc) infection by inducing MDSCs to differentiate to AMs. In rodent models of PcP, we found that 5 weeks of ATRA treatment reduced the number of MDSCs in the lungs and increased the number of AMs which cleared Pc infection. We also found that ATRA in combination with primaquine was as effective as the combination of trimethoprim and sulfamethaxazole for treatment of PcP and completely eliminated MDSCs and Pc organisms in the lungs in two weeks. No relapse of PcP was seen after three weeks of the ATRA-primaquine combination treatment. Prolonged survival of Pc-infected animals was also achieved by this regimen. This is the very first successful development of a therapeutic regimen for PcP that combines an immune modulator with an antibiotic, enabling the hosts to effectively defend the infection. Results of our study may serve as a model for development of novel therapies for other infections with MDSC accumulation.
Related JoVE Video
CPEB4 Knockout Mice Exhibit Normal Hippocampus-Related Synaptic Plasticity and Memory.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Regulated RNA translation is critical to provide proteins needed to maintain persistent modification of synaptic strength, which underlies the molecular basis of long-term memory (LTM). Cytoplasmic polyadenylation element-binding proteins (CPEBs) are sequence-specific RNA-binding proteins and regulate translation in various tissues. All four CPEBs in vertebrates are expressed in the brain, including the hippocampal neurons, suggesting their potential roles in translation-dependent plasticity and memory. Although CPEB1 and CPEB3 have been shown to control specific kinds of hippocampus-related LTM, the role of CPEB2 and CPEB4 in learning and memory remains elusive. Thus, we generated CPEB4 knockout (KO) mice and analyzed them using several behavioral tests. No difference was found in the anxiety level, motor coordination, hippocampus-dependent learning and memory between the KO mice and their wild-type (WT) littermates. Electrophysiological recordings of multiple forms of synaptic plasticity in the Schaffer collateral pathway-CA1 neurons also showed normal responses in the KO hippocampal slices. Morphological analyses revealed that the CPEB4-lacking pyramidal neurons possessed slightly elongated dendritic spines. Unlike its related family members, CPEB1 and CPEB3, CPEB4 seems to be dispensable for hippocampus-dependent plasticity, learning and memory.
Related JoVE Video
Up-regulation of SOX9 in sertoli cells from testiculopathic patients accounts for increasing anti-mullerian hormone expression via impaired androgen receptor signaling.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS) and androgen insensitivity syndrome (AIS), high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling.
Related JoVE Video
Iron deposition following chronic myocardial infarction as a substrate for cardiac electrical anomalies: initial findings in a canine model.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Iron deposition has been shown to occur following myocardial infarction (MI). We investigated whether such focal iron deposition within chronic MI lead to electrical anomalies.
Related JoVE Video
A fast method for computing the centroid of a type-2 fuzzy set.
IEEE Trans Syst Man Cybern B Cybern
PUBLISHED: 12-14-2011
Show Abstract
Hide Abstract
Type reduction does the work of computing the centroid of a type-2 fuzzy set. The result is a type-1 fuzzy set from which a corresponding crisp number can then be obtained through defuzzification. Type reduction is one of the major operations involved in type-2 fuzzy inference. Therefore, making type reduction efficient is a significant task in the application of type-2 fuzzy systems. Liu introduced a horizontal slice representation, called the ?-plane representation, and proposed a type-reduction method for a type-2 fuzzy set. By exploring some useful properties of the ?-plane representation and of the type reduction for interval type-2 fuzzy sets, a fast method is developed for computing the centroid of a type-2 fuzzy set. The number of computations and comparisons involved is greatly reduced. Convergence in each iteration can then speed up, and type reduction can be done much more efficiently. The effectiveness of the proposed method is analyzed mathematically and demonstrated by experimental results.
Related JoVE Video
Activation of orexin 1 receptors in the periaqueductal gray of male rats leads to antinociception via retrograde endocannabinoid (2-arachidonoylglycerol)-induced disinhibition.
J. Neurosci.
PUBLISHED: 10-14-2011
Show Abstract
Hide Abstract
Orexin A and B are hypothalamic peptides known to modulate arousal, feeding, and reward via OX1 and OX2 receptors. Orexins are also antinociceptive in the brain, but their mechanism(s) of action remain unclear. Here, we investigated the antinociceptive mechanism of orexin A in the rat ventrolateral periaqueductal gray (vlPAG), a midbrain region crucial for initiating descending pain inhibition. In vlPAG slices, orexin A (30-300 nm) depressed GABAergic evoked IPSCs. This effect was blocked by an OX1 [1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB 334867)], but not OX2 [N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (compound 29)], antagonist. Orexin A increased the paired-pulse ratio of paired IPSCs and decreased the frequency, but not amplitude, of miniature IPSCs. Orexin A-induced IPSC depression was mimicked by (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a cannabinoid 1 (CB1) receptor agonist. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM 251), a CB1 antagonist, reversed depressant effects by both agonists. Orexin A-induced IPSC depression was prevented by 1-[6-[[(17?)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and tetrahydrolipstatin, inhibitors of phospholipase C (PLC) and diacylglycerol lipase (DAGL), respectively, and enhanced by cyclohexyl[1,1-biphenyl]-3-ylcarbamate (URB602), which inhibits enzymatic degradation of 2-arachidonoylglycerol (2-AG). Moderate DAGL?, but not DAGL?, immunoreactivity was observed in the vlPAG. Orexin A produced an overall excitatory effect on evoked postsynaptic potentials and hence increased vlPAG neuronal activity. Intra-vlPAG microinjection of orexin A reduced hot-plate nociceptive responses in rats in a manner blocked by SB 334867 and AM 251. Therefore, orexin A may produce antinociception by activating postsynaptic OX1 receptors, stimulating synthesis of 2-AG, an endocannabinoid, through a Gq-protein-mediated PLC-DAGL? enzymatic cascade culminating in retrograde inhibition of GABA release (disinhibition) in the vlPAG.
Related JoVE Video
Robust scoring functions for protein-ligand interactions with quantum chemical charge models.
J Chem Inf Model
PUBLISHED: 10-07-2011
Show Abstract
Hide Abstract
Ordinary least-squares (OLS) regression has been used widely for constructing the scoring functions for protein-ligand interactions. However, OLS is very sensitive to the existence of outliers, and models constructed using it are easily affected by the outliers or even the choice of the data set. On the other hand, determination of atomic charges is regarded as of central importance, because the electrostatic interaction is known to be a key contributing factor for biomolecular association. In the development of the AutoDock4 scoring function, only OLS was conducted, and the simple Gasteiger method was adopted. It is therefore of considerable interest to see whether more rigorous charge models could improve the statistical performance of the AutoDock4 scoring function. In this study, we have employed two well-established quantum chemical approaches, namely the restrained electrostatic potential (RESP) and the Austin-model 1-bond charge correction (AM1-BCC) methods, to obtain atomic partial charges, and we have compared how different charge models affect the performance of AutoDock4 scoring functions. In combination with robust regression analysis and outlier exclusion, our new protein-ligand free energy regression model with AM1-BCC charges for ligands and Amber99SB charges for proteins achieve lowest root-mean-squared error of 1.637 kcal/mol for the training set of 147 complexes and 2.176 kcal/mol for the external test set of 1427 complexes. The assessment for binding pose prediction with the 100 external decoy sets indicates very high success rate of 87% with the criteria of predicted root-mean-squared deviation of less than 2 Å. The success rates and statistical performance of our robust scoring functions are only weakly class-dependent (hydrophobic, hydrophilic, or mixed).
Related JoVE Video
Accommodating protein flexibility for structure-based drug design.
Curr Top Med Chem
PUBLISHED: 06-30-2011
Show Abstract
Hide Abstract
Proper incorporation of protein flexibility for prediction of binding poses and affinities of small compounds has attracted increasing attention recently in computational drug design. Various approaches have been proposed to accommodate protein flexibility in the prediction of binding modes and the binding free energy of ligands in an efficient manner. In this review, the significance of incorporating protein flexibility is discussed from the structural biophysical point of view, and then various approaches of generating protein conformation ensembles, as well as their successes and limitations, are introduced and compared. Special emphasis is on how to generate a proper ensemble of conformation for a specific purpose, as well as the computational efficiency of various approaches. Different searching algorithms for the prediction of optimal binding poses of ligands, which are the core engines of docking programs, are accounted for. Scoring functions for evaluation of protein-ligand complexes are compared. Two end-point methods of free energy calculation, Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) and the Linear Interaction Energy (LIE) method, are briefly reviewed. Finally, we also provide an example for the extension of the conventional protein-ligand docking algorithm for prediction of multiple binding sites and ligand translocation pathways.
Related JoVE Video
Naturally transmitted segmented filamentous bacteria segregate with diabetes protection in nonobese diabetic mice.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-27-2011
Show Abstract
Hide Abstract
Vertebrates typically harbor a rich gastrointestinal microbiota, which has coevolved with the host over millennia and is essential for several host physiological functions, in particular maturation of the immune system. Recent studies have highlighted the importance of a single bacterial species, segmented filamentous bacteria (SFB), in inducing a robust T-helper cell type 17 (Th17) population in the small-intestinal lamina propria (SI-LP) of the mouse gut. Consequently, SFB can promote IL-17-dependent immune and autoimmune responses, gut-associated as well as systemic, including inflammatory arthritis and experimental autoimmune encephalomyelitis. Here, we exploit the incomplete penetrance of SFB colonization of NOD mice in our animal facility to explore its impact on the incidence and course of type 1 diabetes in this prototypical, spontaneous model. There was a strong cosegregation of SFB positivity and diabetes protection in females, but not in males, which remained relatively disease-free regardless of the SFB status. In contrast, insulitis did not depend on SFB colonization. SFB-positive, but not SFB-negative, females had a substantial population of Th17 cells in the SI-LP, which was the only significant, repeatable difference in the examined T-cell compartments of the gut, pancreas, or systemic lymphoid tissues. Th17-signature transcripts dominated the very limited SFB-induced molecular changes detected in SI-LP CD4(+) T cells. Thus, a single bacterium, and the gut immune system alterations associated with it, can either promote or protect from autoimmunity in predisposed mouse models, probably reflecting their variable dependence on different Th subsets.
Related JoVE Video
Characteristics transformation of humic acid during ozonation and biofiltration treatment processes.
Water Environ. Res.
PUBLISHED: 06-11-2011
Show Abstract
Hide Abstract
Characteristics transformation of humic acid extracted from natural water during the ozonation and biofiltration treatment processes was investigated based on the analyses of total organic carbon, UV absorbance, infrared spectroscopy, and gel permeation chromatography. With ozone doses of 1, 4, and 7 mg/L, high-molecular-weight humic acid was cleaved into low-molecular-weight particles and accumulated at group 6 (molecular weight < or = 4000 g/mole) on the Sephadex G-75 (Pharmacia Fine Chemicals, Uppsala, Sweden) fraction. Furthermore, the molecular-size distribution of organic compounds by Sephadex G-25 was shifted from groups 2 (molecular weight = 4000 to 400 g/mole) and 3 (molecular weight = 400 to 180 g/mole) to groups 2, 3, and 4 (molecular weight < or = 180 g/mole) under ozone doses of 1 and 4 mg/L. An increased ozone dose destroyed functional groups C=C and C-O of aromatic and phenolic compounds and increased UV-insensitive biodegradable organic carbon for subsequent biofiltration.
Related JoVE Video
Improvement of porphyrins for G-quadruplex DNA targeting.
Biochimie
PUBLISHED: 06-07-2011
Show Abstract
Hide Abstract
G-quadruplex nucleic acids are emerging as therapeutic targets for small molecules referred to as small-molecule G-quadruplex ligands. The porphyrin H(2)-TMPyP4 was early reported to be a suitable motif for G-quadruplex DNA recognition. It probably binds to G-quadruplex nucleic acid through ?-? stacking with the external G-quartets. We explored chemical modifications of this porphyrin such as insertion of various metal ions in the centre of the aromatic core and addition of bulky substituents that may improve the specificity of the compound toward G-quadruplex DNA. Porphyrin metallation, affording a G4-ligand with two symmetric faces, allowed the conclusion that the presence of an axial water molecule perpendicular to the aromatic plane lowered but did not hamper ?-? stacking interactions between the aromatic parts of the ligand on the one hand and the external G-quartet on the other. The charge introduced in the centre of the porphyrin had little influence on binding. Thus, the ionic channel in the centre of G-quadruplex nucleic acids was not found to provide clear additional molecular clues for G-quadruplex nucleic acids targeting by porphyrins tested in the present study. Furthermore, we confirmed the unique G-quadruplex selectivity of a porphyrin modified with four bulky substituents at the meso positions and showed that although the compound is not "drug-like" it was capable of entering cells in culture and mediated some of the typical cellular effects of small-molecule G-quadruplex ligands.
Related JoVE Video
Patients and surgery-related factors that affect time to recovery of consciousness in adult patients undergoing elective cardiac surgery.
J Chin Med Assoc
PUBLISHED: 03-31-2011
Show Abstract
Hide Abstract
Central nervous system dysfunction is a serious complication following cardiac surgery. The prompt and predictable recovery of consciousness (ROC) from anesthesia is essential for neurological evaluations. This retrospective study aimed to determine the factors that were related to ROC time after elective cardiac surgery.
Related JoVE Video
Design and synthesis of novel dual-action compounds targeting the adenosine A(2A) receptor and adenosine transporter for neuroprotection.
ChemMedChem
PUBLISHED: 03-04-2011
Show Abstract
Hide Abstract
A novel compound, N?-(4-hydroxybenzyl)adenosine, isolated from Gastrodia elata and which has been shown to be a potential therapeutic agent for preventing and treating neurodegenerative disease, was found to target both the adenosine A(2A) receptor (A(2A) R) and the equilibrative nucleoside transporter 1 (ENT1). As A(2A) R and ENT1 are proximal in the synaptic crevice of striatum, where the mutant huntingtin aggregate is located, the dual-action compounds that concomitantly target these two membrane proteins may be beneficial for the therapy of Huntingtons disease. To design the desired dual-action compounds, pharmacophore models of the A(2A) R agonists and the ENT1 inhibitors were constructed. Accordingly, potentially active compounds were designed and synthesized by chemical modification of adenosine, particularly at the N? and C? positions, if the predicted activity was within an acceptable range. Indeed, some of the designed compounds exhibit significant dual-action properties toward both A(2A) R and ENT1. Both pharmacophore models exhibit good statistical correlation between predicted and measured activities. In agreement with competitive ligand binding assay results, these compounds also prevent apoptosis in serum-deprived PC12 cells, rendering a crucial function in neuroprotection and potential utility in the treatment of neurodegenerative diseases.
Related JoVE Video
Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.
Cell
PUBLISHED: 02-16-2011
Show Abstract
Hide Abstract
The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-? and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.
Related JoVE Video
N-?-acetyltransferase 10 protein suppresses cancer cell metastasis by binding PIX proteins and inhibiting Cdc42/Rac1 activity.
Cancer Cell
PUBLISHED: 02-03-2011
Show Abstract
Hide Abstract
N-?-acetyltransferase 10 protein, Naa10p, is an N-acetyltransferase known to be involved in cell cycle control. We found that Naa10p was expressed lower in varieties of malignancies with lymph node metastasis compared with non-lymph node metastasis. Higher Naa10p expression correlates the survival of lung cancer patients. Naa10p significantly suppressed migration, tumor growth, and metastasis independent of its enzymatic activity. Instead, Naa10p binds to the GIT-binding domain of PIX, thereby preventing the formation of the GIT-PIX-Paxillin complex, resulting in reduced intrinsic Cdc42/Rac1 activity and decreased cell migration. Forced expression of PIX in Naa10-transfected tumor cells restored the migration and metastasis ability. We suggest that Naa10p functions as a tumor metastasis suppressor by disrupting the migratory complex, PIX-GIT- Paxillin, in cancer cells.
Related JoVE Video
A new drug design targeting the adenosinergic system for Huntingtons disease.
PLoS ONE
PUBLISHED: 02-02-2011
Show Abstract
Hide Abstract
Huntingtons disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The expanded CAG repeats are translated into polyglutamine (polyQ), causing aberrant functions as well as aggregate formation of mutant Htt. Effective treatments for HD are yet to be developed.
Related JoVE Video
H3K9 histone methyltransferase G9a promotes lung cancer invasion and metastasis by silencing the cell adhesion molecule Ep-CAM.
Cancer Res.
PUBLISHED: 10-12-2010
Show Abstract
Hide Abstract
G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression.
Related JoVE Video
Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution.
Curr. Biol.
PUBLISHED: 10-11-2010
Show Abstract
Hide Abstract
Animal behavior is governed by the activity of interconnected brain circuits. Comprehensive brain wiring maps are thus needed in order to formulate hypotheses about information flow and also to guide genetic manipulations aimed at understanding how genes and circuits orchestrate complex behaviors.
Related JoVE Video
Silymarin protects spinal cord and cortical cells against oxidative stress and lipopolysaccharide stimulation.
Neurochem. Int.
PUBLISHED: 08-27-2010
Show Abstract
Hide Abstract
Contusive spinal cord injury (SCI) is a devastating event which leads to a loss of neurological function below the level of injury. A secondary degenerative process is initiated following acute SCI. This secondary cascade provides opportunities for the delivery of therapeutic interventions. Silymarin, a widely used "liver herb", is frequently used for the protection against various hepatobiliary problems. However, the effectiveness of silymarin in central nervous system (CNS), especially in spinal cord, is not firmly established. The present work evaluates the effects of silymarin and its major constituent, silybin, on oxidative stress and lipopolysaccharide (LPS) stimulation in primary neuronal/glial cell cultures and in vivo. Silymarin or silybin inhibited glial cell proliferation in a concentration-dependent manner. Furthermore, it protected glial cells against peroxide-induced reactive oxygen species (ROS) formation, ATP depletion, and cell damage. Interestingly, the inhibition of peroxide-induced ROS by silybin could be partially attenuated by inhibitors of NF?B or protein kinase C (PKC), suggesting an involvement of NF?B and PKC signaling pathways. In mixed neuronal/glial cell cultures from cerebral cortex or spinal cord, silymarin or silybin effectively attenuated peroxide-induced ROS formation, with silymarin being more effective than silybin, implicating other constituents of silymarin that may be involved. Consistently, silymarin reduced LPS-induced injures in spinal neuronal/glial cell cultures. In vivo, intrathecal administration of silymarin immediately after eliciting contusive SCI effectively improved hindlimb locomotor behavior in the rats. Taken together, silymarin or silybin shows promise in protecting the CNS cells from toxin- or injury-induced damages and might be used to treat head- or spinal cord-injuries related to free radical assault.
Related JoVE Video
Orexins/hypocretins: pain regulation and cellular actions.
Curr. Pharm. Des.
PUBLISHED: 07-09-2010
Show Abstract
Hide Abstract
Orexin A and B (also named hypocretin 1 and 2) are 33 and 28 amino acid-containing neuropeptides, respectively, derived from prepro-orexin (prepro-hypocretin) which is localized in the the lateral and perifonical areas of the hypothalamus. Two G-protein coupled receptor subtypes, OX1 and OX2, were identified. Orexin-containing fibers and OX receptors are widely distributed in the central nervous system. Orexins have been implicated in the arousal, rewarding, energy homeostasis, autonomic central control and antinociceptive systems. Subtype-selective peptide agonists and antagonists and non-peptide antagonists, but not non-peptide agonists, have been developed. This review summarizes the studies investigating the antinociceptive effects of orexins in various animal models of pain, including trigeminovascular pain, and their cellular mechanisms. Orexins are antinociceptive at both spinal and supraspinal levels. The antinociceptive effect of orexin A is comparable to opioids but orexin B is less or not effective. This effect is opioid-independent and mainly mediated through OX1 receptors. Some animal studies suggest that endogenous orexins may be released during postoperative and inflammatory, but not acute, pain states, or during some stress conditions, which may contribute to stress-induced analgesia. Purinergic P(2X) and glycine receptors are proposed to be involved in orexin-induced spinal antinociception. The supraspinal sites of actions might involve the posterior hypothalamus, which contributes to the trigeminovascular nociception, and the ventrolateral periaqueductal gray, which mediates descending pain inhibition. Endocannobinoids and nociceptin/orphanin FQ were found to interplay with orexins in nocicpetive processing. Further studies are required to elucidate the receptor subtype-specific mechanism(s) and clinical implications of orexin-induced antinociception.
Related JoVE Video
Effect of neuromuscular electrical stimulation in a patient with Sjogrens syndrome with dysphagia: a real time videofluoroscopic swallowing study.
Chang Gung Med J
PUBLISHED: 06-30-2010
Show Abstract
Hide Abstract
Severe dysphagia in a 54 year-old woman with Sjogrens syndrome with involvement of multiple cranial nerves significantly improved after treatment with neuromuscular electrical stimulation (NMES) in combination with a swallowing rehabilitation program. The swallowing response was assessed in real time using a videofluoroscope. Immediate improvement in the tongue retraction force, clearing of the valleculae, increase in laryngeal elevation and shortening of pharyngeal transit time were noted during stimulation. The patient returned to independent oral feeding after 46 sessions of NMES. After follow-up for 1 year, we found that the patient maintained adequate oral feeding and did not show signs of pulmonary complications.
Related JoVE Video
?-Actin is a downstream effector of the PI3K/AKT signaling pathway in myeloma cells.
Mol. Cell. Biochem.
PUBLISHED: 06-17-2010
Show Abstract
Hide Abstract
Interleukin 6 is the in vivo growth factor of myeloma cells. In response to IL-6 stimulation, the PI3K/AKT signaling pathway is activated in these cells. With comparative proteomic approaches, this study reveals many putative downstream effectors of the PI3K/AKT pathway. Mass spectrometry analysis of excised protein spots from 2-dimensional gel allowed the identification of proteins such as ?-Actin, cyclophilin A, E3 SUMO-protein ligase PIAS-NY protein, HSP 27, PML, and transforming growth factor ?-2. Among these putative effectors, ?-Actin was chosen for further characterization. Phosphorylation of ?-Actin by AKT upon IL-6 stimulation was confirmed by western blotting using a phospho-AKT substrate antibody. Interestingly, IL-6 significantly increased cell migration (P < 0.05) and the content of filamentous actin (P < 0.05). Therefore, IL-6 stimulation could have effects on the migration of myeloma cells, and the phosphorylation of ?-Actin is probably involved in the process.
Related JoVE Video
Effects of temperature and initial pH on biohydrogen production from food-processing wastewater using anaerobic mixed cultures.
Biodegradation
PUBLISHED: 04-11-2010
Show Abstract
Hide Abstract
This study attempted to determine the optimal temperature and initial cultivation pH by conducting a series of batch tests in stirred-tank bioreactor using fructose-producing wastewater as an organic substrate. The bioreactor temperature was controlled at 35-55°C with an initial pH of 4-8. Hydrogen production efficiency was assessed using specific hydrogen production potential (SHPP) and the maximum specific hydrogen production rate (SHPR(m)). Experimental results indicated that temperature and initial pH markedly affected SHPP and SHPR(m), volatile fatty acids distribution as well as the ratio of butyrate/acetate (BHu/HAc). Two-fold higher SHPP and SHPR(m) were obtained at thermophilic condition (55°C) than those at mesophilic condition (35°C). The optimal initial pH was 6 for hydrogen production with peak values of SHPP of 166.8 ml-H(2)/g-COD and SHPR(m) of 26.7 ml-H(2)/g-VSS-h for fructose-processing wastewater. Molasses-processing wastewater had a higher SHPP (187.0 ml-H(2)/g-COD) and SHPR(m) (42.7 ml-H(2)/gVSS-h) than fructose-processing wastewater at pH 6. The DGGE profiles indicated that molasses-processing wastewater is a better substrate than fructose-processing wastewater for growth of hydrogen-producing bacteria due to the high staining intensity of bands.
Related JoVE Video
Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells.
Immunity
PUBLISHED: 03-30-2010
Show Abstract
Hide Abstract
Commensal microbes can have a substantial impact on autoimmune disorders, but the underlying molecular and cellular mechanisms remain largely unexplored. We report that autoimmune arthritis was strongly attenuated in the K/BxN mouse model under germ-free (GF) conditions, accompanied by reductions in serum autoantibody titers, splenic autoantibody-secreting cells, germinal centers, and the splenic T helper 17 (Th17) cell population. Neutralization of interleukin-17 prevented arthritis development in specific-pathogen-free K/BxN mice resulting from a direct effect of this cytokine on B cells to inhibit germinal center formation. The systemic deficiencies of the GF animals reflected a loss of Th17 cells from the small intestinal lamina propria. Introduction of a single gut-residing species, segmented filamentous bacteria, into GF animals reinstated the lamina propria Th17 cell compartment and production of autoantibodies, and arthritis rapidly ensued. Thus, a single commensal microbe, via its ability to promote a specific Th cell subset, can drive an autoimmune disease.
Related JoVE Video
Molecular dynamics simulations of the rotary motor F(0) under external electric fields across the membrane.
Biophys. J.
PUBLISHED: 03-23-2010
Show Abstract
Hide Abstract
The membrane-bound component F(0), which is a major component of the F(0)F(1)-ATP synthase, works as a rotary motor and plays a central role in driving the F(1) component to transform chemiosmotic energy into ATP synthesis. We conducted molecular dynamics simulations of b(2)-free F(0) in a 1-palmitoyl-2-oleoyl-phosphatidylcholine lipid bilayer for tens of nanoseconds with two different protonation states of the cAsp-61 residue at the interface of the a-c complex in the absence of electric fields and under electric fields of +/-0.03 V/nm across the membrane. To our surprise, we observed that the upper half of the N-terminal helix of the c(1) subunit rotated about its axis clockwise by 30 degrees . An energetic analysis revealed that the electrostatic repulsion between this N-terminal helix and subunit c(12) was a major contributor to the observed rotation. A correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. The deuterium order parameter (S(CD)) profile calculated by averaging all the lipids in the F(0)-bound bilayer was not very different from that of the pure bilayer system, in agreement with recent (2)H solid-state NMR experiments. However, by delineating the lipid properties according to their vicinity to F(0), we found that the S(CD) profiles of different lipid shells were prominently different. Lipids close to F(0) formed a more ordered structure. Similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. The lipids in the proximity of F(0) exhibited very significantly reduced diffusional motion. The numerical value of S(CD) was anticorrelated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. Our findings will help elucidate the dynamics of F(0) depending on the protonation state and electric field, and may also shed some light on the interactions between the motor F(0) and its surrounding lipids under physiological conditions, which could help to rationalize its extraordinary energy conversion efficiency.
Related JoVE Video
Significance of the pH-induced conformational changes in the structure of C-reactive protein measured by dual polarization interferometry.
Biosens Bioelectron
PUBLISHED: 02-23-2010
Show Abstract
Hide Abstract
Emerging evidence indicates that the conformation of C-reactive protein (CRP) plays important roles in human inflammation and cardiovascular disease (CVD). The different conformations in the structure of CRP under different pH conditions remain an important issue to be investigated for explaining various functions of CRP under certain physiologic and pathologic conditions. We directly measured the pH-induced conformational changes in the structure of CRP by dual polarization interferometry (DPI). The CRP was attached to an aldehyde-functionalized DPI sensor chip at a concentration of 50 ?g/ml, and attained 2.019 ng/mm2 to form a surface coverage with a 1.71×10(-14) mol/mm2 CRP monolayer. A pentagonal structure with an average monolayer thickness value of 5.70±0.12nm and a layer density of 0.374±0.058 g/cm2 was obtained at pH 7.0. Moreover, the DPI biosensor signals directly reflected the considerable structural parameters and phenomena of conformational changes of CRP in a pH range of 2.0-10.0. The results obtained showed that the pentameric structure of CRP might dissociated into monomers or monomer aggregates as the pH shifts toward both acidic and alkaline conditions, but only partial rearrangements of CRP subunits might occur at extremely acidic physiological conditions. Considering the proinflammatory effect and subclinical chronic inflammation, pH-induced conformational changes in the structure of CRP between monomeric and pentameric formations may strongly relate to vascular atherosclerosis and subsequent CVD.
Related JoVE Video
Inhibition of histone deacetylase activity is a novel function of the antifolate drug methotrexate.
Biochem. Biophys. Res. Commun.
PUBLISHED: 12-08-2009
Show Abstract
Hide Abstract
Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used for treating human cancers, and overexpression of histone deacetylase (HDAC) is usually found in tumors. HDAC inhibitors (HDACi) can reactivate tumor suppressor genes and serve as potential anti-cancer drugs. In this study, we found that MTX shared structural similarity with some HDACi and molecular modeling showed that MTX indeed docks into the active site of HDLP, a bacterial homologue of HDAC. Subsequent in vitro assay demonstrated MTXs inhibition on HDAC activity in human cancer cells. The global acetylation of histone H3 was also induced by MTX. Moreover, MTX inhibited immunoprecipitated HDAC1/2 activity but not their protein levels. This study provides evidence that MTX inhibits HDAC activity.
Related JoVE Video
IL-17-producing T cells can augment autoantibody-induced arthritis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-02-2009
Show Abstract
Hide Abstract
Rheumatoid arthritis is a T lymphocyte-mediated disorder, but the precise nature of T cell involvement remains unclear. In the K/BxN mouse model of inflammatory arthritis, T cells initiate disease by providing help to B cells to produce arthritogenic autoantibodies. Here, we have characterized an additional, nonhumoral role for T cells in promoting autoantibody-induced arthritis. Autoreactive KRN T cells introduced either by direct transfer or bone marrow transplantation into B-cell-deficient hosts enhanced K/BxN serum-transferred arthritis, an effect that was dependent on expression of the cognate MHC-molecule/peptide complex. The T cell influence was dependent on interleukin (IL)-17; in contrast, standard serum-transferred arthritis, unenhanced by the addition of T cells, was unaffected by IL-17 neutralization. An IL-17-producing population of transferred KRN T cells was identified and found to be supported by the cotransfer of arthritogenic autoantibodies. IL-17-producing KRN T cells were enriched in inflamed joints of K/BxN mice, suggesting either selective recruitment or preferential differentiation. These results demonstrate the potential for autoreactive T cells to play two roles in the development of arthritis, both driving the production of pathogenic autoantibodies and bolstering the subsequent inflammatory cascade dependent on the innate immune system.
Related JoVE Video
Anesthetic management of a patient with congenital methemoglobinemia.
Acta Anaesthesiol Taiwan
PUBLISHED: 09-19-2009
Show Abstract
Hide Abstract
Methemoglobinemia occasionally causes cyanosis particularly in congenital methemoglobinemia. Avoidance of exposure to oxidizing agents is important for patients with congenital methemoglobinemia because of their deficient enzymatic pathways and decreased oxygen-carrying capacity. Here, we present a patient with preoperatively undiagnosed congenital methemoglobinemia who underwent uterine myomectomy under general anesthesia. The patient was a 35-year-old woman who displayed a low pulse oximetry reading of 91% prior to induction of anesthesia. Methemoglobinemia was first suspected intraoperatively because of a mismatch of SpO2 of finger pulse oximetry and SaO2 of arterial blood, and was later confirmed by multiple-wavelength CO-oximetry. The pathophysiology, etiology, clinical manifestations, anesthetic considerations, and treatment options of methemoglobinemia are discussed.
Related JoVE Video
CD72, a coreceptor with both positive and negative effects on B lymphocyte development and function.
J. Clin. Immunol.
PUBLISHED: 06-06-2009
Show Abstract
Hide Abstract
B lymphocytes remain in a resting state until activated by antigenic stimuli through interaction with the B cell receptor (BCR). Coreceptors on B cells can modulate the thresholds for signaling through the BCR for growth and differentiation. CD72 is a B cell coreceptor that has been shown to interact with CD100, a semaphorin, and to enhance BCR signaling.
Related JoVE Video
SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.
Nucleic Acids Res.
PUBLISHED: 05-11-2009
Show Abstract
Hide Abstract
Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design.
Related JoVE Video
Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs.
Nucleic Acids Res.
PUBLISHED: 05-06-2009
Show Abstract
Hide Abstract
We developed a simple, direct and cost-effective approach to search for the most likely target genes of a known microRNA (miRNA) in vitro. We term this method labeled miRNA pull-down (LAMP) assay system. Briefly, the pre-miRNA is labeled with digoxigenin (DIG), mixed with cell extracts and immunoprecipitated by anti-DIG antiserum. When the DIG-labeled miRNA and bound mRNA complex are obtained, the total cDNAs are then subcloned and sequenced, or RT-PCR-amplified, to search for the putative target genes of a known miRNA. After successfully identifying the known target genes of Caenorhabditis elegans miRNAs lin-4 and let-7 and zebrafish let-7, we applied LAMP to find the unknown target gene of zebrafish miR-1, which resulted in the identification of hand2. We then confirmed hand2 as a novel target gene of miR-1 by whole-mount in situ hybridization and luciferase reporter gene assay. We further validated this target gene by microarray analysis, and the results showed that hand2 is the top-scoring among 302 predicted putative target genes. We concluded that LAMP is an experimental approach for high-throughput identification of the target gene of known miRNAs from both C. elegans and zebrafish, yielding fewer false positive results than those produced by using only the bioinformatics approach.
Related JoVE Video
The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish.
Dev. Biol.
PUBLISHED: 04-15-2009
Show Abstract
Hide Abstract
Transcription factor Six1a plays important roles in morphogenesis, organogenesis, and cell differentiation. However, the role of Six1a during zebrafish cranial muscle development is still unclear. Here, we demonstrated that Six1a was required for sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscle development. Although Six1a was also necessary for myod and myogenin expression in head muscles, it did not affect myf5 expression in cranial muscles that originate from head mesoderm. Overexpression of myod enabled embryos to rescue all the defects in cranial muscles induced by injection of six1a-morpholino (MO), suggesting that myod is directly downstream of six1a in controlling craniofacial myogenesis. However, overexpression of six1a was unable to rescue arch muscle defects in the tbx1- and myf5-morphants, suggesting that six1a is only involved in myogenic maintenance, not its initiation, during arch muscle myogenesis. Although the craniofacial muscle defects caused by pax3-MO phenocopied those induced by six1a-MO, injection of six1a, myod or myf5 mRNA did not rescue the cranial muscle defects in pax3 morphants, suggesting that six1a and pax3 do not function in the same regulatory network. Therefore, we proposed four putative regulatory pathways to understand how six1a distinctly interacts with either myf5 or myod during zebrafish craniofacial muscle development.
Related JoVE Video
Effects of chloramphenicol, florfenicol, and thiamphenicol on growth of algae Chlorella pyrenoidosa, Isochrysis galbana, and Tetraselmis chui.
Ecotoxicol. Environ. Saf.
PUBLISHED: 03-24-2009
Show Abstract
Hide Abstract
This study investigated the growth inhibition effects of three phenicol antibiotics on microalgae used in aquaculture. Different dose levels of chloramphenicol (CAP), florfenicol (FF), and thiamphenicol (TAP) were added to cultures of one freshwater green alga, Chlorella pyrenoidosa, and two marine algae, Isochrysis galbana and Tetraselmis chui. For the two marine algae, FF showed higher toxicity levels (EC50, 1.3-8 mg l(-1)) than CAP (4-41 mg l(-1)) and TAP (38-158 mg l(-1)). CAP was more toxic to the freshwater algae (EC50, 14 mg l(-1)) than FF (215 mg l(-1)) and TAP (1283 mg l(-1)). TAP was the least toxic to the three algae, but maintained the highest stability during the test period. Among the tested algae, T. chui was the species most sensitive to the three antibiotics. This study demonstrates that all three phenicol antibiotics can inhibit growth of the three microalgae and should be carefully used in aquaculture.
Related JoVE Video
Human-specific histone methylation signatures at transcription start sites in prefrontal neurons.
PLoS Biol.
Show Abstract
Hide Abstract
Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5-1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans.
Related JoVE Video
Target prediction of small molecules with information of key molecular interactions.
Curr Top Med Chem
Show Abstract
Hide Abstract
The knowledge about to which biomolecules a small molecule binds is highly valuable in the drug development process. Although analytical methods to dissect ligand-binding proteome have made substantial progress in the past decades, it is generally too costly, if not infeasible, to know where a small molecule binds at very high resolution. Computational prediction of binding partners of small chemical molecules has become a useful approach to evaluate their potential therapeutic applications or adverse effects. In this article two computational approaches that were adopted to perform target identification, namely, molecular docking and pharmacophore fitting, are reviewed. Both approaches enable the identification of key interactions between the biomolecules and the small molecules. Databases that can be used to further improve the implementation and the computational methods and to benchmark their performances are also included.
Related JoVE Video
Dietary intake, glucose metabolism and sex hormones in women with polycystic ovary syndrome (PCOS) compared with women with non-PCOS-related infertility.
Br. J. Nutr.
Show Abstract
Hide Abstract
The present study investigated dietary intake, glucose metabolism and sex hormones in women with polycystic ovary syndrome (PCOS). A total of forty-five women (aged 25–40 years) with PCOS and 161 control women (aged 25–43 years) with non-PCOS-related infertility were recruited. Anthropometry, glucose tolerance and sex hormones were determined and dietary intake was assessed. Women with PCOS had lower serum sex hormone-binding globulin and increased BMI, waist:hip ratio, luteinising hormone, ratio of luteinising hormone: follicle-stimulating hormone, testosterone and free androgen index (FAI). Postprandial glucose, fasting insulin and insulin resistance were elevated in women with PCOS. Women with PCOS had reduced energy and carbohydrate intake but higher fat intake. Serum sex hormone-binding globulin level was negatively associated with BMI in both groups and negatively correlated with macronutrient intake in the PCOS group with hyperandrogenism. However, FAI was positively correlated with BMI, waist circumference and glucose metabolic parameters in both groups. Therefore, women with PCOS consume lower energy and carbohydrate compared with those with non-PCOS-related infertility and macronutrient intake is only negatively associated with serum sex hormone-binding globulin level in the PCOS group with hyperandrogenism.
Related JoVE Video
Dynamic instability--a common denominator in prokaryotic and eukaryotic DNA segregation and cell division.
Cell. Mol. Biol. Lett.
Show Abstract
Hide Abstract
Dynamic instability is an essential phenomenon in eukaryotic nuclear division and prokaryotic plasmid R1 segregation. Although the molecular machines used in both systems differ greatly in composition, strong similarities and requisite nuances in dynamics and segregation mechanisms are observed. This brief examination of the current literature provides a functional comparison between prokaryotic and eukaryotic dynamically unstable filaments, specifically ParM and microtubules. Additionally, this mini-review should support the notion that any dynamically unstable filament could serve as the molecular machine driving DNA segregation, but these machines possess auxiliary features to adapt to temporal and spatial disparities in either system.
Related JoVE Video
Agreement of cardiac output measurement between pulse contour analysis and thermodilution in various body positions: a porcine study.
J. Surg. Res.
Show Abstract
Hide Abstract
We elucidated the effects of various body positions on the agreement of cardiac output (CO) measurement between pulse contour analysis with the PiCCO monitor and thermodilution with pulmonary artery catheterization.
Related JoVE Video
Accumulation of myeloid-derived suppressor cells in the lungs during Pneumocystis pneumonia.
Infect. Immun.
Show Abstract
Hide Abstract
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of hematopoietic precursors with the ability to adversely affect host immunity. They have been shown to accumulate in pathological conditions, such as cancer and some microbial diseases. In the mouse and rat models of Pneumocystis pneumonia (PcP), we found a distinct population of cells with MDSC-like morphology in the bronchoalveolar lavage (BAL) fluid, constituting up to 50% of the total cells in BAL fluid. These cells were not seen in the BAL fluid from normal animals or from Pneumocystis-infected animals that had been successfully treated for PcP with a combination of trimethoprim and sulfamethoxazole. With flow cytometry, these cells were found to express the characteristic MDSC surface markers Gr-1 and CD11b in mice or CD11bc and His48 in rats. Using reverse transcription-PCR, we demonstrated that these cells produced high levels of arginase-1 and inducible nitric oxide synthase (iNOS) mRNA. These cells were shown to suppress CD4(+) T-cell proliferation in response to stimulation by anti-CD3 and anti-CD28 antibodies. Adoptive transfer of these cells to normal mice caused lung damage, as indicated by elevated levels of albumin and lactate dehydrogenase in the BAL fluid. These experiments provide evidence of the presence of MDSCs in the lungs during PcP. Further studies on the roles of MDSCs in PcP are warranted in order to develop treatment strategies which can reduce the number of MDSCs and the damage caused by these cells.
Related JoVE Video
Clerodendrum inerme Leaf Extract Alleviates Animal Behaviors, Hyperlocomotion, and Prepulse Inhibition Disruptions, Mimicking Tourette Syndrome and Schizophrenia.
Evid Based Complement Alternat Med
Show Abstract
Hide Abstract
Previously, we found a patient with intractable motor tic disorder, a spectrum of Tourette syndrome (TS), responsive to the ground leaf juice of Clerodendrum inerme (CI). Here, we examined the effect of the ethanol extract of CI leaves (CI extract) on animal behaviors mimicking TS, hyperlocomotion, and sensorimotor gating deficit. The latter is also observed in schizophrenic patients and can be reflected by a disruption of prepulse inhibition of acoustic startle response (PPI) in animal models induced by methamphetamine and NMDA channel blockers (ketamine or MK-801), based on hyperdopaminergic and hypoglutamatergic hypotheses, respectively. CI extract (10-300?mg/kg, i.p.) dose-dependently inhibited hyperlocomotion induced by methamphetamine (2?mg/kg, i.p.) and PPI disruptions induced by methamphetamine, ketamine (30?mg/kg, i.p.), and MK-801 (0.3?mg/kg, i.p.) but did not affect spontaneous locomotor activity, rotarod performance, and grip force. These results suggest that CI extract can relieve hyperlocomotion and improve sensorimotor gating deficit, supporting the therapeutic potential of CI for TS and schizophrenia.
Related JoVE Video
Relationship between the head deviation angle and hemineglect in patients with right hemisphere stroke.
Eur. Neurol.
Show Abstract
Hide Abstract
Patients with hemineglect have been reported to have abnormal head posture. We attempted to determine the extent to which the angles are correlated with the severity of hemineglect.
Related JoVE Video
SOX1 functions as a tumor suppressor by antagonizing the WNT/?-catenin signaling pathway in hepatocellular carcinoma.
Hepatology
Show Abstract
Hide Abstract
Oncogenic activation of the Wnt/?-catenin signaling pathway is common in hepatocellular carcinoma (HCC). Our recent studies have demonstrated that SRY (sex determining region Y)-box 1 (SOX1) and secreted frizzled-related proteins are concomitantly promoter-hypermethylated, and this might lead to abnormal activation of the Wnt signaling pathway in HCC. SOX1 encodes a transcription factor involved in the regulation of embryonic development and cell fate determination. However, the expression and functional role of SOX1 in HCC remains unclear. In this study, we confirmed via quantitative methylation-specific polymerase chain reaction that SOX1 was frequently downregulated through promoter hypermethylation in HCC cells and tissues. Overexpression of SOX1 by a constitutive or inducible approach could suppress cell proliferation, colony formation, and invasion ability in HCC cell lines, as well as tumor growth in nonobese diabetic/severe combined immunodeficiency mice. Conversely, knockdown of SOX1 by withdrawal of doxycycline could partially restore cell proliferation and colony formation in HCC cells. We used a T cell factor (TCF)-responsive luciferase reporter assay and western blot analysis to prove that SOX1 could regulate TCF-responsive transcriptional activity and inhibit the expression of Wnt downstream genes. Furthermore, we used glutathione S-transferase pull-down, co-immunoprecipitation, and confocal microscopy to demonstrate that SOX1 could interact with ?-catenin but not with the ?-catenin/TCF complex. Moreover, restoration of the expression of SOX1 induces significant cellular senescence in Hep3B cells.
Related JoVE Video
AutoBind: automatic extraction of protein-ligand-binding affinity data from biological literature.
Bioinformatics
Show Abstract
Hide Abstract
Determination of the binding affinity of a protein-ligand complex is important to quantitatively specify whether a particular small molecule will bind to the target protein. Besides, collection of comprehensive datasets for protein-ligand complexes and their corresponding binding affinities is crucial in developing accurate scoring functions for the prediction of the binding affinities of previously unknown protein-ligand complexes. In the past decades, several databases of protein-ligand-binding affinities have been created via visual extraction from literature. However, such approaches are time-consuming and most of these databases are updated only a few times per year. Hence, there is an immediate demand for an automatic extraction method with high precision for binding affinity collection.
Related JoVE Video
When cytokinin, a plant hormone, meets the adenosine A2A receptor: a novel neuroprotectant and lead for treating neurodegenerative disorders?
PLoS ONE
Show Abstract
Hide Abstract
It is well known that cytokinins are a class of phytohormones that promote cell division in plant roots and shoots. However, their targets, biological functions, and implications in mammalian systems have rarely been examined. In this study, we show that one cytokinin, zeatin riboside, can prevent pheochromocytoma (PC12) cells from serum deprivation-induced apoptosis by acting on the adenosine A(2A) receptor (A(2A)-R), which was blocked by an A(2A)-R antagonist and a protein kinase A (PKA) inhibitor, demonstrating the functional ability of zeatin riboside by mediating through A(2A)-R signaling event. Since the A(2A)-R was implicated as a therapeutic target in treating Huntingtons disease (HD), a cellular model of HD was applied by transfecting mutant huntingtin in PC12 cells. By using filter retardation assay and confocal microscopy we found that zeatin riboside reversed mutant huntingtin (Htt)-induced protein aggregations and proteasome deactivation through A(2A)-R signaling. PKA inhibitor blocked zeatin riboside-induced suppression of mutant Htt aggregations. In addition, PKA activated proteasome activity and reduced mutant Htt protein aggregations. However, a proteasome inhibitor blocked both zeatin riboside-and PKA activator-mediated suppression of mutant Htt aggregations, confirming mediation of the A(2A)-R/PKA/proteasome pathway. Taken together, zeatin riboside might have therapeutic potential as a novel neuroprotectant and a lead for treating neurodegenerative disorders.
Related JoVE Video
idTarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach.
Nucleic Acids Res.
Show Abstract
Hide Abstract
Identification of possible protein targets of small chemical molecules is an important step for unravelling their underlying causes of actions at the molecular level. To this end, we construct a web server, idTarget, which can predict possible binding targets of a small chemical molecule via a divide-and-conquer docking approach, in combination with our recently developed scoring functions based on robust regression analysis and quantum chemical charge models. Affinity profiles of the protein targets are used to provide the confidence levels of prediction. The divide-and-conquer docking approach uses adaptively constructed small overlapping grids to constrain the searching space, thereby achieving better docking efficiency. Unlike previous approaches that screen against a specific class of targets or a limited number of targets, idTarget screen against nearly all protein structures deposited in the Protein Data Bank (PDB). We show that idTarget is able to reproduce known off-targets of drugs or drug-like compounds, and the suggested new targets could be prioritized for further investigation. idTarget is freely available as a web-based server at http://idtarget.rcas.sinica.edu.tw.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

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.