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In JoVE (1)
Other Publications (20)
- Journal of Immunology (Baltimore, Md. : 1950)
- Hinyokika Kiyo. Acta Urologica Japonica
- Neurology
- Surgical Endoscopy
- Proceedings of the National Academy of Sciences of the United States of America
- The International Journal of Tuberculosis and Lung Disease : the Official Journal of the International Union Against Tuberculosis and Lung Disease
- The Journal of Pharmacology and Experimental Therapeutics
- Langmuir : the ACS Journal of Surfaces and Colloids
- Journal of Molecular and Cellular Cardiology
- Proceedings of the National Academy of Sciences of the United States of America
- International Journal of Toxicology
- The Journal of the Acoustical Society of America
- Assay and Drug Development Technologies
- Biomedical Optics Express
- Leukemia : Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
- Pediatric Research
- Journal of Immunology (Baltimore, Md. : 1950)
- Journal of Colloid and Interface Science
- Proceedings of the National Academy of Sciences of the United States of America
- Perfusion
Articles by Alfredo Quiñones-Hinojosa in JoVE
JoVE Neuroscience
Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants
1 Neuroscience Lab/ Fac. Psicologia, University of Colima, 2Department of Neurosurgery, Johns Hopkins University
In this article, we show a method to make glass capillary needles with a 50-μm lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.
Other articles by Alfredo Quiñones-Hinojosa on PubMed
The E3 Ubiquitin Ligase Cbl-b Regulates Expansion but Not Functional Activity of Self-reactive CD4 T Cells
Cbl-b is an E3 ubiquitin ligase that limits Ag responsiveness in T cells by targeting TCR-inducible signaling molecules. Cbl-b deficiency thus renders T cells hyperresponsive to antigenic stimulation and predisposes individuals toward developing autoimmunity. In part because Cbl-b(-/-) T cells do not require CD28 costimulation to become activated, and insufficient costimulation is a critical parameter that confers anergy induction over effector differentiation, it has been hypothesized that Cbl-b(-/-) T cells are resistant to anergy. This possibility has been supported in models in which anergy is normally induced in vitro, or in vivo following exposure to soluble Ag boluses. In the current study, we characterized the response of Cbl-b(-/-) CD4 T cells in an in vivo system in which anergy is normally induced by a constitutively expressed peripheral self-Ag. Cbl-b expression increased in self-Ag-induced anergic wild-type CD4 T cells, and Cbl-b(-/-) CD4 T cells underwent more robust proliferation and expansion upon initially encountering cognate self-Ag compared with wild-type counterparts. Nevertheless, both wild-type and Cbl-b(-/-) CD4 T cells ultimately developed the same impaired ability to respond to antigenic restimulation. The more extensive expansion that occurred during the initial induction of anergy did, however, allow the anergic CD4 T cells to expand to greater numbers when they were functionally resuscitated following replacement of the initial source of tolerizing self-Ag with a viral form of the same Ag.
[A Case of Primary Testicular Malignant Lymphoma Associated with Contralateral Testicular Recurrence After a Nine-year Interval: a Case Report]
A 69-year-old man visited our hospital in February 1996 with a chief complain of left scrotal swelling. The left scrotal content was hard by palpation and ultrasonography showed a hypo-echoic lesion. Left high orchiectomy was performed with suspicion of a testicular tumor. Pathological examination demonstrated a diffuse large B-cell lymphoma (DLBCL) originating from the left testis, and then he underwent 5 courses of chemotherapy consisting of THP-COP (THP-adriamycin, cyclophosphamide, vincristin, predonisone). Following the treatment for five years, he had no evidence of recurrence. Nine years later, in October 2005, he noticed right scrotal swelling. He underwent right high orchiectmy with suspicion of a contralateral testicular malignant lymphoma. Pathological examination revealed DLBCL. He underwent chemotherapy (rituximab-THP-COP) and achieved complete remission again. He is doing well without recurrence of disease for three years after the last treatment.
25-Hydroxyvitamin D, Dementia, and Cerebrovascular Pathology in Elders Receiving Home Services
Vitamin D deficiency has potential adverse effects on neurocognitive health and subcortical function. However, no studies have examined the association between vitamin D status, dementia, and cranial MRI indicators of cerebrovascular disease (CVD).
A Population-based Analysis of Emergent Vs. Elective Hospital Admissions for an Intrathoracic Stomach
Large-scale, population-based analyses of the demographics, management, and healthcare resource utilization of patients with an intrathoracic stomach are largely unknown, an issue which has become more important with the aging of the population. Our objective was to understand the magnitude of the problem and to assess clinical outcomes and hospital costs in elective and emergent admissions of patients with an intrathoracic stomach in a large population-based study.
Neuroprotection by Selective Allosteric Potentiators of the EP2 Prostaglandin Receptor
Activation of the Galphas-coupled EP2 receptor for prostaglandin E2 (PGE(2)) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 microM (i) potentiated the cAMP response to a low concentration of PGE(2) by > 50%; (ii) had no effect on EP4 or beta2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE(2) on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE(2) on EP2 receptors 4- to 5-fold at 10 to 20 microM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE(2) receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE(2) released in a cell-injury setting is neuroprotective.
Decrease in Risk of Tuberculosis Infection Despite Increase in Tuberculosis Among Young Adults in Urban Vietnam
To assess whether the increase in tuberculosis (TB) notification rates among young adults in Vietnam reflects increased transmission in the population at large.
Implementation of a Fluorescence-based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit As Subunit-selective N-methyl-D-aspartate Receptor Antagonists
N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.
Effect of UV-ozone Treatment on Poly(dimethylsiloxane) Membranes: Surface Characterization and Gas Separation Performance
A thin SiO(x) selective surface layer was formed on a series of cross-linked poly(dimethylsiloxane) (PDMS) membranes by exposure to ultraviolet light at room temperature in the presence of ozone. The conversion of the cross-linked polysiloxane to SiO(x) was monitored by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray (EDX) microanalysis, contact angle analysis, and atomic force microscopy (AFM). The conversion of the cross-linked polysiloxane to SiO(x) increased with UV-ozone exposure time and cross-linking agent content, and the surface possesses highest conversion. The formation of a SiO(x) layer increased surface roughness, but it decreased water contact angle. Gas permeation measurements on the UV-ozone exposure PDMS membranes documented interesting gas separation properties: the O(2) permeability of the cross-linked PDMS membrane before UV-ozone exposure was 777 barrer, and the O(2)/N(2) selectivity was 1.9; after UV-ozone exposure, the permeability decreased to 127 barrer while the selectivity increased to 5.4. The free volume depth profile of the SiO(x) layer was investigated by novel slow positron beam. The results show that free volume size increased with the depth, yet the degree of siloxane conversion to SiO(x) does not affect the amount of free volume.
S100A1 Gene Therapy for Heart Failure: a Novel Strategy on the Verge of Clinical Trials
Representing the common endpoint of various cardiovascular disorders, heart failure (HF) shows a dramatically growing prevalence. As currently available therapeutic strategies are not capable of terminating the progress of the disease, HF is still associated with a poor clinical prognosis. Among the underlying molecular mechanisms, the loss of cardiomyocyte Ca(2+) cycling integrity plays a key role in the pathophysiological development and progression of the disease. The cardiomyocyte EF-hand Ca(2+) sensor protein S100A1 emerged as a regulator both of sarcoplasmic reticulum (SR), sarcomere and mitochondrial function implicating a significant role in cardiac physiology and dysfunction. In this review, we aim to recapitulate the translation of S100A1-based investigation from first clinical observations over basic research experiments back to a near-clinical setting on the verge of clinical trials today. We also address needs for further developments towards "second-generation" gene therapy and discuss the therapeutic potential of S100A1 gene therapy for HF as a promising novel strategy for future cardiologists. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy".
Reversing Chemoresistance by Small Molecule Inhibition of the Translation Initiation Complex EIF4F
Deregulation of cap-dependent translation is associated with cancer initiation and progression. The rate-limiting step of protein synthesis is the loading of ribosomes onto mRNA templates stimulated by the heterotrimeric complex, eukaryotic initiation factor (eIF)4F. This step represents an attractive target for anticancer drug discovery because it resides at the nexus of the TOR signaling pathway. We have undertaken an ultra-high-throughput screen to identify inhibitors that prevent assembly of the eIF4F complex. One of the identified compounds blocks interaction between two subunits of eIF4F. As a consequence, cap-dependent translation is inhibited. This compound can reverse tumor chemoresistance in a genetically engineered lymphoma mouse model by sensitizing cells to the proapoptotic action of DNA damage. Molecular modeling experiments provide insight into the mechanism of action of this small molecule inhibitor. Our experiments validate targeting the eIF4F complex as a strategy for cancer therapy to modulate chemosensitivity.
Sodium Pyruvate Modulates Cell Death Pathways in HaCaT Keratinocytes Exposed to Half-mustard Gas
2-Chloroethyl ethyl sulfide (CEES) or half-mustard gas, a sulfur mustard (HD) analog, is a genotoxic agent that causes oxidative stress and induces both apoptotic and necrotic cell death. Sodium pyruvate induced a necrosis-to-apoptosis shift in HaCaT cells exposed to CEES levels ≤ 1.5 mmol/L and lowered markers of DNA damage, oxidative stress, and inflammation. This study provides a rationale for the future development of multicomponent therapies for HD toxicity in the skin. We hypothesize that a combination of pyruvates with scavengers/antioxidants encapsulated in liposomes for optimal local delivery should be therapeutically beneficial against HD-induced skin injury. However, the latter suggestion should be verified in animal models exposed to HD.
Classification of Echolocation Clicks from Odontocetes in the Southern California Bight
This study presents a system for classifying echolocation clicks of six species of odontocetes in the Southern California Bight: Visually confirmed bottlenose dolphins, short- and long-beaked common dolphins, Pacific white-sided dolphins, Risso's dolphins, and presumed Cuvier's beaked whales. Echolocation clicks are represented by cepstral feature vectors that are classified by Gaussian mixture models. A randomized cross-validation experiment is designed to provide conditions similar to those found in a field-deployed system. To prevent matched conditions from inappropriately lowering the error rate, echolocation clicks associated with a single sighting are never split across the training and test data. Sightings are randomly permuted before assignment to folds in the experiment. This allows different combinations of the training and test data to be used while keeping data from each sighting entirely in the training or test set. The system achieves a mean error rate of 22% across 100 randomized three-fold cross-validation experiments. Four of the six species had mean error rates lower than the overall mean, with the presumed Cuvier's beaked whale clicks showing the best performance (<2% error rate). Long-beaked common and bottlenose dolphins proved the most difficult to classify, with mean error rates of 53% and 68%, respectively.
A Dual-readout F2 Assay That Combines Fluorescence Resonance Energy Transfer and Fluorescence Polarization for Monitoring Bimolecular Interactions
Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.
Gold Nanorods for Intravital Vascular Imaging of Preneoplastic Oral Mucosa
We explore the feasibility of using gold nanorods with efficient two-photon luminescence properties as contrast agents for intravital imaging of neoplasia. This investigation spanned ex vivo characterization in cells/tissue to in vivo implementation in an oral carcinogenesis model. GNRs were >40 times brighter than surrounding tissue. Intravital imaging revealed 3D microvasculature, and in dysplasia, abnormal vessels (dense and tortuous) compared to normal. GNRs were diffusely distributed in lesions after 24 hours. No known previous study has revealed abnormal vessel structure in dysplasia by imaging. Results suggest GNRs can function as high-contrast agents for in vivo visualization of carcinogenesis features.
Low Expression of PP2A Regulatory Subunit B55α is Associated with T308 Phosphorylation of AKT and Shorter Complete Remission Duration in Acute Myeloid Leukemia Patients
The regulation of protein kinase B (AKT) is a dynamic process that depends on the balance between phosphorylation by upstream kinases for activation and inactivation by dephosphorylation by protein phosphatases. Phosphorylated AKT is commonly found in acute myeloid leukemia (AML) and confers an unfavorable prognosis. Understanding the relative importance of upstream kinases and AKT phosphatase in the activation of AKT is relevant for the therapeutic targeting of this signaling axis in AML. The B55α subunit of protein phosphatase 2A (PP2A) has been implicated in AKT dephosphorylation, but its role in regulating AKT in AML is unknown. We examined B55α protein expression in blast cells derived from 511 AML patients using reverse phase protein analysis. B55α protein expression was lower in AML cells compared with normal CD34+ cells. B55α protein levels negatively correlated with threonine 308 phosphorylation levels. Low levels of B55α were associated with shorter complete remission duration, demonstrating that decreased expression is an adverse prognostic factor in AML. These findings suggest that decreased B55α expression in AML is at least partially responsible for increased AKT signaling in AML and suggests that therapeutic targeting of PP2A could counteract this.
Characterization of Neural Breathing Pattern in Spontaneously Breathing Preterm Infants
The aim was to characterize the neural breathing pattern in nonintubated preterm infants. The diaphragm electrical activity (EAdi) and heart rate were simultaneously measured repeatedly for 1 h over several days using a modified feeding tube equipped with miniaturized sensors. The EAdi waveform was quantified for phasic and tonic activity, neural timings, and prevalence of recurring patterns, including central apnea. Ten infants with mean age 7 d (range, 3-13 d) were studied. Their birth weight was 1512 g (1158-1800 g) and GA at birth 31 wk (28-36 wk). Neural inspiratory and expiratory times were 278 ms (195-450 ms) and 867 ms (668-1436 ms) and correlated with GA (p < 0.001). Tonic EAdi represented 29.5% of phasic EAdi (16-40%) and was related to GA (r = 0.61, p < 0.001). For the group, 68% of the time was regular phasic breathing (without tonic activity) and 29% of the time with elevated tonic activity. Central apneas >5 s occurred on average 10 times per hour (2-29). Heart rate reductions were correlated to central apnea duration. In conclusion, esophageal recordings of the EAdi waveform demonstrate that neural breathing pattern is variable, with regards to timing, amplitude, and pattern with a distinct amount of tonic diaphragm activity.
CD134 Plus CD137 Dual Costimulation Induces Eomesodermin in CD4 T Cells to Program Cytotoxic Th1 Differentiation
Cytotoxic CD4 Th1 cells are emerging as a therapeutically useful T cell lineage that can effectively target tumors, but until now the pathways that govern their differentiation have been poorly understood. We demonstrate that CD134 (OX40) costimulation programs naive self- and virus-reactive CD4 T cells to undergo in vivo differentiation into cytotoxic Th1 effectors. CD137 (4-1BB) costimulation maximized clonal expansion, and IL-2 was necessary for cytotoxic Th1 differentiation. Importantly, the T-box transcription factor Eomesodermin was critical for inducing the cytotoxic marker granzyme B. CD134 plus CD137 dual costimulation also imprinted a cytotoxic phenotype on bystanding CD4 T cells. Thus, to our knowledge, the current study identifies for the first time a specific costimulatory pathway and an intracellular mechanism relying on Eomesodermin that induces both Ag-specific and bystander cytotoxic CD4 Th1 cells. This mechanism might be therapeutically useful because CD134 plus CD137 dual costimulation induced CD4 T cell-dependent tumoricidal function in a mouse melanoma model.
Using Colloid Lithography to Fabricate Silicon Nanopillar Arrays on Silicon Substrates
In this study, we partially grafted geminal silanol groups in the protecting organic shells on the surfaces of gold nanoparticles (AuNPs) and then assembled the alkyl-AuNP-Si(OH)(4) particles onto the surfaces of silicon (Si) wafers. The density of assembled AuNPs on the Si surface was adjusted by varying the geminal silanol group content on the AuNP surface; at its optimal content, it approached the high assembly density (0.0254 particles/nm(2)) of an AuNP assembled monolayer. Using reactive-ion etching (RIE) with the templates as masks, we transferred the patterned AuNP assemblies to form large-area, size-tunable, Si nanopillar arrays, the assembly density of which was controlled by the dimensions of the AuNPs. Using this colloidal lithography (CL) process, we could generate Si nanopillars having sub-10-nm diameters and high aspect ratios. The water contact angles of the high-aspect-ratio Si nanopillars approached 150°. We used another fabrication process, involving electron beam lithography and oxygen plasma treatment, to generate hydrophilic 200-nm-resolution line patterns on a Si surface to assemble the AuNPs into 200-nm-resolution dense lines for use as an etching mask. Subsequent CL provided a patterned Si nanopillar array having a feature size of 200 nm on the Si surface. Using this approach, it was possible to pattern sub-10-nm Si nanopillar arrays having densities as high as 0.0232 nm(-2).
Small Molecule Antagonist Reveals Seizure-induced Mediation of Neuronal Injury by Prostaglandin E2 Receptor Subtype EP2
With interest waning in the use of cyclooxygenase-2 (COX-2) inhibitors for inflammatory disease, prostaglandin receptors provide alternative targets for the treatment of COX-2-mediated pathological conditions in both the periphery and the central nervous system. Activation of prostaglandin E2 receptor (PGE(2)) subtype EP2 promotes inflammation and is just beginning to be explored as a therapeutic target. To better understand physiological and pathological functions of the prostaglandin EP2 receptor, we developed a suite of small molecules with a 3-aryl-acrylamide scaffold as selective EP2 antagonists. The 12 most potent compounds displayed competitive antagonism of the human EP2 receptor with K(B) 2-20 nM in Schild regression analysis and 268- to 4,730-fold selectivity over the prostaglandin EP4 receptor. A brain-permeant compound completely suppressed the up-regulation of COX-2 mRNA in rat cultured microglia by EP2 activation and significantly reduced neuronal injury in hippocampus when administered in mice beginning 1 h after termination of pilocarpine-induced status epilepticus. The salutary actions of this novel group of antagonists raise the possibility that selective block of EP2 signaling via small molecules can be an innovative therapeutic strategy for inflammation-related brain injury.
Evaluation of Quadrox-i and Capiox FX Neonatal Oxygenators with Integrated Arterial Filters in Eliminating Gaseous Microemboli and Retaining Hemodynamic Properties During Simulated Cardiopulmonary Bypass
Perfusion quality during cardiopulmonary bypass (CPB) procedures can contribute to postoperative neurological complications and influence patient recovery and outcome. Gaseous microemboli generated in the circuit and hemodynamic properties of blood reaching the patient can be monitored during CPB to optimize perfusion. Oxygenators that oxygenate the blood during CPB can significantly influence the quality of blood reaching the patient by their manufacturing designs. New hollow-fiber membrane oxygenators are developed with integrated arterial filters to reduce priming volume and eliminate a separate arterial filter in the circuit. To evaluate the performance of these new oxygenators, we used a simulated model to compare the Quadrox-i Neonatal and the Capiox Baby FX05 neonatal oxygenators and to provide a review of these oxygenators with their respective counterparts which have separate arterial filters. We found that microemboli counts for the new Quadrox-i and Capiox FX05 oxygenators are similar in the arterial line, but different across the oxygenator for all experimental conditions. The arterial purge line diverting blood from the patient reduces microemboli count for the Capiox FX05, but is inconsistent for the Quadrox-i Neonatal. While hemodynamic energy delivered to the patient is similar for both oxygenators, shunted blood flow for the Quadrox-i Neonatal oxygenator is three times higher than the Capiox FX05 (103.6 mL/min vs 33.0 mL/min at 400 mL/min and 35°C) (p<0.001).
