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1.  Metabolic effects of the contraceptive skin patch and subdermal contraceptive implant in Mexican women: A prospective study 
Reproductive Health  2014;11:33.
The contraceptive skin patch (CSP) accepted by the U.S. FDA in 2001 includes ethinylestradiol and norelgestromine, whereas the subdermal contraceptive implant (SCI) has etonogestrel and is also approved by the FDA. In Mexico, both are now widely used for contraception but their effects on Mexican population are unknown. The objective of the study was to evaluate if these treatments induce metabolic changes in a sample of indigenous and mestizo Mexican women.
An observational, prospective, longitudinal, non-randomized study of women between 18 and 35 years of age assigned to CSP or SCI. We performed several laboratory tests: clinical chemistry, lipid profile, and liver and thyroid function tests. Also, serum levels of insulin, C-peptide, IGF-1, leptin, adiponectin, and C reactive protein were assayed.
Sixty-two women were enrolled, 25 used CSP (0 indigenous; 25 mestizos) and 37 used SCI (18 indigenous; 19 mestizos). Clinical symptoms were relatively more frequent in the SCI group. Thirty-four contraceptive users gained weight without other clinical significant changes. After 4 months of treatment, significant changes were found in some biochemical parameters in both treatment groups. Most were clinically irrelevant. Interestingly, the percentage of users with an abnormal atherogenic index diminished from 75% to 41.6% after follow-up.
The CSP slightly modified the metabolic variables. Most changes were nonsignificant, whereas for SCI users changes were more evident and perhaps beneficial. Results of this attempt to evaluate the effects of contraceptives in mestizo and native-American populations show that clinical symptoms are frequent in Mexican users of CSP and SCI. Although these medications may affect some metabolic variables, these changes seem clinically irrelevant. Induction of abnormalities in other physiological pathways cannot be ruled out.
PMCID: PMC4044294  PMID: 24767248
Contraceptive skin patch; Subdermal contraceptive implant; Metabolic effects; Metabolic changes; Contraception; Parche cutáneo anticonceptivo; Implante contraceptivo subdérmico; Efectos metabólicos; Cambios metabólicos; Contracepción
2.  A clinical exploratory study with itolizumab, an anti-CD6 monoclonal antibody, in patients with rheumatoid arthritis 
Results in Immunology  2012;2:204-211.
T cells are involved in the pathogenesis of rheumatoid arthritis (RA). CD6 is a co-stimulatory molecule, predominantly expressed on lymphocytes, that has been linked to autoreactive responses. The purpose of this study was to evaluate the safety, immunogenicity and preliminary efficacy of itolizumab, a humanized anti-CD6 monoclonal antibody, in patients with active rheumatoid arthritis. Fifteen patients were enrolled in a phase I, open-label, dose-finding study. Five cohorts of patients received a weekly antibody monotherapy with a dose-range from 0.1 to 0.8 mg/kg. Itolizumab showed a good safety profile, with no severe or serious adverse events reported so far. No signs or symptoms associated with immunosuppression were observed in the study. Objective clinical responses were achieved in more than 80% of patients after treatment completion, and these responses tend to be sustained afterwards. This clinical study constitutes the first evidence of the safety and positive clinical effect of a monotherapy using an anti-CD6 antibody in patients with rheumatoid arthritis.
▸ Exploratory study to assess itolizumab in rheumatoid arthritis patients. ▸ Good safety profile, neither severe or serious adverse events, nor immunosuppression. ▸ Objective clinical response achieved in more than 80% of patients. ▸ Clinical response trends to be sustained after itolizumab withdrawal.
PMCID: PMC3862386  PMID: 24371585
Rheumatoid arthritis; Exploratory study; T lymphocyte; CD6; ACR, American College of Rheumatology; AE, adverse events; CRP, C reactive protein; iv, intravenous; DMARD, disease-modifying antirheumatic drug; ESR, eritrosedimentation rate; mAbs, monoclonal antibodies; NSAIDs, nonsteroidal antiinflammatory drugs; RA, rheumatoid arthritis; RF, rheumatoid factor; SAE, serious adverse event.
3.  A handy approximation for a mediated bioelectrocatalysis process, related to Michaelis-Menten equation 
SpringerPlus  2014;3:162.
In this article, Perturbation Method (PM) is employed to obtain a handy approximate solution to the steady state nonlinear reaction diffusion equation containing a nonlinear term related to Michaelis-Menten of the enzymatic reaction. Comparing graphics between the approximate and exact solutions, it will be shown that the PM method is quite efficient.
PMCID: PMC3982037  PMID: 24741477
Michaelis-Menten kinetics; Perturbation method; Reaction/diffusion equation; Mediated bioelectrocatalysis
4.  Clinical and microbiological profile of infectious keratitis in children 
BMC Ophthalmology  2013;13:54.
Infectious keratitis is a sight-threatening condition for children. The purpose of this study was to describe the clinical profile, risk factors and microbiological profile of infectious keratitis in children.
Retrospective review of clinical records of patients under 16 years of age with history of microbial keratitis seen at a tertiary referral center. Clinical characteristics, risk factors, visual and surgical outcomes as well as the microbiological profile are analyzed.
Forty-one eyes of 41 patients. Mean age was 8.7 years. Time between the onset of symptoms and ophthalmological examination was 12.7 days. Predisposing factors were found in 78%; ocular trauma was the most common (25%). Visual acuity equal or worse than 20/200 at admission correlated positively with a poorer visual outcome, p=0.002. Positivity of cultures was 34%. Gram-positive bacteria were isolated in 78.5%; Staphylococcus epidermidis (28.6%) was the most common microorganism.
Our study emphasizes the importance of a prompt diagnosis and treatment of infectious corneal ulcers in children. Trauma and contact lenses were the main predisposing factors. Gram-positive organisms were isolated in the vast majority of cases and visual outcomes are usually poor.
PMCID: PMC4015831  PMID: 24131681
Paediatrics; Children; Drug-resistance; Microbial; Risk factors
5.  Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers 
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.
PMCID: PMC3722562  PMID: 23894227
RNA aptamers; targeted delivery; siRNA delivery; cell-SELEX; cell-internalizing aptamers
6.  A pathway-based analysis provides additional support for an immune-related genetic susceptibility to Parkinson's disease 
Holmans, Peter | Moskvina, Valentina | Jones, Lesley | Sharma, Manu | Vedernikov, Alexey | Buchel, Finja | Sadd, Mohamad | Bras, Jose M. | Bettella, Francesco | Nicolaou, Nayia | Simón-Sánchez, Javier | Mittag, Florian | Gibbs, J. Raphael | Schulte, Claudia | Durr, Alexandra | Guerreiro, Rita | Hernandez, Dena | Brice, Alexis | Stefánsson, Hreinn | Majamaa, Kari | Gasser, Thomas | Heutink, Peter | Wood, Nicholas W. | Martinez, Maria | Singleton, Andrew B. | Nalls, Michael A. | Hardy, John | Morris, Huw R. | Williams, Nigel M. | Arepalli, Sampath | Barker, Roger | Barrett, Jeffrey | Ben-Shlomo, Yoav | Berendse, Henk W. | Berg, Daniela | Bhatia, Kailash | de Bie, Rob M.A. | Biffi, Alessandro | Bloem, Bas | Brice, Alexis | Bochdanovits, Zoltan | Bonin, Michael | Bras, Jose M. | Brockmann, Kathrin | Brooks, Janet | Burn, David J. | Charlesworth, Gavin | Chen, Honglei | Chinnery, Patrick F. | Chong, Sean | Clarke, Carl E. | Cookson, Mark R. | Cooper, Jonathan M. | Corvol, Jen-Christophe | Counsell, Carl | Damier, Philippe | Dartigues, Jean Francois | Deloukas, Panagiotis | Deuschl, Günther | Dexter, David T. | van Dijk, Karin D. | Dillman, Allissa | Durif, Frank | Durr, Alexandra | Edkins, Sarah | Evans, Jonathan R. | Foltynie, Thomas | Gao, Jianjun | Gardner, Michelle | Gasser, Thomas | Gibbs, J. Raphael | Goate, Alison | Gray, Emma | Guerreiro, Rita | Gústafsson, Ómar | Hardy, John | Harris, Clare | Hernandez, Dena G. | Heutink, Peter | van Hilten, Jacobus J. | Hofman, Albert | Hollenbeck, Albert | Holmans, Peter | Holton, Janice | Hu, Michele | Huber, Heiko | Hudson, Gavin | Hunt, Sarah E. | Huttenlocher, Johanna | Illig, Thomas | Langford, Cordelia | Lees, Andrew | Lesage, Suzanne | Lichtner, Peter | Limousin, Patricia | Lopez, Grisel | Lorenz, Delia | Martinez, Maria | McNeill, Alisdair | Moorby, Catriona | Moore, Matthew | Morris, Huw | Morrison, Karen E. | Moskvina, Valentina | Mudanohwo, Ese | Nalls, Michael A. | Pearson, Justin | Perlmutter, Joel S. | Pétursson, Hjörvar | Plagnol, Vincent | Pollak, Pierre | Post, Bart | Potter, Simon | Ravina, Bernard | Revesz, Tamas | Riess, Olaf | Rivadeneira, Fernando | Rizzu, Patrizia | Ryten, Mina | Saad, Mohamad | Sawcer, Stephen | Schapira, Anthony | Scheffer, Hans | Sharma, Manu | Shaw, Karen | Sheerin, Una-Marie | Shoulson, Ira | Schulte, Claudia | Sidransky, Ellen | Simón-Sánchez, Javier | Singleton, Andrew B. | Smith, Colin | Stefánsson, Hreinn | Stefánsson, Kári | Steinberg, Stacy | Stockton, Joanna D. | Sveinbjornsdottir, Sigurlaug | Talbot, Kevin | Tanner, Carlie M. | Tashakkori-Ghanbaria, Avazeh | Tison, François | Trabzuni, Daniah | Traynor, Bryan J. | Uitterlinden, André G. | Velseboer, Daan | Vidailhet, Marie | Walker, Robert | van de Warrenburg, Bart | Wickremaratchi, Mirdhu | Williams, Nigel | Williams-Gray, Caroline H. | Winder-Rhodes, Sophie | Wood, Nicholas
Human Molecular Genetics  2012;22(5):1039-1049.
Parkinson's disease (PD) is the second most common neurodegenerative disease affecting 1–2% in people >60 and 3–4% in people >80. Genome-wide association (GWA) studies have now implicated significant evidence for association in at least 18 genomic regions. We have studied a large PD-meta analysis and identified a significant excess of SNPs (P < 1 × 10−16) that are associated with PD but fall short of the genome-wide significance threshold. This result was independent of variants at the 18 previously implicated regions and implies the presence of additional polygenic risk alleles. To understand how these loci increase risk of PD, we applied a pathway-based analysis, testing for biological functions that were significantly enriched for genes containing variants associated with PD. Analysing two independent GWA studies, we identified that both had a significant excess in the number of functional categories enriched for PD-associated genes (minimum P = 0.014 and P = 0.006, respectively). Moreover, 58 categories were significantly enriched for associated genes in both GWA studies (P < 0.001), implicating genes involved in the ‘regulation of leucocyte/lymphocyte activity’ and also ‘cytokine-mediated signalling’ as conferring an increased susceptibility to PD. These results were unaltered by the exclusion of all 178 genes that were present at the 18 genomic regions previously reported to be strongly associated with PD (including the HLA locus). Our findings, therefore, provide independent support to the strong association signal at the HLA locus and imply that the immune-related genetic susceptibility to PD is likely to be more widespread in the genome than previously appreciated.
PMCID: PMC3561909  PMID: 23223016
7.  Loss of FTO Antagonises Wnt Signaling and Leads to Developmental Defects Associated with Ciliopathies 
PLoS ONE  2014;9(2):e87662.
Common intronic variants in the Human fat mass and obesity-associated gene (FTO) are found to be associated with an increased risk of obesity. Overexpression of FTO correlates with increased food intake and obesity, whilst loss-of-function results in lethality and severe developmental defects. Despite intense scientific discussions around the role of FTO in energy metabolism, the function of FTO during development remains undefined. Here, we show that loss of Fto leads to developmental defects such as growth retardation, craniofacial dysmorphism and aberrant neural crest cells migration in Zebrafish. We find that the important developmental pathway, Wnt, is compromised in the absence of FTO, both in vivo (zebrafish) and in vitro (Fto−/− MEFs and HEK293T). Canonical Wnt signalling is down regulated by abrogated β-Catenin translocation to the nucleus whilst non-canonical Wnt/Ca2+ pathway is activated via its key signal mediators CaMKII and PKCδ. Moreover, we demonstrate that loss of Fto results in short, absent or disorganised cilia leading to situs inversus, renal cystogenesis, neural crest cell defects and microcephaly in Zebrafish. Congruently, Fto knockout mice display aberrant tissue specific cilia. These data identify FTO as a protein-regulator of the balanced activation between canonical and non-canonical branches of the Wnt pathway. Furthermore, we present the first evidence that FTO plays a role in development and cilia formation/function.
PMCID: PMC3913654  PMID: 24503721
8.  A Collusion-Resistant Fingerprinting System for Restricted Distribution of Digital Documents 
PLoS ONE  2013;8(12):e81976.
Digital fingerprinting is a technique that consists of inserting the ID of an authorized user in the digital content that he requests. This technique has been mainly used to trace back pirate copies of multimedia content such as images, audio, and video. This study proposes the use of state-of-the-art digital fingerprinting techniques in the context of restricted distribution of digital documents. In particular, the system proposed by Kuribayashi for multimedia content is investigated. Extensive simulations show the robustness of the proposed system against average collusion attack. Perceptual transparency of the fingerprinted documents is also studied. Moreover, by using an efficient Fast Fourier Transform core and standard computer machines it is shown that the proposed system is suitable for real-world scenarios.
PMCID: PMC3859583  PMID: 24349165
9.  The Transacting Factor CBF-A/Hnrnpab Binds to the A2RE/RTS Element of Protamine 2 mRNA and Contributes to Its Translational Regulation during Mouse Spermatogenesis 
PLoS Genetics  2013;9(10):e1003858.
During spermatogenesis, mRNA localization and translation are believed to be regulated in a stage-specific manner. We report here that the Protamine2 (Prm2) mRNA transits through chromatoid bodies of round spermatids and localizes to cytosol of elongating spermatids for translation. The transacting factor CBF-A, also termed Hnrnpab, contributes to temporal regulation of Prm2 translation. We found that CBF-A co-localizes with the Prm2 mRNA during spermatogenesis, directly binding to the A2RE/RTS element in the 3′ UTR. Although both p37 and p42 CBF-A isoforms interacted with RTS, they associated with translationally repressed and de-repressed Prm2 mRNA, respectively. Only p42 was found to interact with the 5′cap complex, and to co-sediment with the Prm2 mRNA in polysomes. In CBF-A knockout mice, expression of protamine 2 (PRM2) was reduced and the Prm2 mRNA was prematurely translated in a subset of elongating spermatids. Moreover, a high percentage of sperm from the CBF-A knockout mouse showed abnormal DNA morphology. We suggest that CBF-A plays an important role in spermatogenesis by regulating stage-specific translation of testicular mRNAs.
Author Summary
During eukaryotic gene expression, a fraction of newly exported mRNA molecules is transported to the cellular periphery for translation. The underlying mechanisms are not fully understood even though they likely affect specialized functions in many cell types including oligodendrocyets, neurons and germ cells. We discovered that the heterogeneous nuclear ribonucleoprotein CBF-A, interacts with a conserved sequence, the RNA trafficking sequence (RTS), located in the untranslated region of transported mRNAs. This interaction facilitates transport of myelin basic protein mRNA and dendritic mRNAs in oligodendrocytes and neurons, respectively. Here we investigated whether RTS-recognition by CBF-A coordinates transport and localized translation of the Protamine 2 mRNA in spermatogenic cells. During spermatogenesis the Protamine 2 mRNAs is synthesized and kept in a silent form to be translated at later stages. We show that by interacting with the RTS of the Protamine 2 mRNA both CBF-A isoforms contribute to regulate the transcript at the translational level. In a CBF-A knockout mouse model, we demonstrate that the interplay between the CBF-A isoforms in translation regulation of the Protamine 2 mRNA and other testicular transcripts has an impact on spermatogenesis.
PMCID: PMC3798277  PMID: 24146628
10.  Unsupervised Cardiac Image Segmentation via Multiswarm Active Contours with a Shape Prior 
This paper presents a new unsupervised image segmentation method based on particle swarm optimization and scaled active contours with shape prior. The proposed method uses particle swarm optimization over a polar coordinate system to perform the segmentation task, increasing the searching capability on medical images with respect to different interactive segmentation techniques. This method is used to segment the human heart and ventricular areas from datasets of computed tomography and magnetic resonance images, where the shape prior is acquired by cardiologists, and it is utilized as the initial active contour. Moreover, to assess the performance of the cardiac medical image segmentations obtained by the proposed method and by the interactive techniques regarding the regions delineated by experts, a set of validation metrics has been adopted. The experimental results are promising and suggest that the proposed method is capable of segmenting human heart and ventricular areas accurately, which can significantly help cardiologists in clinical decision support.
PMCID: PMC3807539  PMID: 24198850
11.  Mannheim Carotid Intima-Media Thickness and Plaque Consensus (2004–2006–2011): An Update on Behalf of the Advisory Board of the 3rd and 4th Watching the Risk Symposium 13th and 15th European Stroke Conferences, Mannheim, Germany, 2004, and Brussels, Belgium, 2006 
Intima-media thickness (IMT) provides a surrogate end point of cardiovascular outcomes in clinical trials evaluating the efficacy of cardiovascular risk factor modification. Carotid artery plaque further adds to the cardiovascular risk assessment. It is defined as a focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the surrounding IMT value or demonstrates a thickness >1.5 mm as measured from the media-adventitia interface to the intima-lumen interface. The scientific basis for use of IMT in clinical trials and practice includes ultrasound physics, technical and disease-related principles as well as best practice on the performance, interpretation and documentation of study results. Comparison of IMT results obtained from epidemiological and interventional studies around the world relies on harmonization on approaches to carotid image acquisition and analysis. This updated consensus document delineates further criteria to distinguish early atherosclerotic plaque formation from thickening of IMT. Standardized methods will foster homogenous data collection and analysis, improve the power of randomized clinical trials incorporating IMT and plaque measurements and facilitate the merging of large databases for meta-analyses. IMT results are applied to individual patients as an integrated assessment of cardiovascular risk factors. However, this document recommends against serial monitoring in individual patients.
PMCID: PMC3760791  PMID: 23128470
Intima-media thickness; Carotid plaque; Vascular ultrasound; Randomized clinical trials; Carotid disease; Reference values
12.  Molecular Modeling Optimization of Anticoagulant Pyridine Derivatives 
Intravascular clotting remains a major health problem in the United States, the most prominent being deep vein thrombosis, pulmonary embolism and thromboembolic stroke. Previous reports on the use of pyridine derivatives in cardiovascular drug development encourage us to pursue new types of compounds based on a pyridine scaffold. Eleven pyridine derivatives (oximes, semicarbazones, N-oxides) previously synthesized in our laboratories were tested as anticoagulants on pooled normal plasma using the prothrombin time (PT) protocol. The best anticoagulant within the oxime series was compound AF4, within the oxime N-oxide series was compound AF4-N-Oxide, and within the semicarbazone series, compound MD1-30Y. We also used a molecular modeling approach to guide our efforts, and found that there was good correlation between coagulation data and computational energy scores. Molecular docking was performed to target the active site of thrombin with the DOCK v5.2 package. The results of molecular modeling indicate that improvement in anticoagulant activities can be expected by functionalization at the 3-position of the pyridine ring and by N-oxide formation. Results reported here prove the suitability of DOCK in the lead optimization process.
PMCID: PMC3752647  PMID: 18372200
pyridine oximes; pyridine semicarbazones; anticoagulants; molecular modeling; DOCK; thrombin
13.  Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice 
Memórias do Instituto Oswaldo Cruz  2013;108(5):616-622.
The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.
PMCID: PMC3970608  PMID: 23903978
Giardia lamblia; immunogenic antigen; monoclonal antibody
14.  Role of the Plasma Membrane Transporter of Organic Cations OCT1 and Its Genetic Variants in Modern Liver Pharmacology 
BioMed Research International  2013;2013:692071.
Changes in the uptake of many drugs by the target cells may dramatically affect the pharmacological response. Thus, downregulation of SLC22A1, which encodes the organic cation transporter type 1 (OCT1), may affect the response of healthy hepatocytes and liver cancer cells to cationic drugs, such as metformin and sorafenib, respectively. Moreover, the overall picture may be modified to a considerable extent by the preexistence or the appearance during the pathogenic process of genetic variants. Some rare OCT1 variants enhance transport activity, whereas other more frequent variants impair protein maturation, plasma membrane targeting or the function of this carrier, hence reducing intracellular active drug concentrations. Here, we review current knowledge of the role of OCT1 in modern liver pharmacology, which includes the use of cationic drugs to treat several diseases, some of them of great clinical relevance such as diabetes and primary liver cancer (cholangiocarcinoma and hepatocellular carcinoma). We conclude that modern pharmacology must consider the individual evaluation of OCT1 expression/function in the healthy liver and in the target tissue, particularly if this is a tumor, in order to predict the lack of response to cationic drugs and to be able to design individualized pharmacological treatments with the highest chances of success.
PMCID: PMC3747481  PMID: 23984399
17.  Analysis and Prediction of Pathways in HeLa Cells by Integrating Biological Levels of Organization with Systems-Biology Approaches 
PLoS ONE  2013;8(6):e65433.
It has recently begun to be considered that cancer is a systemic disease and that it must be studied at every level of complexity using many of the currently available approaches, including high-throughput technologies and bioinformatics. To achieve such understanding in cervical cancer, we collected information on gene, protein and phosphoprotein expression of the HeLa cell line and performed a comprehensive analysis of the different signaling pathways, transcription networks and metabolic events in which they participate. A total expression analysis by RNA-Seq of the HeLa cell line showed that 19,974 genes were transcribed. Of these, 3,360 were over-expressed, and 2,129 under-expressed when compared to the NHEK cell line. A protein-protein interaction network was derived from the over-expressed genes and used to identify central elements and, together with the analysis of over-represented transcription factor motifs, to predict active signaling and regulatory pathways. This was further validated by Metal-Oxide Affinity Chromatography (MOAC) and Tandem Mass Spectrometry (MS/MS) assays which retrieved phosphorylated proteins. The 14-3-3 family members emerge as important regulators in carcinogenesis and as possible clinical targets. We observed that the different over- and under-regulated pathways in cervical cancer could be interrelated through elements that participate in crosstalks, therefore belong to what we term “meta-pathways”. Additionally, we highlighted the relations of each one of the differentially represented pathways to one or more of the ten hallmarks of cancer. These features could be maintained in many other types of cancer, regardless of mutations or genomic rearrangements, and favor their robustness, adaptations and the evasion of tissue control. Probably, this could explain why cancer cells are not eliminated by selective pressure and why therapy trials directed against molecular targets are not as effective as expected.
PMCID: PMC3680226  PMID: 23785426
18.  Bardet–Biedl syndrome proteins control the cilia length through regulation of actin polymerization 
Human Molecular Genetics  2013;22(19):3858-3868.
Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet–Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanisms by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4- and Bbs6-deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, fewer mutant cells were ciliated [48% ± 6 for wild-type (WT) cells versus 23% ± 7 for Bbs4 null cells; P < 0.0001] and their cilia were shorter (2.55 μm ± 0.41 for WT cells versus 2.16 μm ± 0.23 for Bbs4 null cells; P < 0.0001). While the microtubular cytoskeleton and cortical actin were intact, actin stress fibre formation was severely disrupted, forming abnormal apical stress fibre aggregates. Furthermore, we observed over-abundant focal adhesions (FAs) in Bbs4-, Bbs6- and Bbs8-deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerization, we showed that RhoA-GTP levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4-deficient cells with chemical inhibitors of RhoA, we were able to restore the cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control the cilia length through alteration of RhoA levels.
PMCID: PMC3766180  PMID: 23716571
19.  Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers 
Pharmaceuticals  2013;6(3):295-319.
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.
PMCID: PMC3722562  PMID: 23894227
RNA aptamers; targeted delivery; siRNA delivery; cell-SELEX; cell-internalizing aptamers
20.  Chronic Stress Induces Structural Alterations in Splenic Lymphoid Tissue That Are Associated with Changes in Corticosterone Levels in Wistar-Kyoto Rats 
BioMed Research International  2013;2013:868742.
Major depressive disorder patients present chronic stress and decreased immunity. The Wistar-Kyoto rat (WKY) is a strain in which the hypothalamic-pituitary-adrenal axis is overactivated. To determine whether chronic stress induces changes in corticosterone levels and splenic lymphoid tissue, 9-week-old male rats were subject to restraint stress (3 h daily), chemical stress (hydrocortisone treatment, 50 mg/Kg weight), mixed stress (restraint plus hydrocortisone), or control treatment (without stress) for 1, 4, and 7 weeks. The serum corticosterone levels by RIA and spleens morphology were analyzed. Corticosterone levels as did the structure, size of the follicles and morphology of the parenchyma (increase in red pulp) in the spleen, varied depending on time and type of stressor. These changes indicate that chronic stress alters the immune response in the spleen in WKY rats by inducing morphological changes, explaining in part the impaired immunity that develops in organisms that are exposed to chronic stress.
PMCID: PMC3582072  PMID: 23533999
21.  Global landscape of HIV–human protein complexes 
Nature  2011;481(7381):365-370.
Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host’s cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry1-3 to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV–human protein–protein interactions involving 435 individual human proteins, with ~40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.
PMCID: PMC3310911  PMID: 22190034
22.  Tectonic DSAEK for the Management of Impending Corneal Perforation 
Purpose. To report a case of severe corneal thinning secondary to dry eye treated with a tectonic Descemet stripping automated lamellar keratoplasty (DSAEK) and amniotic membrane graft. Methods. A 72-year-old man with a history of long standing diabetes mellitus type 2 and dry eye presented with 80% corneal thinning and edema on the right eye and no signs of infectious disease, initially managed with topical unpreserved lubrication and 20% autologous serum drops. Eight weeks after, the defect advanced in size and depth until Descemetocele was formed. Thereafter, he underwent DSAEK for tectonic purposes. One month after the procedure, the posterior lamellar graft was well adhered but a 4 mm epithelial defect was still present. A multilayered amniotic membrane graft was then performed. Results. Ocular surface healed quickly and reepithelization occurred over a 2-week period. Eight months after, the ocular surface remained stable and structurally adequate. Conclusion. Tectonic DSAEK in conjunction with multilayered amniotic graft may not only provide structural support and avoid corneal perforation, but may also promote reepithelization and ocular surface healing and decrease concomitant inflammation.
PMCID: PMC3521400  PMID: 23259100
23.  Delayed-Onset Post-Keratoplasty Endophthalmitis Caused by Vancomycin-Resistant Enterococcus faecium 
Case Reports in Ophthalmology  2012;3(3):370-374.
Vancomycin-resistant Enterococcus (VRE) endophthalmitis after penetrating keratoplasty (PKP) is very rare, the management is a challenge due to both the pattern of antibiotic resistance and the aggressive nature of the infectious process. We report the first delayed-onset case of VRE endophthalmitis after PKP.
Materials and Methods
Case report of a 51-year-old female with a 7-week history of PKP who arrived at the emergency room with signs and symptoms of endophthalmitis. Initial visual acuity was light perception, and a posterior pole exam was not possible due to the intense vitreous reaction. Mode B ultrasound was used to assess the posterior pole. The patient underwent pars plana vitrectomy and received intravitreous antibiotics.
Vitreous stains and cultures were positive for Enterococcus faecium resistant to vancomycin. Donor rim cultures and viral PCR were negative. Treatment was carried out by repeated intravitreal antibiotics and systemic linezolid. Clinical improvement was seen after the second dose of intravitreous antibiotics and systemic linezolid, but visual acuity remained at light perception consistent with the ischemic changes observed in the posterior pole.
VRE endophthalmitis might be associated with positive donor rim cultures. Prompt use of systemic linezolid in addition to intravitreous antibiotics is recommendable, but even with prompt treatment, visual prognosis is guarded.
PMCID: PMC3506059  PMID: 23185179
Delayed-onset endophthalmitis; Penetrating keratoplasty; Vancomycin-resistant Enterococcus; Intravitreal antibiotics; Systemic linezolid
24.  African American Race and Prevalence of Atrial Fibrillation:A Meta-Analysis 
Background. It has been observed that African American race is associated with a lower prevalence of atrial fibrillation (AF) compared to Caucasian race. To better quantify the association between African American race and AF, we performed a meta-analysis of published studies among different patient populations which reported the presence of AF by race. Methods. A literature search was conducted using electronic databases between January 1999 and January 2011. The search was limited to published studies in English conducted in the United States, which clearly defined the presence of AF in African American and Caucasian subjects. A meta-analysis was performed with prevalence of AF as the primary endpoint. Results. In total, 10 studies involving 1,031,351 subjects were included. According to a random effects analysis, African American race was associated with a protective effect with regard to AF as compared to Caucasian race (odds ratio 0.51, 95% CI 0.44 to 0.59, P < 0.001). In subgroup analyses, African American race was significantly associated with a lower prevalence of AF in the general population, those hospitalized or greater than 60 years old, postcoronary artery bypass surgery patients, and subjects with heart failure. Conclusions. In a broad sweep of subjects in the general population and hospitalized patients, the prevalence of AF in African Americans is consistently lower than in Caucasians.
PMCID: PMC3328147  PMID: 22548197
25.  Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers 
Nucleic Acids Research  2012;40(13):6319-6337.
Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2+-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating ‘cell-type specific’, ‘cell-internalizing RNA ligands (aptamers)’ capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2+-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2+-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.
PMCID: PMC3401474  PMID: 22467215

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