Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of ...functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor-host nuclear or cell-cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders.
Retinal degenerative diseases are a major cause of untreatable blindness. Stem cell therapy to replace lost photoreceptors represents a feasible future treatment. We previously demonstrated that ...postmitotic photoreceptor precursors expressing an NrlGFP transgene integrate into the diseased retina and restore some light sensitivity. As genetic modification of precursor cells derived from stem cell cultures is not desirable for therapy, we have tested cell selection strategies using fluorochrome‐conjugated antibodies recognizing cell surface antigens to sort photoreceptor precursors. Microarray analysis of postnatal NrlGFP‐expressing precursors identified four candidate genes encoding cell surface antigens (Nt5e, Prom1, Podxl, and Cd24a). To test the feasibility of using donor cells isolated using cell surface markers for retinal therapy, cells selected from developing retinae by fluorescence‐activated cell sorting based on Cd24a expression (using CD24 antibody) and/or Nt5e expression (using CD73 antibody) were transplanted into the wild‐type or Crb1rd8/rd8 or Prph2rd2/rd2 mouse eye. The CD73/CD24‐sorted cells migrated into the outer nuclear layer, acquired the morphology of mature photoreceptors and expressed outer segment markers. They showed an 18‐fold higher integration efficiency than that of unsorted cells and 2.3‐fold higher than cells sorted based on a single genetic marker, NrlGFP, expression. These proof‐of‐principle studies show that transplantation competent photoreceptor precursor cells can be efficiently isolated from a heterogeneous mix of cells using cell surface antigens without loss of viability for the purpose of retinal stem cell therapy. Refinement of the selection of donorphotoreceptor precursor cells can increase the number of integrated photoreceptor cells,which is a prerequisite for the restoration of sight. STEM CELLS 2011;29:1391–1404
Hydrogen peroxide (H
2
O
2
) plays a role in many facets — a household item, an important industrial chemical, a biomarker in vivo, and several others. For this reason, its measurement and ...quantification in a variety of media are important. While spectroscopic detection is primarily used for H
2
O
2
, electrochemical methods offer advantages in versatility, cost, and sensitivity. In this work, we investigate a 2-step surface metal nanoparticle (NP) modification for platinum (Pt) and palladium (Pd) on boron-doped diamond (BDD) electrodes for the detection of H
2
O
2
. Several parameters such as the metal salt concentration and electrodeposition charge in the 2-step modification were varied to find an optimum. Using cyclic voltammetry (CV), the BDD-PdNP electrode types were found to yield a sharper, more well-resolved H
2
O
2
oxidation peak compared to the BDD-PtNP electrodes. Both metal NP electrode types significantly improved the response compared to the bare BDD electrode; a 150–200× improvement in sensitivity was observed across all modified electrode types. Calibration experiments were completed at both low and high concentration ranges in stagnant and flow-based solutions. The lowest limit of detection (LOD) obtained was 50 nM (5E-08 M) on a BDD-PdNP electrode modified with 1.0 mM PdCl
2
to 5.0 mC in the wet chemical seeding and electrodeposition steps. 0.25 mM PdCl
2
to 3.23 mC and 0.25 mM HPtCl
6
−
to 3.23 mC also yielded a sufficient response for low-level H
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O
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, with LODs around 100 nM (1E-07 M). Overall, this work exemplifies the wide applicability of BDD and achieves sub-μM H
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O
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LODs with a non-enzymatic electrode material.
Leber congenital amaurosis is a group of inherited retinal dystrophies that cause severe sight impairment in childhood; RPE65-deficiency causes impaired rod photoreceptor function from birth and ...progressive impairment of cone photoreceptor function associated with retinal degeneration. In animal models of RPE65 deficiency, subretinal injection of recombinant adeno-associated virus (AAV) 2/2 vectors carrying RPE65 cDNA improves rod photoreceptor function, and intervention at an early stage of disease provides sustained benefit by protecting cone photoreceptors against retinal degeneration. In affected humans, administration of these vectors has resulted to date in relatively modest improvements in photoreceptor function, even when retinal degeneration is comparatively mild, and the duration of benefit is limited by progressive retinal degeneration. We conclude that the demand for RPE65 in humans is not fully met by current vectors, and predict that a more powerful vector will provide more durable benefit. With this aim we have modified the original AAV2/2 vector to generate AAV2/5-OPTIRPE65. The new configuration consists of an AAV vector serotype 5 carrying an optimized hRPE65 promoter and a codon-optimized hRPE65 gene. In mice, AAV2/5-OPTIRPE65 is at least 300-fold more potent than our original AAV2/2 vector.
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•The software framework jMetalSP for Big Data Optimization is presented.•jMetalSP has been deployed on a Hadoop/Spark cluster to solve the dynamic TSP.•Three different data sources ...have been considered: Open Data, Twitter, and Kafka.•Obtained solutions are able to adapt to changing traffic conditions in NY.•jMetalSP is open source and free available to academic and industry communities.
Multi-objective metaheuristics have become popular techniques for dealing with complex optimization problems composed of a number of conflicting functions. Nowadays, we are in the Big Data era, so metaheuristics must be able to solve dynamic problems that may vary over time due to the processing and analysis of several streaming data sources. As this is a new field, there is a need for software platforms to solve dynamic multi-objective Big Data optimization problems. In this paper, we present jMetalSP, which combines the multi-objective optimization features of the jMetal framework with the streaming facilities of the Apache Spark cluster computing system. Thus, existing state-of-the-art multi-objective metaheuristics can be easily adapted to deal with dynamic optimization problems that are fed by multiple streaming data sources. Moreover, these algorithms can take advantage of the parallel computing features of Spark. We describe the architecture of jMetalSP and show how it can be used to solve a dynamic bi-objective instance of the Traveling Salesman Problem (TSP) based on New York City's real-time traffic data. We have also carried out an experimental study to assess the performance of the resultant jMetalSP application in a Hadoop cluster composed of 100 nodes.
The aim of this study is to explore the effectiveness and safety of oral corticosteroids (prednisone) in the treatment of early stage SARS-Cov-2 pneumonia in patients who do not yet meet hospital ...admission criteria.
Randomized clinical trial, controlled, open, parallel group, to evaluate the effectiveness of steroids in adult patients with confirmed COVID-19, with incipient pulmonary involvement, without hospital admission criteria. Patients will be stratified by the presence or not of radiological data on pneumonia.
We will include patients with early stage SARS-Cov-2 pneumonia who do not meet hospital admission criteria from the reference hospital, the Hospital Universitario de Burgos, in the region of Castilla y León, Spain. Patients will be followed-up by specialist physicians and Primary Health Care professionals.
- Men and women. - Age between 18 and 75 years old. - Diagnosed SARS-CoV-2 infection, by PCR and/or IgM+ antibody test and/or antigen test. - Clinical diagnosis of lung involvement: (respiratory symptoms +/- pathological auscultation +/- O2 desaturation) - Chest X-ray with mild-moderate alterations or normal. - Patients who give their verbal informed consent in front of witnesses, which will be reflected in the patients' medical records.
- Oxygen desaturation below 93% or P0
< 62. - Moderate-severe dyspnea or significant respiratory or general deterioration that makes admission advisable. - Chest X-ray with multifocal infiltrates. - Insulin-dependent diabetes with poor control or glycaemia in the emergency room test greater than 300 mg/ml (fasting or not). - Other significant comorbidities: Severe renal failure (creatinine clearance < 30 mL/min); cirrhosis or chronic liver disease, poorly controlled hypertension. - Heart rhythm disturbances (including prolonged QT). - Severe immunosuppression (HIV infection, long-term use of immunosuppressive agents); cancer. - Pregnant or breast-feeding women. - Patients under use of glucocorticoids for other diseases. - History of allergy or intolerance to any of the drugs in the study (prednisone, azithromycin or hydroxychloroquine). - Patients who took one or more of the study drugs in the 7 days prior to study inclusion. - Patients taking non-suppressible drugs with risk of QT prolongation or significant interactions. - Patients unwilling or unable to participate until study completion. - Participation in another study.
Eligible patients will be randomized to receive standard outpatient treatment only (group 1) or standard outpatient treatment plus prednisone (group 2). - Group 1: paracetamol 1 g/8 h (on demand) + hydroxychloroquine 400 mg/12h the first day, 200 mg/12 h for 4 days + azithromycin 500 mg/24h for 5 days. - Group 2: paracetamol 1 g/8 h (on demand) + hydroxychloroquine 400 mg/12h the first day, 200 mg/12 h for 4 days + azithromycin 500 mg/24h for 5 days + prednisone 60 mg / 24 h for 3 days, 30 mg / 24 h for 3 days and 15 mg / 24 h for 3 days.
If the patient requires ambulatory observation, according to the protocol established in this respect in the Emergency Department, meets all the criteria for inclusion and none for exclusion, data will be taken by the person responsible on the data collection sheet. The main result is admission after 30 days. Secondary outcomes are 30-day ICU admission and hospital stay. The safety variable will be the occurrence of clinical symptoms or delirium related to the steroids. Also, the possible decompensations of diabetes will be measured. All tests will be on an intention-to-treat basis.
Treatment will be assigned according to stratified randomization by the presence or absence of radiological data of lung involvement (previously performed by random sequence 1:1 generated with Epidat and kept hidden by opaque, sealed envelopes, which will only be opened after inclusion and basal measurement).
Participants, caregivers and personnel responsible for outcomes measurement will not be blinded to group assignment, once the patient is included and the basal measurement performed, as per protocol design.
The percentage of patients with incipient lung involvement is unknown, but given that pulmonary involvement already exists it is estimated to be around 20%. We consider that the intervention could reduce this percentage to 5%, so the necessary sample size would be 200 subjects (100 per group), with a power of 80% and an estimated loss percentage of 10%.
The protocol with code TAC-COVID-19, version 2.0 on date: April 16, 2020 is approved by the Spanish Drug Agency (AEMPS) and the local Ethics Committee. The trial is in the recruitment phase. Recruitment began 19 April, 2020 and is anticipated to be complete by April 2021.
The trial was registered under the title "OUTPATIENT TREATMENT OF EARLY PULMONARY COVID19 WITH CORTICOSTEROIDS AS AN OPPORTUNITY TO MODIFY THE COURSE OF THE DISEASE" with EudraCT number 2020-001622-64 , registered on 3 April 2020.
The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.
Two families of regioisomeric 1,4-benzodiazepines, 4-benzyl-3H-benzoe1,4diazepin-5-ones and 4-benzoyl-4,5-dihydro-3H-benzoe1,4diazepines, have been synthesized through a similar Ugi/reduction ...cyclization sequence. Their conformation and stability depend on the position of the tautomeric imine/enamine equilibrium present in the diazepine nucleus, which in turn depends on the relative position of the carbonyl group adjacent to the nitrogen at the 4-position in the benzodiazepine system. Moreover, the electrophilic center on the imine tautomer is essential for the antitumor activity of some benzodiazepines as a DNA binding position. The mechanism of tautomerization in the presence or absence of the oxo group has been studied computationally using DFT methods (B3LYP/6-31G** level).
Degeneration of the neural retina is the leading cause of untreatable blindness in the developed world. Stem cell replacement therapy offers a novel strategy for retinal repair. Postmitotic ...photoreceptor precursors derived from the early postnatal (P) retina are able to migrate and integrate into the adult mouse retina following transplantation into the subretinal space, but it is likely that a large number of these cells would be required to restore vision. The adult recipient retina presents a very different environment to that from which photoreceptor precursor donor cells isolated from the developing postnatal retina are derived. Here we considered the possibility that modulation of the recipient environment by ectopic expression of developmentally regulated growth factors, normally present during photoreceptor development, might enhance the migration and integration of transplanted cells into the adult neural retina. Adeno-associated viral (AAV) vectors were used to introduce three growth factors previously reported to play a role in photoreceptor development, IGF1, FGF2, and CNTF, into the adult retina, prior to transplantation of P4 cells derived from the Nrl.GFP+ve neural retina. At 3 weeks posttransplantation the number of integrated, differentiated photoreceptor cells present in AAV-mediated neurotrophic factor-treated eyes was assessed and compared to control treated contralateral eyes. We show, firstly, that it is possible to manipulate the recipient retinal microenvironment via rAAV-mediated gene transfer with respect to these developmentally relevant growth factors. Moreover, when combined with cell transplantation, AAV-mediated expression of IGF1 led to significantly increased levels of cell integration, while overexpression of FGF2 had no significant effect on integrated cell number. Conversely, expression of CNTF led to a significant decrease in cell integration and an exacerbated glial response that led to glial scarring. Together, these findings demonstrate the importance of the extrinsic environment of the recipient retina for photoreceptor cell transplantation and show for the first time that it is possible to manipulate this environment using viral vectors to influence photoreceptor transplantation efficiency.
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