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1.
Article in English | MEDLINE | ID: mdl-38047355

ABSTRACT

Occupational ApplicationsAn understanding of fluency in human-robot teaming from a physiological standpoint is still incomplete. In our experimental study involving 24 participants, we designed a scenario for shared-space human-robot collaboration (HRC) for a material sorting task. When compared to a sequential mode of interaction, the simultaneous mode resulted in significantly higher perceptions of fluency and engagement, primarily by reducing human idle time. These observations were complemented by significant changes in physiological responses, such as ECG entropy and low frequency power. These responses could predict fluency and engagement with accuracies of 90 and 97%, respectively. Notably, the perception of fluency and preferred mode of interaction were influenced by individual preferences. Hence, it is crucial to consider both physiological responses and user preferences when designing HRC systems, to ensure a positive experience with the robot teammate and to foster engagement in long-term teamwork. Furthermore, these signals can be obtained using a single robust, low-cost, and comfortable sensor.


Background In the current industry, a key enabler of flexible manufacturing is human-robot collaboration (HRC), a scenario wherein a human and a robot interact and work together in a shared space to accomplish a common task. In HRC, the timing and coordination between the human and robot are crucial factors that impact the fluency, efficacy, and acceptance of human-robot teams.Purpose Experimental research on the physiological indicators of fluency in human-robot collaborative tasks in a shared workspace is still in its infancy. We posit that by relating the mental perceptions of fluency to features from physiological signals, we could bring more clarity to the complex mapping between subjective and objective measures of fluency.Methods Twenty-four participants (12 males and 12 females), with mean (SD) age = 25.7 (2.9) years, completed an experimental study. We investigated the effects of interaction mode (sequential, simultaneous) and level of human involvement (low, medium, high) on perceived fluency, engagement, performance, and physiological response (heart rate variability = HRV) in a collaborative item sorting task.Results The simultaneous mode of interaction and a higher level of human involvement led to higher ratings for fluency and engagement, along with ECG changes, specifically an 11.6% increase in low-frequency power and a 3% reduction in information entropy. Using machine learning, these HRV features could predict perceived fluency and engagement with 90 and 97% accuracy, respectively.Conclusion Our results indicate that a human operator's perceived fluency in human-robot collaborative tasks can be measured using HRV metrics. Our findings expand the current fluency framework from a physiological perspective and offer additional objective measures derived from HRV, which could be practically applied to improve the design and optimization of HRC systems.

2.
J Neuroeng Rehabil ; 20(1): 43, 2023 04 12.
Article in English | MEDLINE | ID: mdl-37046310

ABSTRACT

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease without known cure. However, early medical treatment can help control its progression and postpone intellectual decay. Since AD is preceded by a period of cognitive deterioration, the effective assessment of cognitive capabilities is crucial to develop reliable screening procedures. For this purpose, cognitive tests are extensively used to evaluate cognitive areas such as language, attention, or memory. METHODS: In this work, we analyzed the potential of a visual dynamics evaluation, the rapid serial visual presentation task (RSVP), for the detection of cognitive impairment in AD. We compared this evaluation with two of the most extended brief cognitive tests applied in Spain: the Clock-drawing test (CDT) and the Phototest. For this purpose, we assessed a group of patients (mild AD and mild cognitive impairment) and controls, and we evaluated the ability of the three tests for the discrimination of the two groups. RESULTS: The preliminary results obtained suggest the RSVP performance is statistically higher for the controls than for the patients (p-value = 0.013). Furthermore, we obtained promising classification results for this test (mean accuracy of 0.91 with 95% confidence interval 0.72, 0.97). CONCLUSIONS: Since the RSVP is a computerized, auto-scored, and potentially self-administered brief test, it could contribute to speeding-up cognitive impairment screening and to reducing the associated costs. Furthermore, this evaluation could be combined with other tests to augment the efficiency of cognitive impairment screening protocols and to potentially monitor patients under medical treatment.


Subject(s)
Alzheimer Disease , Cognition Disorders , Cognitive Dysfunction , Neurodegenerative Diseases , Humans , Alzheimer Disease/diagnosis , Feasibility Studies , Cognitive Dysfunction/diagnosis , Neuropsychological Tests
3.
Cureus ; 14(10): e30793, 2022 Oct.
Article in English | MEDLINE | ID: mdl-36447686

ABSTRACT

Cecal appendix tumors are unusual and have a low frequency of presentation, which is reported between 0.2 and 0.5% of all digestive tract tumors. From the mentioned tumors, the carcinoid ones are the most common neoplasms of the cecal appendix and are characterized in most cases by slow growth and an asymptomatic clinical course. However, in some cases, they can present as a metastatic disease with fatal outcomes. We report the case of a 24-year-old female patient with a typical case of acute appendicitis, in whom an additional diagnosis of a carcinoid tumor is obtained upon receipt of the pathology report. The prognosis of appendicular carcinoid tumors is good, with a five-year survival rate of 95%-100% and a recurrence rate of less than 1%.

4.
Comput Methods Programs Biomed ; 220: 106841, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35523023

ABSTRACT

Early detection is critical to control Alzheimer's disease (AD) progression and postpone cognitive decline. Traditional medical procedures such as magnetic resonance imaging are costly, involve long waiting lists, and require complex analysis. Alternatively, for the past years, researchers have successfully evaluated AD detection approaches based on machine learning and electroencephalography (EEG). Nonetheless, these approaches frequently rely upon manual processing or involve non-portable EEG hardware. These aspects are suboptimal regarding automated diagnosis, since they require additional personnel and hinder portability. In this work, we report the preliminary evaluation of a self-driven AD multi-class discrimination approach based on a commercial EEG acquisition system using sixteen channels. For this purpose, we recorded the EEG of three groups of participants: mild AD, mild cognitive impairment (MCI) non-AD, and controls, and we implemented a self-driven analysis pipeline to discriminate the three groups. First, we applied automated artifact rejection algorithms to the EEG recordings. Then, we extracted power, entropy, and complexity features from the preprocessed epochs. Finally, we evaluated a multi-class classification problem using a multi-layer perceptron through leave-one-subject-out cross-validation. The preliminary results that we obtained are comparable to the best in literature (0.88 F1-score), what suggests that AD can potentially be detected through a self-driven approach based on commercial EEG and machine learning. We believe this work and further research could contribute to opening the door for the detection of AD in a single consultation session, therefore reducing the costs associated to AD screening and potentially advancing medical treatment.


Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Wearable Electronic Devices , Alzheimer Disease/diagnostic imaging , Cognitive Dysfunction/diagnosis , Electroencephalography/methods , Humans , Machine Learning
5.
Infectio ; 25(4): 212-240, oct.-dic. 2021. tab, graf
Article in English | LILACS, COLNAL | ID: biblio-1286716

ABSTRACT

Abstract Intra-abdominal infections are frequent at all levels of health care, therefore, it is necessary to maintain a high level of clinical suspicion, performing the fastest and most cost-effective measures to confirm the diagnosis and offer a precise and targeted multidisciplinary therapy, this being the only way to have an impact on the morbidity of this infection, reducing mortality and minimizing the complications and costs of health care. Intra-abdominal infections are linked to the appearance and selection of resistant mutants in both bacteria and fungi, becoming currently a major public health problem. Increasing bacterial resistance when associated with a greater possibility of difficulties in antimicrobial treatment increases mortality. This evidence-based consensus brings together the recommendations for the diagnosis and treatment of intra-abdominal infections in the pediatric and adult population. With strict monitoring of bacterial resistance and stimulating the control of the risk factors that have the greatest impact on the appearance of this phenomenon, this consensus is intended to be a practical guide that is easy to implement, and with periodic updates it will favor and facilitate multidisciplinary and the adequacy of the therapeutic management of intra-abdominal infections.


Resumen Las infecciones intrabdominales son frecuentes en todos los niveles de atención en salud, por ende, es necesario mantener un alto nivel de sospecha clínica, realizando las medidas más rápidas y costoefectivas para confirmar el diagnóstico y así ofrecer de una forma precisa y dirigida la terapéutica multidisciplinaria, siendo esta la única manera de tener impacto en la morbilidad de esta infección, disminuyendo la mortalidad y minimizando las complicaciones y los costos de la atención en salud. Las infecciones intrabdominales se encuentran ligadas a la aparición y selección de las mutantes resistentes tanto en las bacterias como en los hongos, convirtiéndose en la actualidad en una gran problemática en la salud pública. La creciente resistencia bacteriana al asociarse a mayor posibilidad de dificultades en el tratamiento antimicrobiano incrementa la mortalidad. Este consenso basado en la evidencia, reúne las recomendaciones en el diagnóstico y en el tratamiento de las infecciones intrabdominales en la población pediátrica y de adultos. Con un estricto seguimiento de la resistencia bacteriana y estimulando el control de los factores de riesgo que tienen mas impacto en la aparición de este fenómeno, este consenso pretende ser una practica guía de fácil implementación, y con periódicas actualizaciones favorecerá y facilitará el manejo multidisciplinario y la adecuación del manejo terapéutico de las infecciones intrabdominales.


Subject(s)
Humans , Child , Adult , Intraabdominal Infections , Peritonitis , Bacteria , Risk Factors , Mortality , Colombia , Sepsis , Delivery of Health Care , Infections , Anti-Bacterial Agents
6.
Sci Rep ; 11(1): 16323, 2021 08 11.
Article in English | MEDLINE | ID: mdl-34381080

ABSTRACT

Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.


Subject(s)
Cornea/cytology , Adult , Cell Differentiation/physiology , Conjunctiva/cytology , Cornea/pathology , Corneal Diseases/pathology , Epithelial Cells/cytology , Epithelium, Corneal/cytology , Humans , Limbus Corneae/cytology , Sequence Analysis, RNA/methods , Stem Cell Niche/physiology , Stem Cells/cytology , Transcriptome/physiology
7.
Transl Vis Sci Technol ; 9(3): 21, 2020 02 18.
Article in English | MEDLINE | ID: mdl-32714647

ABSTRACT

Purpose: Early in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retinal progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist. Methods: CRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the VSX2 (Progenitor), BRN3b (Ganglion), and RCVRN (Photoreceptor) stop codons with sequences encoding a viral P2A peptide fused to Cerulean, green fluorescent protein, and mCherry reporter genes, respectively, to generate a triple transgenic reporter hiPSC line called PGP1. This was accomplished by co-electroporating HDR templates and sgRNA/Cas9 vectors into hiPSCs followed by antibiotic selection. Functional validation of the PGP1 hiPSC line included the ability to generate retinal organoids, with all major retinal cell types, displaying the expression of the three fluorescent reporters consistent with the onset of target gene expression. Disaggregated organoids were also analyzed by fluorescence-activated cell sorting and fluorescent populations were tested for the expression of the targeted gene. Results: Retinal organoids formed from the PGP1 line expressed appropriate fluorescent proteins consistent with the differentiation of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types. Conclusions and Translational Relevance: The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.


Subject(s)
Induced Pluripotent Stem Cells , Animals , Cell Differentiation , Humans , Organoids , Photoreceptor Cells , Retina
8.
Int J Infect Control ; 16(3): 1-6, 2020 Jul 23.
Article in English | MEDLINE | ID: mdl-34135996

ABSTRACT

Infection control and antimicrobial stewardship programs (ICASPs) are essential to reduce the emergence and spread of antimicrobial resistance. The primary objective of this study was to assess the feasibility of extending a commercial off-the-shelf (COTS) software for ICASPs in low- and middle-income countries (LMICs). This project involved three hospitals in Colombia, including Centro Médico Imbanaco, Clínica San Francisco, and DIME Clínica Neurocardiovascular. A COTS platform (ILÚM Health Solutions™ Kenilworth, NJ) was extended to function in a range of technology settings, and translatable to almost any language. ICASP features were added, including clinical practice guidelines, hand hygiene (HH) documentation, and isolation precaution (IP) documentation. The platform was delivered as a smartphone mobile application ("app") for both iOS and Android. The app was successfully implemented at all sites, however, full back-end data integration was not feasible at any site. In contrast to the United States, a suite of surveillance tools and physician-focused decision support without patient data proved to be valuable. Language translation processing occurred quickly and incurred minimal costs. HH and IP compliance tracking were the most used features among ICASP staff; treatment guidelines were most often used by physicians. Use of the app streamlined activities and reduced the time spent on ICASP tasks. Users consistently reported positive impressions including simplicity of design, ease of navigation, and improved efficiency. This ICASP app was feasible in limited-resource settings, highly acceptable to users, and represents an innovative approach to antimicrobial resistance prevention.

9.
Prog. obstet. ginecol. (Ed. impr.) ; 62(6): 575-578, nov.-dic. 2019. ilus
Article in Spanish | IBECS | ID: ibc-192146

ABSTRACT

El síndrome de Ballantyne o síndrome en "espejo" es una rara patología caracterizada por presentar características clínicas similares entre feto y madre; no existe una incidencia aproximada reportada; hasta el 2010 se habían reportado aproximadamente 56 casos; en una última revisión sistemática en 2017 se informa casi el doble de casos reportados. El síndrome de Ballantyne fue descrito inicialmente en los casos de hidrops fetalis por isoinmunizacíon rhesus, pero se encuentra con mayor frecuencia asociado a hidrops fetalis no autoinmune de etiología desconocida. Este síndrome está asociado a múltiples complicaciones maternas como: edema pulmonar, síndrome de distres respiratorio del adulto, derrame pericárdico y falla renal. Presentamos el caso de una paciente de 34 años con síndrome de Ballantyne y preeclampsia severa; así mismo se identificó inmunoglobulina M para virus herpes


Ballantyne's syndrome or syndrome "in mirror" is a rare pathology characterized by presenting similar clinical characteristics between fetus and mother; there is no reported incidence; until 2010 approximately 56 cases had been reported; a last systematic review in 2017 reports almost twice as many cases. The Ballantyne's syndrome was initially described in the cases of hydrops fetalis by rhesus isoimmunization, but it is more frequently found associated with non-autoimmune hidrops fetalis of unknown etiology. This syndrome is associated with multiple maternal complications such as: pulmonary edema, adult respiratory distress syndrome, pericardial effusion and renal failure. We present the case of a 34-year-old patient with Ballantyne's syndrome and severe preeclampsia, as well as immunoglobulin M for herpes virus


Subject(s)
Humans , Female , Pregnancy , Infant, Newborn , Young Adult , Pregnancy Complications , Pre-Eclampsia , Fetal Death , Hydrops Fetalis , Edema , Severity of Illness Index , Immunoglobulin M/blood , Syndrome
10.
Article in Spanish | LILACS-Express | LILACS | ID: biblio-1010179

ABSTRACT

La angina de Ludwig es una celulitis potencialmente mortal, rápidamente progresiva que involucra el piso de boca y la región cervico-facial. Se caracteriza por una induración y afectación del espacio submandibular, sublingual y submental. Cursa con alteraciones sistémicas y metabólicas. Por lo general, no se observan abscesos o linfadenopatías en la descripción clásica, sin embargo la progresión a la formación de abscesos dentro del espacio involucrado y los espacios contiguos es frecuente. La etiología en la mayoría de casos es odontogénica. El tratamiento debe priorizar el manejo de la vía aérea, drenaje quirúrgico, eliminación de la causa de infección, antibioticoterapia rápida agresiva y el control metabólico con soporte hídrico electrolítico. Se presenta el caso de una paciente mujer con antecedentes de anemia y odontalgia de pieza dentaria del maxilar inferior. Acudió por el servicio de emergencia con un aumento de volumen de tercio inferior facial, con predominio de región submandibular bilateral, submental y sublingual. Mal estado general, fiebre, taquicardia, disfagia, odinofagia y leucocitosis. Se diagnosticó angina de Ludwig. Se realizó el manejo de la vía aérea, drenaje intraoral, drenaje extraoral, exodoncia, antibioticoterapia inmediata y soporte hídrico electrolítico. Evolucionó favorablemente y posteriormente fue dada de alta sin complicaciones. Palabras clave: Angina de Ludwig; Celulitis; Mandíbula.


Ludwig's angina is a life-threatening, rapidly progressive cellulitis that involves the floor of the mouth and the cervico-facial region. With induration and involvement of bilateral submandibular space, sublingual bilateral and submental. It presents with systemic and metabolic alterations. In general, no abscess or lymphadenopathy is seen in the classic description However, progression to abscess formation within the space involved and contiguous spaces is common. The etiology in most cases is odontogenic. Treatment should prioritize the management of the airway, surgical drainage, elimination of the cause of infection, aggressive rapid antibiotic therapy and metabolic control with electrolytic water support. A female patient with a history of anemia presented odontalgia of the lower jaw teeth. She came the emergency room with an increase in volume of the lower third of the face. Predominantly on submandibular bilateral, submental and sublingual regions, as well as scanted general condition, fever, tachycardia, dysphagia, odynophagia and leukocytosis. The diagnosis of Ludwig's angina was reached. Management of the airway, intraoral drainage, extraoral drainage, exodontia, immediate antibiotic therapy and electrolytic water support; were performed. She evolved favorably and was later discharged without complications. Keywords: Cellulitis; Ludwig's angina; Mandible.

11.
Dev Biol ; 428(1): 88-100, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28576690

ABSTRACT

Complement components have been implicated in a wide variety of functions including neurogenesis, proliferation, cell migration, differentiation, cancer, and more recently early development and regeneration. Following our initial observations indicating that C3a/C3aR signaling induces chick retina regeneration, we analyzed its role in chick eye morphogenesis. During eye development, the optic vesicle (OV) invaginates to generate a bilayer optic cup (OC) that gives rise to the retinal pigmented epithelium (RPE) and neural retina. We show by immunofluorescence staining that C3 and the receptor for C3a (the cleaved and active form of C3), C3aR, are present in chick embryos during eye morphogenesis in the OV and OC. Interestingly, C3aR is mainly localized in the nuclear compartment at the OC stage. Loss of function studies at the OV stage using morpholinos or a blocking antibody targeting the C3aR (anti-C3aR Ab), causes eye defects such as microphthalmia and defects in the ventral portion of the eye that result in coloboma. Such defects were not observed when C3aR was disrupted at the OC stage. Histological analysis demonstrated that microphthalmic eyes were unable to generate a normal optic stalk or a closed OC. The dorsal/ventral patterning defects were accompanied by an expansion of the ventral markers Pax2, cVax and retinoic acid synthesizing enzyme raldh-3 (aldh1a3) domains, an absence of the dorsal expression of Tbx5 and raldh-1 (aldh1a1) and a re-specification of the ventral RPE to neuroepithelium. In addition, the eyes showed overall decreased expression of Gli1 and a change in distribution of nuclear ß-catenin, suggesting that Shh and Wnt pathways have been affected. Finally, we observed prominent cell death along with a decrease in proliferating cells, indicating that both processes contribute to the microphthalmic phenotype. Together our results show that C3aR is necessary for the proper morphogenesis of the OC. This is the first report implicating C3aR in eye development, revealing an unsuspected hitherto regulator for proper chick eye morphogenesis.


Subject(s)
Body Patterning/physiology , Complement C3a/metabolism , Gene Expression Regulation, Developmental , Receptors, Complement/metabolism , Retinal Pigment Epithelium/embryology , Aldehyde Dehydrogenase/metabolism , Animals , Apoptosis/physiology , Cell Proliferation/physiology , Chick Embryo , Hedgehog Proteins/metabolism , Microphthalmos/embryology , Morphogenesis/physiology , PAX2 Transcription Factor/metabolism , Receptors, Complement/genetics , Retinal Dehydrogenase/metabolism , T-Box Domain Proteins/metabolism , Wnt Signaling Pathway/physiology , Zinc Finger Protein GLI1/biosynthesis , beta Catenin/metabolism
12.
Rev. peru. ginecol. obstet. (En línea) ; 62(4): 467-468, oct. 2016.
Article in Spanish | LILACS | ID: biblio-991531
13.
Development ; 143(23): 4368-4380, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27660325

ABSTRACT

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.


Subject(s)
Cell Differentiation/physiology , Cell Self Renewal/physiology , Embryonic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Tankyrases/antagonists & inhibitors , Wnt Signaling Pathway/physiology , Animals , Bone Morphogenetic Protein 4/metabolism , Cells, Cultured , Cellular Reprogramming/physiology , Germ Layers/embryology , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Humans , Janus Kinases/metabolism , Leukemia Inhibitory Factor/metabolism , Mice , STAT3 Transcription Factor/metabolism
14.
J Vis Exp ; (105): e52002, 2015 Nov 02.
Article in English | MEDLINE | ID: mdl-26556302

ABSTRACT

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.


Subject(s)
Electroporation/methods , Retina/physiology , Transfection/methods , Animals , Chick Embryo , Photoreceptor Cells, Vertebrate/cytology , Photoreceptor Cells, Vertebrate/physiology , Plasmids/administration & dosage , Plasmids/genetics , Primary Cell Culture , Reproducibility of Results , Retina/cytology
15.
Nat Commun ; 5: 4047, 2014 Jun 10.
Article in English | MEDLINE | ID: mdl-24915161

ABSTRACT

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies.


Subject(s)
Induced Pluripotent Stem Cells/cytology , Photoreceptor Cells, Vertebrate/physiology , Retina/cytology , Cell Differentiation , Cell Line , Humans , Retina/physiology
16.
Exp Eye Res ; 109: 40-50, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23370269

ABSTRACT

Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22-25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner.


Subject(s)
Electroporation/instrumentation , Electroporation/methods , Retina/cytology , Retina/physiology , Transfection/instrumentation , Transfection/methods , Animals , Cell Count , Chick Embryo , Chickens , Eye Proteins/genetics , Gene Expression Regulation, Developmental/genetics , Green Fluorescent Proteins/genetics , Homeodomain Proteins/genetics , PAX6 Transcription Factor , Paired Box Transcription Factors/genetics , Plasmids/genetics , Plasmids/pharmacokinetics , Primary Cell Culture/methods , Repressor Proteins/genetics
17.
Lab Chip ; 13(4): 554-61, 2013 Feb 21.
Article in English | MEDLINE | ID: mdl-23160191

ABSTRACT

A Parylene C neural probe with a three dimensional sheath structure was designed, fabricated, and characterized. Multiple platinum (Pt) electrodes for recording neural signals were fabricated on both inner and outer surfaces of the sheath structure. Thermoforming of Parylene was used to create the three dimensional sheath structures from flat surface micromachined microchannels using solid microwires as molds. Benchtop electrochemical characterization was performed on the thin film Pt electrodes using cyclic voltammetry and electrochemical impedance spectroscopy and showed that electrodes possessed low impedances suitable for neuronal recordings. A procedure for implantation of the neural probe was developed and successfully demonstrated in vitro into an agarose brain tissue model. The electrode-lined sheath will be decorated with eluting neurotrophic factors to promote in vivo neural tissue ingrowth post-implantation. These features will enhance tissue integration and improve recording quality towards realizing reliable chronic neural interfaces.


Subject(s)
Electrodes, Implanted , Neurons/physiology , Polymers/chemistry , Xylenes/chemistry , Electric Stimulation , Platinum/chemistry
18.
Rev. Fac. Med. (Bogotá) ; 60(3): 5-29, set.-dic. 2012. ilus, graf, tab
Article in Spanish | LILACS | ID: lil-669255

ABSTRACT

Antecedentes. Por ser la enfermedad hipertensiva de carácter asintomático, el problema de la adherencia al tratamiento de pacientes hipertensos puede ser grave. Objetivo. Estudiar la adherencia al tratamiento de los pacientes hipertensos atendidos en la ciudad de Manizales (Colombia) por el programa ASSBASALUD E.S.E en el año 2011. Materiales y métodos. Estudio de corte transversal en el cual se empleó una población de 200 personas hipertensas (73,5% femenino, edad media 63,76 años) atendidas en ASSBASALUD ESE, Manizales, en el segundo semestre de 2011. Se empleó el cuestionario Martín-Bayarre-Grau (MBG) y el cuestionario de Morinsky-Green para evaluar la red de apoyo social y se empleó el cuestionario Medical OutcomesStudy (MOS). Resultados. Adherentes totales 45% según Morinsky-Green, adherentes totales 51% según MBG, respecto al cuestionario MOS se tiene, 12,29 personas en promedio en su red de apoyo social, apoyo emocional de 74,83%, ayuda material 80,45%, relaciones sociales de ocio y distracción de 78,61%, apoyo afectivo de 83,28%, el fármaco más empleado fue enalapril 17,9%, seguido de verapamilo 10,1%. La adherencia según el cuestionario MBG mostró dependencia significativa entre variables como: educación (p=0,000), conocimientos de la patología (p=0,032), y con los resultados del cuestionario de apoyo social, MOS (p=0,000). La adherencia según Morinsky-Green mostró muy pocas relaciones significativas. Conclusión. La población en estudio presenta bajos niveles de adherencia asociada a baja educación, bajos conocimientos de la patología, bajo apoyo social, se hace necesario una intervención efectiva de ASSBASALUD ESE, a través de su personal de salud. El cuestionario MBG mostró mayor consistencia en la descripción de la adherencia que el cuestionario de Morinsky-Green.


Background. Difficulties regarding patient adherence when treating hypertension could be extremely serious as one is dealing with an asymptomatic hypertensive disease. Objective. Studying adherence to treatment concerning hypertensive patients being attended in Manizales, Colombia, by the state-run Assbasalud programme in 2011. Materials and Methods. This was a cross-sectional study involving a population of 200 hypertensive people (73.5% were female, average age was 63.76 years) being attended by the state-run Assbasalud ESE, Manizales, during the second half of 2011. The Martín-Bayarre-Grau (MBG) and Morisky-Green (MG) questionnaires were used for evaluating the social support network, as well as the Medical Outcomes Study (MOS) questionnaire. Results.45% of patients were totally adherent according to MG and 51% totally adherent according to MBG. Regarding the MOS questionnaire, 12.29 people on average were in a patient's social support network, 74.83% received emotional support, 80.45% material aid, 78.61% were involved in leisure and entertainment-related activities, 83.28% were receiving affective support and enalapril was the drug most used in treatment (17.9%), followed by verapamil (10.1%). According to the MBG questionnaire, adherence significantly depended on variables such as education (p=0.000), knowledge about the disease (p=0.032) and MOS social support questionnaire results (p=0.000). The MG questionnaire revealed very few significant relationships for treatment adherence. Conclusion. The study revealed low adherence levels associated with having a low educational level, poor knowledge regarding the disease and poor social support, thereby making it necessary that Assbasalud ESE take more effective action, especially through its healthcare personnel. The MBG questionnaire had greater consistency regarding a description of adherence than the MG questionnaire.

19.
Article in English | MEDLINE | ID: mdl-23367082

ABSTRACT

We present the preliminary electrochemical characterization of 3D Parylene C sheath microelectrode array probes towards realizing reliable chronic neuroprosthetic recordings. Electrochemical techniques were used to verify electrode integrity after our novel post-fabrication thermoforming process was applied to flat surface micromachined structures to achieve a hollow sheath probe shape. Characterization of subsequent neurotrophic coatings was performed and accelerated life testing was used to simulate six months in vivo. Prior to probe implantation, crosstalk was measured and electrode surface properties were evaluated through the use of electrochemical impedance spectroscopy.


Subject(s)
Coated Materials, Biocompatible/chemistry , Electrodes, Implanted , Microelectrodes , Polymers/chemistry , Xylenes/chemistry , Electric Impedance , Electrochemistry/methods , Equipment Design , Equipment Failure Analysis , Materials Testing , Prosthesis Implantation , Reproducibility of Results , Sensitivity and Specificity
20.
BMC Dev Biol ; 11: 46, 2011 Jul 22.
Article in English | MEDLINE | ID: mdl-21781303

ABSTRACT

BACKGROUND: Understanding the mechanisms governing cell fate specification remains one of the main challenges in the study of retinal development. In this context, molecular markers that identify specific cell types become crucial tools for the analysis and interpretation of these phenomena. In studies using the developing chick retina, expression of the mid-size neurofilament (NF-M) and a chick-specific microtubule associated protein recognized by the RA4 antibody (MAP(RA4)), have been broadly used to selectively identify ganglion cells and their committed precursors. However, observations in our laboratory suggested that the expression of these proteins may not be restricted to cells of the ganglion cell lineage. Because of its potential significance in the field, we pursued a detailed analysis of the expression of these two molecules in combination with an array of proteins that allowed precise identification of all retinal cell-type precursors throughout the development of the chick retina. RESULTS: Both, NF-M and MAP(RA4) proteins, showed a dynamic pattern of expression coincident with the progression of retinal cell differentiation. Both proteins were coexpressed spatially and temporally in postmitotic neuronal precursors throughout development. Expression of both proteins was seen in ganglion cell precursors and adult differentiated ganglion cells, but they were also transiently expressed by precursors of the photoreceptor, horizontal, bipolar and amacrine cell lineages. CONCLUSIONS: We have clearly demonstrated that, contrary to the generally accepted paradigm, expression of NF-M and MAP(RA4) proteins is not exclusive to ganglion cells. Rather, both proteins are transiently expressed by all neuronal retinal progenitors in a developmentally-regulated manner. In addition, MAP(RA4) and NF-M are the first molecules so far characterized that may allow unambiguous identification of postmitotic precursors from the pool of mitotically active progenitors and/or the differentiated cell population during retinogenesis. These results are of significant impact for the field of developmental biology of the retina, since they provide novel and important information for the appropriate design and interpretation of studies on retinal cell differentiation, as well as for the reinterpretation of previously published studies.


Subject(s)
Cytoskeletal Proteins/metabolism , Neural Stem Cells/metabolism , Retinal Ganglion Cells/metabolism , Amacrine Cells/cytology , Amacrine Cells/metabolism , Animals , Cell Differentiation , Cell Lineage , Chickens , Cytoskeleton/metabolism , Intermediate Filaments/metabolism , Neurons/cytology , Neurons/metabolism , Retina/cytology , Retina/metabolism , Retinal Ganglion Cells/cytology
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