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.
► We describe a high efficiency electroporation method for chick retinal cell cultures. ► It yields 5-fold higher transfection efficiencies than currently available protocols. ► It can be coupled to high-throughput, single cell analysis. ► Its features provide high-power detection for cell-autonomous gene function studies.
In 2013, the Pan American Health Organization established a multi-site, multi-country network to evaluate influenza vaccine effectiveness (VE). We pooled data from five consecutive seasons in five ...countries to conduct an analysis of southern hemisphere VE against laboratory-confirmed influenza hospitalizations in young children and older adults.
We used a test-negative design to estimate VE against laboratory-confirmed influenza in hospitalized young children (aged 6─24 months) and older adults (aged ≥60 years) in Argentina, Brazil, Chile, Colombia, and Paraguay. Following country-specific influenza surveillance protocol, hospitalized persons with severe acute respiratory infections (SARI) at 48 sentinel hospitals (March 2013–December 2017) were tested for influenza virus infection by rRT-PCR. VE was estimated for young children and older adults using logistic random effects models accounting for cluster (country), adjusting for sex, age (months for children, and age-in-year categories for adults), calendar year, country, preexisting conditions, month of illness onset and prior vaccination as an effect modifier for the analysis in adults.
We included 8426 SARI cases (2389 children and 6037 adults) in the VE analyses. Among young children, VE against SARI hospitalization associated with any influenza virus was 43% (95%CI: 33%, 51%) for children who received two doses, but was 20% (95%CI: −16%, 45%) and not statistically significant for those who received one dose in a given season. Among older adults, overall VE against SARI hospitalization associated with any influenza virus was 41% (95%CI: 28%, 52%), 45% (95%CI: 34%, 53%) against A(H3N2), 40% (95%CI: 18%, 56%) against A(H1N1)pdm09, and 20% (95%CI: −40%, 54%) against influenza B viruses.
Our results suggest that over the five-year study period, influenza vaccination programs in five South American countries prevented more than one-third of laboratory confirmed influenza-associated hospitalizations in young children receiving the recommended two doses and vaccinated older adults.
What is already known about this topic? Influenza transmission has changed during the COVID-19 pandemic. What is added by this report? In 2022, influenza A(H3N2) virus, clade 3C.2a1b.2a.2, circulated ...in Chile months earlier than during prepandemic influenza seasons and was associated with 1,002 hospitalizations. Influenza vaccination reduced risk for A(H3N2) virus hospitalization by 49%. What are the implications for public health practice? Like certain Southern Hemisphere countries during the 2022 influenza season, Northern Hemisphere countries might face influenza activity with atypical timing and intensity during the 2022–23 season. Health authorities should encourage all eligible persons to seek influenza vaccination and take precautions to reduce transmission of influenza (e.g., avoiding close contact with persons who are ill).
Retinal regeneration research holds potential for providing new avenues for the treatment of degenerative diseases of the retina. Various animal models have been used to study retinal regeneration ...over the years, providing insights into different aspects of this process. However the mechanisms that drive this important phenomenon remain to be fully elucidated. In the present study, we introduce and characterize a new model system for retinal regeneration research that uses the tadpole of the African clawed frog, Xenopus laevis.
The neural retina was surgically removed from Xenopus laevis tadpoles at stages 51-54, and a heparin-coated bead soaked in fibroblast growth factor 2 (FGF-2) was introduced in the eyes to induce regeneration. Histological and immunohistochemical analyses as well as DiI tracing were performed to characterize the regenerate. A similar surgical approach but with concomitant removal of the anterior portion of the eye was used to assess the capacity of the retinal pigmented epithelium (RPE) to regenerate a retina. Immunohistochemistry for FGF receptors 1 and 2 and phosphorylated extracellular signal-regulated protein kinase (pERK) was performed to start elucidating the intracellular mechanisms involved in this process. The role of the mitogen activated protein kinase (MAPK) pathway was confirmed through a pharmacological approach using the MAPK kinase (MEK) inhibitor U0126.
We observed that Xenopus laevis tadpoles were able to regenerate a neural retina upon induction with FGF-2 in vivo. The regenerated tissue has the characteristics of a differentiated retina, as assessed by the presence and distribution of different retinal cell markers, and DiI tracing indicated that it is able to form an optic nerve. We also showed that retinal regeneration in this system could take place independently of the presence of the anterior eye tissues. Finally, we demonstrated that FGF-2 treatment induces ERK phosphorylation in the pigmented epithelia 10 days after retinectomy, and that inhibition of the MAPK pathway significantly decreases the amount of retina regenerated at 30 days post-operation.
Regeneration of a complete neural retina can be achieved in larval Xenopus laevis through activation of the MAPK signaling pathway by administering exogenous FGF-2. This mechanism is conserved in other animal models, which can regenerate their retina via pigmented epithelium transdifferentiation. Our results provide an alternative approach to retinal regeneration studies, capitalizing on the advantages of the Xenopus laevis tadpole as a model system.
Background
Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), a hematopoietic growth factor, has shown therapeutic and neuroprotective effects in animal models of Alzheimer’s disease (AD), ...Parkinson’s disease, Down syndrome, and stroke. In a retrospective study, we reported that recombinant human GM‐CSF (sargramostim) treatment led to improved cognition in patients with cognitive impairment due to chemotherapy. Our recently completed Phase 2 clinical trial in AD showed that sargramostim treatment increased toward normal both MMSE scores and plasma biomarkers of neurodegeneration. Cohen et al. (2013) developed a transgenic rat model (TgF344‐AD) that expresses the Swedish mutant human APP (APPsw) and mutant human presenilin 1 (PSEN1) genes that cause familial AD. Interestingly, in addition to cerebral amyloidosis, neuronal loss, and cognitive deficits, TgF344‐AD rats also show age‐dependent tauopathy including overexpression of phosphorylated tau and neurofibrillary tangle formation, which do not arise in mouse models of AD. To evaluate the potential ability of GM‐CSF to reverse and/or prevent the full range of AD pathology, we treated ∼20‐month‐old TgF344‐AD rats with GM‐CSF or saline, and investigated AD pathology in the brain and retina. We also analyzed biomarkers in plasma using Meso Scale Discovery (MSD) and SIMOA neuroinflammation panels.
Method
TgF344‐AD rats were injected subcutaneously with GM‐CSF (83.3 ug/kg/day; 5 days/week) or with saline (200 ul/day) for 24 injections total over 32 days. On day 32, blood samples were collected, plasma samples were isolated, and brain and retina tissues were processed for immunohistochemistry and immunoblotting.
Result
TgF344‐AD rats treated with GM‐CSF showed a significant reduction in amyloid plaque deposition in the cortex compared to saline‐treated TgF344‐AD rats. We also observed a trend of reduced amyloid plaques in the CA region of the hippocampus (p=0.06) in GM‐CSF‐treated TgF344‐AD rats. In ongoing experiments, we are investigating the effects of GM‐CSF treatment on tauopathy and neuroinflammation in the brain and retina and on plasma biomarkers.
Conclusion
Although experiments are in progress, our preliminary findings indicate that GM‐CSF treatment rescues some AD pathology in TgF344‐AD rats. Complete results will determine the therapeutic potential for GM‐CSF/sargramostim/Leukine® not only for reducing amyloidosis, but also on other relevant pathological manifestations of AD.
In this study, we propose a predictive model for maceral discrimination based on Raman spectroscopic analyses of dispersed organic matter. Raman micro-spectroscopy was coupled with optical and ...Rock-Eval pyrolysis analyses on a set of seven samples collected from Mesozoic and Cenozoic successions of the Outer sector of the Carpathian fold and thrust belt. Organic petrography and Rock-Eval pyrolysis evidence a type II/III kerogen with complex organofacies composed by the coal maceral groups of the vitrinite, inertinite, and liptinite, while thermal maturity lies at the onset of the oil window spanning between 0.42 and 0.61 Ro%. Micro-Raman analyses were performed, on approximately 30–100 spectra per sample but only for relatively few fragments was it possible to perform an optical classification according to their macerals group. A multivariate statistical analysis of the identified vitrinite and inertinite spectra allows to define the variability of the organofacies and develop a predictive PLS-DA model for the identification of vitrinite from Raman spectra. Following the first attempts made in the last years, this work outlines how machine learning techniques have become a useful support for classical petrography analyses in thermal maturity assessment.
Abstract only
The development of stem cell technologies has opened up new opportunities for research and therapeutic development that, until recently, were unavailable. In particular, the development ...of human stem cell derived 3D tissues or “organoids” that recapitulate the native histoarchitecture, and to some degree the native functionality, have provided incredible advancements in our understanding of human physiology and disease. In this study we take advantage of retinal organoids as a new model to study the mechanisms of cell death affecting retinal ganglion cells during human development.
Human induced pluripotent stem cells (hiPSC) were directed to follow a neuronal lineage and were then further differentiated into 3D retinal tissue. Retinal organoids were collected at various time points during development and analyzed using Western blot and immunofluorescent staining for retinal ganglion cells and programmed cell death markers. Our results show the role of programmed cell death as a mechanism to control retinal ganglion cell numbers during development. This has important consequences for understanding not only basic developmental processes, but also the basis of congenital retinal abnormalities that can lead to diseases of vision. Moreover, this knowledge has potential impact for translational research using retinal organoid models.
Support or Funding Information
This work was supported, in part, by a Challenge Grant to the Department of Ophthalmology at the University of Colorado from Research to Prevent Blindness.
Electrospinning was used to produce fibrous membranes, in single and multiple layers, from poly(ε-caprolactone), pullulan, and from mixtures of poly(ε-caprolactone) with potato modified starch and ...β-glucan. It was possible to obtain single-layer membranes from solutions of pullulan in water, poly(ε-caprolactone) in chloroform, and from mixtures of poly(ε-caprolactone)/β-glucan and poly(ε-caprolactone)/potato modified starch in chloroform. Scanning electron microscopy images showed the formation of ultrathin homogeneous fibers from electrospun poly(ε-caprolactone) and pullulan, whereas the fibers obtained from mixtures of poly(ε-caprolactone)/ β -glucan and poly(ε-caprolactone)/potato modified starch had different sizes and morphologies, as well as irregular microstructures, characterized by the presence of beads. Contact angle analyses showed that pullulan membranes were extremely hydrophilic, while poly(ε-caprolactone) membranes were predominantly hydrophobic. Subsequently, poly(ε-caprolactone)-pullulan-poly(ε-caprolactone) multilayer membranes, with intermediate wettability, were prepared by successive electrospinning steps. Infrared spectroscopy and calorimetric analyses showed the presence of both polymers and the absence of changes in their structure and stability due to electrospinning, indicating adequate compatibility between the two polymers. We foresee that the polyester-polysaccharide multilayer membrane might be used as a biodegradable vehicle for active agents with different hydrophobicity, with applications as food packaging and biocompatible scaffold materials.
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.
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