Transgenic Overexpression of Myocilin Leads to Variable Ocular Anterior Segment and Retinal Alterations Associated with Extracellular Matrix Abnormalities in Adult Zebrafish

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.This research was funded by research grants from the “Instituto de Salud Carlos III/European Regional Development Fund (ERDF)” (PI19/00208 and RD16/0008/0019, OFTARED), the Regional Ministry of Science and Technology of the Board of the Communities of “Castilla-La Mancha” (SBPLY/17/180501/000404; http://www.educa.jccm.es/idiuniv/es, accessed on 3 March 2022) and research funds from Universidad de Castilla-La Mancha (2019-GRIN-26945). A.T. was recipient of a predoctoral contract from Castilla-La Mancha University (Ref.: 2020-PREDUCLM-16605)

Coartación aórtica ± hipoplasia del arco en neonatos y lactantes, ¿abordaje por esternotomía o toracotomía? Una decisión compleja

ResumenObjetivosLa reparación de la coartación aórtica con/sin hipoplasia de arco en neonatos/lactantes es controvertida. Los abordajes quirúrgicos mediante toracotomía o esternotomía, respectivamente, evitan o requieren circulación extracorpórea. Presentamos nuestros resultados, analizando si existen diferencias significativas entre ambas técnicas.MétodosEstudio retrospectivo; 132 lactantes con anatomía de corazón biventricular y cirugía del arco aórtico durante el período 2004-2014: 65-esternotomías y 67 toracotomías. En las esternotomías usamos rutinariamente perfusión cerebral selectiva (PCS). La parada circulatoria es excepcional.ResultadosGrupos no homogéneos: el abordaje esternal asocia más cardiopatía, más hipoplasia de arco y mayor complejidad. Técnicas quirúrgicas: por esternotomía 83% pacientes recibieron anastomosis término-lateral y por toracotomía 87% anastomosis termino-terminal. Mortalidad hospitalaria: 10,7% esternotomía y 4,5% toracotomía (ns). Morbilidad hospitalaria: sin diferencias en complicaciones neurológicas ni en recoartación precoz, pero las esternotomías tienen más lesión del nervio recurrente laríngeo y mayores estancias hospitalarias que las toracotomías (p<0,05). Seguimiento medio: 37±26 meses. Tres pacientes mueren tardíamente (1 esternotomía, 2 toracotomía). Las toracotomías presentan más recoartación tardía (30%) que las esternotomías (17%) (ns) y habitualmente se soluciona con angioplastia percutánea.ConclusionesLa PCS en cirugía del arco por esternotomía permite realizar la anastomosis con precisión. Respecto a la toracotomía, no hay diferencia significativa en la mortalidad hospitalaria ni en las secuelas neurológicas, pero sí asocia mayor lesión recurrencial y un postoperatorio prolongado. La permeabilidad del arco a largo plazo es mejor con la esternotomía pero esta diferencia no resultó significativa. Recomendamos esternotomía media con PCS si existe hipoplasia del arco proximal y/o si hay cardiopatía asociada que requiera cirugía.AbstractObjectivesSurgical approach of infants with coarctation±hypoplastic aortic arch is a challenge. Choosing between sternotomy or thoracotomy is associated, respectively, with the need of extracorporeal circulation or not. We present our results and compare both surgical techniques.MethodsA retrospective analysis was conducted on 132 infants with biventricular heart and surgical reconstruction of aortic arch during 2004-2014: 65-sternotomy and 67-thoracotomy. Selective cerebral perfusion (SCP) was used in the sternotomy group instead of circulatory arrest in order to minimize cerebral injury.ResultsBoth groups are different: sternotomy-group has more associated cardiac diseases, more hypoplastic arch, and higher complexity. Surgical techniques: sternotomy patients received an end-to-side anastomosis (83%), and thoracotomy patients received an end-to-end anastomosis (87%). Hospital mortality: 10.7% in sternotomy and 4.5% in thoracotomy (ns). Hospital morbidity: there are no differences in neurological complications or early re-coarctation, but sternotomy-group presents more recurrent laryngeal nerve lesion and longer postoperative stay than thoracotomies (P<.05). Mean follow-up: 37±26 months. Three patients died in the follow-up (1-sternotomy, 2-thoracotomy). Late re-coarctation is more frequent in thoracotomy (30%) than in sternotomy (17%) (ns) and is generally treated with percutaneous angioplasty.ConclusionsSCP in sternotomy approach of arch surgery allows the surgeon to perform the anastomosis in a bloodless field, and preserves neurological function. Sternotomy, in comparison with thoracotomy, has similar mortality, a higher recurrent nerve lesion, and prolonged hospital stay. Sternotomy seems to be associated with a lower incidence of re-coarctation in the follow-up. Sternotomy with SCP is recommended if there is hypoplastic aortic arch and/or associated cardiac diseases needing surgery

Local root ABA/cytokinin status and aquaporins regulate poplar responses to mild drought stress independently of the ectomycorrhizal fungus Laccaria bicolor

The relatively better performance of mycorrhizal plants subjected to drought stress has commonly been linked to improved root water uptake through the fungal regulation of plant aquaporins and hormones. In this study, we examined the role of ectomycorrhizal fungi in plant water relations and plant hormonal balance under mild drought using split-root seedlings of Populus trichocarpa x deltoides either with or without inoculation with Laccaria bicolor. The root compartments where the drought treatment was applied had higher ABA and lower cytokinin tZR contents, and greater expression of the plant aquaporins PtPIP1;1, PtPIP1;2, PtPIP2;5, and PtPIP2;7. On the other hand, the presence of L. bicolor within the roots down-regulated PtPIP1;4, PtPIP2;3, and PtPIP2;10, and reduced the abundance of PIP2 proteins. In addition, expression of the fungal aquaporins JQ585595 and JQ585596 were positively correlated with root ABA content, while tZR content was positively correlated with PtPIP1;4 and negatively correlated with PtPIP2;7. The results demonstrate a coordinated plant-fungal system that regulates the different mechanisms involved in water uptake in ectomycorrhizal poplar plants

Detection of Pathogens Using Microfluidics and Biosensors

Point-of-care devices technology are a promising way towards the recognition of pathogens in early-stage diagnosis, which is critical for the success of inexpensive treatments as opposed to the high costs of managing the disease. The integration of immunoassays with read out circuitry allows the implementation of diagnostic devices for their use by untrained personnel, without compromising reliability. In the following chapter, three different biosensors based on lab-on-a-chip (LoC) and microfluidic technologies were designed, assembled and tested for pathogen diagnosis. The devices allowed the effective detection of the human papilloma virus, Mycobacterium tuberculosis and Chagas parasite in shorter times and with smaller sample volumes than those required by current clinical diagnosis techniques. All devices were benchmarked against commercial techniques in terms of cost and time requirement per test

Validation of a diagnostic support system for diabetic retinopathy based on clinical parameters

Purpose: To validate a clinical decision support system (CDSS) that estimates risk of diabetic retinopathy (DR) and to personalize screening protocols in type 2 diabetes mellitus (T2DM) patients. Methods: We utilized a CDSS based on a fuzzy random forest, integrated by fuzzy decision trees with the following variables: current age, sex, arterial hypertension, diabetes duration and treatment, HbA1c, glomerular filtration rate, microalbuminuria, and body mass index. Validation was made using the electronic health records of a sample of 101,802 T2DM patients. Diagnosis was made by retinal photographs, according to EURODIAB guidelines and the International Diabetic Retinopathy Classification. Results: The prevalence of DR was 19,759 patients (19.98%). Results yielded 16,593 (16.31%) true positives, 72,617 (71.33%) true negatives, 3165 (3.1%) false positives, and 9427 (9.26%) false negatives, with an accuracy of 0.876 (95% confidence interval [CI], 0.858–0.886), sensitivity of 84% (95% CI, 83.46–84.49), specificity of 88.5% (95% CI, 88.29–88.72), positive predictive value of 63.8% (95% CI, 63.18–64.35), negative predictive value of 95.8% (95% CI, 95.68–95.96), positive likelihood ratio of 7.30, and negative likelihood ratio of 0.18. The type 1 error was 0.115, and the type 2 error was 0.16. Conclusions: We confirmed a good prediction rate for DR from a representative sample of T2DM in our population. Furthermore, the CDSS was able to offer an individualized screening protocol for each patient according to the calculated risk confidence value. Translational Relevance: Results from this study will help to establish a novel strategy for personalizing screening for DR according to patient risk factors.The authors thank Phil Hoddy for his language assistance and for editing and correcting the English text. Supported by Instituto de Investigaciones Carlos III (research projects PI18/00169, PI12/01535, and PI15/01150) and by the European Regional Development Fund (FEDER).The authors thank Phil Hoddy for his language assistance and for editing and correcting the English text. Supported by Instituto de Investigaciones Carlos III (research projects PI18/00169, PI12/01535, and PI15/01150) and by the European Regional Development Fund (FEDER)

Involvement of plant endogenous ABA in Bacillus megaterium PGPR activity in tomato plants

BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria which benefit plants by improving plant productivity and immunity. The mechanisms involved in these processes include the regulation of plant hormone levels such as ethylene and abscisic acid (ABA). The aim of the present study was to determine whether the activity of Bacillus megaterium PGPR is affected by the endogenous ABA content of the host plant. The ABA-deficient tomato mutants flacca and sitiens and their near-isogenic wild-type parental lines were used. Growth, stomatal conductance, shoot hormone concentration, competition assay for colonization of tomato root tips, and root expression of plant genes expected to be modulated by ABA and PGPR were examined. RESULTS: Contrary to the wild-type plants in which PGPR stimulated growth rates, PGPR caused growth inhibition in ABA-deficient mutant plants. PGPR also triggered an over accumulation of ethylene in ABA-deficient plants which correlated with a higher expression of the pathogenesis-related gene Sl-PR1b. CONCLUSIONS: Positive correlation between over-accumulation of ethylene and a higher expression of Sl-PR1b in ABA-deficient mutant plants could indicate that maintenance of normal plant endogenous ABA content may be essential for the growth promoting action of B. megaterium by keeping low levels of ethylene production