Caracterización de glicosidasas y permeasas fúngicas implicadas en el transporte y metabolismo de azúcares

En el presente trabajo se caracterizaron glicosil hidrolasas (GHs) y permeasas fúngicas implicadas en el metabolismo y transporte de azúcares, con dos aplicaciones diferentes: la producción de etanol y la síntesis de isomaltooligosacáridos (IMOS). Se han abordado tres objetivos específicos: 1) desarrollar un proceso eficiente de sacarificación y fermentación simultánea (SSF) de celulosa; 2) comparar distintas estrategias para la fermentación de celobiosa, paso crítico en la fermentación de celulosa; 3) sintetizar IMOS utilizando como material catalítico células de levadura que producen una ¿-glucosidasa de Aspergillus niger. En el proceso propuesto de sacarificación y fermentación simultánea celulosa, el material de partida (papel de filtro) se digirió con una preparación enzimática de Trichodema reesei, y se fermentó con una cepa recombinante de Saccharomyces cerevisiae (T500) que secreta una ß-glucosidasa de Saccharomycopsis fibuligera. La actividad ß-glucosidasa, deficitaria en el cóctel celulolítico de T. reesei, mejoró el progreso de la hidrólisis de la celulosa y la fermentación, ya que disminuye el efecto inhibitorio causado por la acumulación de celobiosa. Con este proceso se alcanzaron rendimientos de etanol por encima de 70 g/L. Se han comparado estrategias de hidrólisis extracelular o intracelular de celobiosa. Para ello, se utilizó la cepa T500 y nuevas cepas recombinantes generadas en este estudio. En una primera aproximación para la hidrólisis intracelular, se ensayaron tres ß-glucosidasas distintas y una permeasa de celobiosa de Penicillium oxalicum. En los transformantes resultantes, la tasa de crecimiento en celobiosa se vio limitada por ß-glucosidasas con baja actividad celobiasa, pero por encima de cierto valor de actividad el principal cuello de botella fue el transporte del azúcar. Por esta razón, buscamos nuevos transportadores de celobiosa procedentes de T. reesei. De 107 secuencias designadas como transportadores de azúcares en el genoma de T. reesei, se seleccionaron diez por su mayor similitud de secuencia con permeasas de celobiosa de otros hongos caracterizadas funcionalmente. Solo una de ellas (Tr_StrC) fue capaz de facilitar el transporte de celobiosa y permitir el crecimiento de S. cerevisiae. Finalmente, se comparó la capacidad de fermentar celobiosa de los dobles transformantes de levadura, con capacidad de transportar e hidrolizar intracelularmente el disacárido, con la del transformante T500. La estrategia extracelular permitió una tasa de fermentación más rápida y mayores rendimientos de etanol en comparación con la estrategia intracelular. La cepa T500 también fue más eficiente en un sistema SSF de celulosa. Se han construido y analizado cepas recombinantes de S. cerevisiae que expresan un gen (aglA) que codifica una ¿-glucosidasa de A. niger. Los transformantes de levadura produjeron actividad ¿-glucosidasa extracelularmente, la mitad de la cual quedó asociada a células y la otra mitad fue liberada al medio de cultivo. Usando maltosa como único sustrato de partida, tras 8 h de incubación, el principal producto de transglicosilación fue panosa, pero tras 24 h el producto predominante fue isomaltosa. La isomaltosa también predominó a tiempos cortos de reacción, si en lugar de sólo maltosa se utilizaba de partida una mezcla de maltosa y glucosa. Para facilitar la síntesis de IMOS se diseñó un proceso en el cual las células de levadura pueden ser utilizadas directamente como material catalítico. Para ello, se expresaron en S. cerevisiae construcciones génicas del gen aglA fusionado con el gen de levadura SED1, en versión completa o truncada, que posee la secuencia GPI (glicosilfosfatidil inositol) de anclaje a pared celular. Las enzimas híbridas resultantes se fijaron de forma estable a la superficie celular. Las células provenientes de los cultivos recombinantes que expresan las construcciones aglA-SED1 se pudieron reciclar pIn the present work, glycosyl hydrolases (GHs) and fungal permeases involved in the metabolism and transport of sugars were characterized, with two different applications: the production of ethanol and the synthesis of isomaltooligosaccharides (IMOS). Three specific objectives have been addressed in order to: 1) develop an efficient process of saccharification and simultaneous fermentation (SSF) of cellulose; 2) compare different strategies for cellobiose fermentation, a critical step in cellulose fermentation; 3) synthesize IMOS using, as catalytic material, yeast cells that produce Aspergillus niger ¿-glucosidase. Strategies for extracellular or intracellular hydrolysis of cellobiose have been compared. To this end, the T500 strain and new recombinant strains generated in this study were used. In a first approach for intracellular hydrolysis, three different ß-glucosidases and a cellobiose permease from Penicillium oxalicum were tested. In the resulting transformants, growth rate in cellobiose was limited by ß-glucosidases with low cellobiase activity, but above a certain activity value the main bottleneck was sugar transport. For this reason, we searched novel cellobiose transporters from T. reesei. Among 107 sequences designated as sugar transporters in the genome of T. reesei, ten were selected based on their higher sequence similarity with functionally characterized cellobiose permeases from other fungi. Only one of them (Tr_StrC) was able to facilitate cellobiose transport and to allow growth of S. cerevisiae. Finally, the ability to ferment cellobiose of the double yeast transformants, capable of transporting and hydrolyzing the disaccharide intracellularly, was compared with that of the transformant T500. The extracellular strategy allowed a faster fermentation rate and higher ethanol yields compared to the intracellular approach. The T500 strain was also more efficient in a cellulose SSF system. Recombinant strains of S. cerevisiae expressing a gene (aglA) encoding an ¿-glucosidase from A. niger have been constructed and analyzed. The yeast transformants produced ¿-glucosidase activity extracellularly, half of which was cell-associated and the other half was released into the culture medium. Using maltose as the only initial substrate, after 8 h of incubation, the main product of transglycosylation was panose, but after 24 h the predominant product was isomaltose. Isomaltose also predominated at short reaction times, if a mixture of maltose and glucose was used instead of maltose alone. In order to facilitate the synthesis of IMOS, a process was designed in which the yeast cells can be used directly as catalytic material. For this purpose, the aglA coding region was fused to full-length or truncated versions of the yeast gene SED1, containing the GPI (glycosylphosphatidyl inositol) sequence for anchoring to the cell wall, and expressed in S. cerevisiae. The resulting hybrid enzymes were fixed stably to the cell surface. Cells from the recombinant cultures expressing the aglA-SED1 constructs could be recycled to produce IMOS in successive reactions.En aquest treball es van caracteritzar glicosil hidrolases (GHs) i permeases fúngiques implicades en el metabolisme i transport de sucres, amb dos aplicacions diferents: la producció d'etanol i la síntesi d'isomaltooligosacàrids (IMOS). S'han abordat tres objectius específics: 1) desenvolupar un procés eficient de sacarificació i fermentació simultània (SSF) de cel·lulosa; 2) comparar distintes estratègies per a la fermentació de celobiosa, pas crític en la fermentació de cel·lulosa; 3) sintetitzar IMOS utilizant com a material catalític cèl·lules de llevat que produeixen una ¿-glucosidasa de Aspergillus niger. En el procés proposat de sacarificació i fermentació simultània de cel·lulosa, el material de partida (paper de filtre) es va digerir amb una preparació enzimàtica de Trichodema reesei i es va fermentar amb una soca recombinant de Saccharomyces cerevisiae (T500), la qual secreta una ß-glucosidasa de Saccharomycopsis fibuligera. L'activitat ß-glucosidasa, deficitària en el còctel cel·lulolític de T. reesei, va millorar el progrés de la hidròlisi de la cel·lulosa i la fermentació, ja que disminuiex l'efecte inhibitori causat per l'acumulació de celobiosa. Amb aquest procés es van assolir rendiments d'etanol superiors a 70 g/L. S'han comparat estratègies d'hidròlisi extracel·lular o intracel·lular de celobiosa. Per a això, es va emprar la soca T500 i noves soques recombinants generades en aquest estudi. En una primera aproximació per a l'hidròlisi intracel·lular, s'assajaren tres ß-glucosidases distintes i una permeasa de celobiosa de Penicillium oxalicum. Als transformants resultants, la tasa de creixement amb celobiosa va estar limitada per ß-glucosidases amb baixa activitat celobiasa, però per damunt d'un cert valor d'activitat el principal coll d'ampolla va ser el transport del sucre. Per aquesta raó, cercàrem nous transportadors de celobiosa procedents de T. reesei. De 107 seqüències designades com a transportadors de sucres en el genoma de T. reesei, es van seleccionar deu per la seua major similitut de seqüència amb permeases de celobiosa d'altres fongs caracteritzades funcionalment. Només una d'elles (Tr_StrC) va ser capaç de facilitar el transport de celobiosa i permetre el creixement de S. cerevisiae. Finalment, es va comparar la capacitat de fermentar celobiosa dels dobles transformants de llevat, amb capacitat de transportar i hidrolitzar intracel·lularment el disacàrid, amb la del transformant T500. La estratègia extracel·lular va permetre una tasa de fermentació més ràpida i majors rendiments d'etanol en comparació amb la estratègia intracel·lular. La soca T500 també va ser més eficient en un sistema SSF de cel·lulosa. S'han construït i analitzat soques recombinants de S. cerevisiae que expresen un gen (aglA) que codifica una ¿-glucosidasa de A. niger. Els transformants de llevat produiren activitat ¿-glucosidasa extracelul·larment, la meitat de la qual va quedar associada a cèl·lules i l'altra meitat va ser alliberada al medi de cultiu. Utilitzant maltosa com a únic substrat de partida, després de 8 h d'incubació, el principal producte de transglicosilació va serpanosa, però després de 24 h el producte predominant va ser isomaltosa. La isomaltosa també va predominar a tiemps curts de reacció, si en lloc de només maltosa s'utilizava de partida una combinació de maltosa i glucosa. Per a facilitar la síntesi de IMOS es va dissenyar un procés en el qual les cèl·lules de llevat poden ser utilitzades directament com a material catalític. Per a això, s'expresaren en S. cerevisiae construccions gèniques del gen aglA fusionat amb el gen de llevat SED1, en versió completa o truncada, el qual conté la seqüència GPI (glicosilfosfatidil inositol) d'ancoratge a paret cel·lular. Els enzims híbrids resultants es van fixar de forma estable a la superfície cel·lular. Les cèl·lules provinents dels cultius recombinants que expCasa Villegas, MF. (2018). Caracterización de glicosidasas y permeasas fúngicas implicadas en el transporte y metabolismo de azúcares [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/106344TESI

Novel Insights into the Transmission of SARS-CoV-2 Through the Ocular Surface and its Detection in Tears and Conjunctival Secretions: A Review

SARS-CoV-2 is a highly transmissible virus that spreads mainly via person-to-person contact through respiratory droplets, or through contact with contaminated objects or surfaces from an infected person. At present we are passing through a phase of slow and painful understanding of the origin, epidemiological profile, clinical spectrum, and risk profile of the virus. To the best of our knowledge there is only limited and contradictory evidence concerning SARS-CoV-2 transmission through other routes. Importantly, the eye may constitute not only a potential site of virus replication but also an alternative transmission route of the virus from the ocular surface to the respiratory and gastrointestinal tract. It is therefore imperative to gain a better insight into the potential ophthalmological transmission route of the virus and establish directions on best practice and future models of care for ophthalmological patients. This review article critically evaluates available evidence on the ophthalmological mode of viral transmission and the value of earlier identification of the virus on the eye. More evidence is urgently needed to better evaluate the need for protective measures and reliable ocular diagnostic tests to diminish further pandemic spread

Tear and aqueous humour cytokine profile in primary open‐angle glaucoma

Purpose: To evaluate the concentrations of pro‐inflammatory cytokines in tear and aqueous humour of patients with primary open‐angle glaucoma (POAG), relative to healthy controls. Method: Tear and aqueous humour samples were collected from 29 healthy controls and 27 POAG patients. Twenty‐seven inflammatory cytokines were analysed: interleukin (IL)‐1β, IL‐1ra, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐8, IL9, IL‐10, IL‐12, IL‐13, IL‐15, IL‐17, eotaxin, fibroblast growth factor (FGF) basic, granulocyte colony‐stimulating factor, granulocyte‐monocyte colony‐stimulating factor, interferon (IFN)‐γ, interferon gamma‐induced protein, monocyte chemo‐attractant protein‐1, macrophage inflammatory protein (MIP)‐1α, MIP‐1β, platelet‐derived growth factor, regulated on activation normal T cell expressed and secreted, tumour necrosis factor (TNF)‐α and vascular endothelial growth factor (VEGF). Results: In tear samples of glaucoma patients, an increase in IL‐4, IL‐12, IL‐15, FGF‐basic and VEGF was observed, as well as a decrease in MIP‐1a relative to the control group (p < 0.05). IL‐5, IL‐12, IL‐15, IFN‐γ and MIP‐1a were significantly higher in aqueous humour of glaucoma eyes (p < 0.05). A poor correlation between cytokine levels in tear and aqueous humour was observed. Conclusion: The different profiles of inflammatory marker expression of patients with POAG and healthy controls confirm the inflammatory activity of the pathology, indicating that some of them could be used as potential biomarkers of this disease

Proinflammatory cytokine profile differences between primary open angle and pseudoexfoliative glaucoma

Introduction: Few studies have investigated glaucoma biomarkers in aqueous humor and tear and have found elevations of proinflammatory cytokines in patients with primary open-angle glaucoma (POAG) and pseudoexfoliative glaucoma (PXG). In this study we investigate differences in inflammatory cytokines between POAG and PXG patients to find specific disease biomarkers. Methods: For this purpose, tear and aqueous humor samples of 14 eyes with POAG and 15 eyes with PXG undergoing cataract surgery were immunoassayed for 27 pro-inflammatory cytokines. The concentrations of cytokines in tear and aqueous humor and their association with clinical variables were analysed, correlated and compared between the groups. Results: We found that the levels of three cytokines differed significantly in the aqueous humor of POAG and PXG patients: IL-12 and IL-13 were higher in the POAG group, while MCP-1(MCAF) was higher in the PXG group. The number of topical hypotensive medications was correlated with diminished levels of two cytokines (IL-7 and basic fibroblast growth factor) in aqueous humor in the POAG group and with diminished levels of IL-12 in tear in the PXG group. Conclusion: We conclude that both POAG and PXG show elevated concentrations of proinflammatory cytokines in tear and aqueous humor that could be used as biomarkers for these types of glaucoma and that the concentrations in aqueous humor of three cytokines: IL-12, IL-13 and MCP-1(MCAF) could be used to differentiate POAG and PXG

Optic nerve and macular optical coherence tomography in recovered COVID-19 patients

Purpose: To investigate the peripapillary retinal nerve fiber layer thickness (RNFLT), macular RNFLT, ganglion cell layer (GCL), and inner plexiform layer (IPL) thickness in recovered COVID-19 patients compared to controls. Methods: Patients previously diagnosed with COVID-19 were included, while healthy patients formed the historic control group. All patients underwent an ophthalmological examination, including macular and optic nerve optical coherence tomography. In the case group, socio-demographic data, medical history, and neurological symptoms were collected. Results: One hundred sixty patients were included; 90 recovered COVID-19 patients and 70 controls. COVID-19 patients presented increases in global RNFLT (mean difference 4.3; CI95% 0.8 to 7.7), nasal superior (mean difference 6.9; CI95% 0.4 to 13.4), and nasal inferior (mean difference 10.2; CI95% 2.4 to 18.1) sectors of peripapillary RNFLT. Macular RNFL showed decreases in COVID-19 patients in volume (mean difference −0.05; CI95% −0.08 to −0.02), superior inner (mean difference −1.4; CI95% −2.5 to −0.4), nasal inner (mean difference −1.1; CI95% −1.8 to −0.3), and nasal outer (mean difference −4.7; CI95% −7.0 to −2.4) quadrants. COVID-19 patients presented increased GCL thickness in volume (mean difference 0.04; CI95% 0.01 to 0.07), superior outer (mean difference 2.1; CI95% 0.8 to 3.3), nasal outer (mean difference 2.5; CI95% 1.1 to 4.0), and inferior outer (mean difference1.2; CI95% 0.1 to 2.4) quadrants. COVID-19 patients with anosmia and ageusia presented an increase in peripapillary RNFLT and macular GCL compared to patients without these symptoms. Conclusions: SARS-CoV-2 may affect the optic nerve and cause changes in the retinal layers once the infection has resolved

Neither 1 G nor 2 G fuel ethanol: setting the ground for a sugarcane-based biorefinery using an iSUCCELL yeast platform

First-generation (1 G) fuel ethanol production in sugarcane-based biorefineries is an established economic enterprise in Brazil. Second-generation (2 G) fuel ethanol from lignocellulosic materials, though extensively investigated, is currently facing severe difficulties to become economically viable. Some of the challenges inherent to these processes could be resolved by efficiently separating, and partially hydrolysing the cellulosic fraction of the lignocellulosic materials into the disaccharide cellobiose. Here we propose an alternative biorefinery, where the sucrose-rich stream from the 1 G process is mixed with a cellobiose-rich stream in the fermentation step. The advantages of mixing are threefold: 1) decreased concentrations of metabolic inhibitors that are typically produced during pretreatment and hydrolysis of lignocellulosic materials; 2) decreased cooling times after enzymatic hydrolysis prior to fermentation; 3) decreased availability of free glucose for contaminating microorganisms and undesired glucose repression effects. The iSUCCELL platform will be built upon the robust Saccharomyces cerevisiae strains currently present in 1 G biorefineries, which offer competitive advantage in non-aseptic environments, and into which intracellular hydrolyses of sucrose and cellobiose will be engineered. It is expected that high yields of ethanol can be achieved in a process with cell recycling, lower contamination levels and decreased antibiotic use, when compared to current 2 G technologies

Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger α-Glucosidase

The α-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular α-glucosidase activity about half of which appeared cell bound whereas the other half was released into the culture medium. With maltose as the substrate, panose is the main transglycosylation product after 8 h of incubation, whereas isomaltose is predominant after 24 h. Isomaltose also becomes predominant at shorter times if a mixture of maltose and glucose is used instead of maltose. To facilitate IMO production, we have designed a procedure by which yeast cells can be used directly as the catalytic agent. For this purpose, we expressed in S. cerevisiae gene constructs in which the aglA gene is fused to glycosylphosphatidylinositol anchor sequences, from the yeast SED1 gene, that determine the covalent binding of the hybrid protein to the cell membrane. The resulting hybrid enzymes were stably attached to the cell surface. The cells from cultures of recombinant yeast strains expressing aglA-SED1 constructions can be used to produce IMOs in successive batches.This work was funded by grants AGL2016-75245-R and BIO2013-48779-C4-3-R from Spain’s “Secretaría de Estado de Investigación, Desarrollo e Innovación”. M.C.-V. was supported by a SENESCYT predoctoral fellowship from the Government of República del Ecuador.Peer reviewe

Systemic treatment with 7,8-Dihydroxiflavone activates TtkB and affords protection of two different retinal ganglion cell populations against axotomy in adult rats

Purpose: To analyze responses of different RGC populations to left intraorbital optic nerve transection (IONT) and intraperitoneal (i.p.) treatment with 7,8-Dihydroxyflavone (DHF), a potent selective TrkB agonist. Methods: Adult albino Sprague-Dawley rats received, following IONT, daily i.p. injections of vehicle (1%DMSO in 0.9%NaCl) or DHF. Group-1 (n = 58) assessed at 7days (d) the optimal DHF amount (1–25 mg/kg). Group-2, using freshly dissected naïve or treated retinas (n = 28), investigated if DHF treatment was associated with TrkB activation using Western-blotting at 1, 3 or 7d. Group-3 (n = 98) explored persistence of protection and was analyzed at survival intervals from 7 to 60d after IONT. Groups 2–3 received daily i.p. vehicle or DHF (5 mg/kg). Retinal wholemounts were immunolabelled for Brn3a and melanopsin to identify Brn3a+RGCs and m+RGCs, respectively. Results: Optimal neuroprotection was achieved with 5 mg/kg DHF and resulted in TrkB phosphorylation. The percentage of surviving Brn3a+RGCs in vehicle treated rats was 60, 28, 18, 13, 12 or 8% of the original value at 7, 10, 14, 21, 30 or 60d, respectively, while in DHF treated retinas was 94, 70, 64, 17, 10 or 9% at the same time intervals. The percentages of m+RGCs diminished by 7d–13%, and recovered by 14d–38% in vehicle-treated and to 48% in DHF-treated retinas, without further variations. Conclusions: DHF neuroprotects Brn3a + RGCs and m + RGCs; its protective effects for Brn3a+RGCs are maximal at 7 days but still significant at 21d, whereas for m+RGCs neuroprotection was significant at 14d and permanent

Analyses of pseudoexfoliation aqueous humor lipidome

Pseudoexfoliation syndrome (PEX) is a systemic disorder that manifests as a fluffy, proteinaceous fibrillar material throughout the body. In the eye, such deposits result in glaucoma (PEXG), due to impeding aqueous humor outflow. Serum lipid alterations and increased lipid peroxidation have been reported in PEX. We report the first ever comprehensive lipid profiling of the aqueous humor (AH) of PEXG. Our untargeted lipidomic analysis of 23 control, 19 primary open angle glaucoma (POAG), 9 PEX, and 14 PEXG AH patients resulted in the identification of 489 lipid species within 26 lipid classes across PEX, PEXG, POAG, and control AH samples. Multiple cholesterol esters (ChEs), phosphatidylcholines (PCs), triglycerides (TGs), and ceramides (Cers) were present in higher concentrations in the PEXG AH than in all other groups. CerG2GNAc1(d34 : 1) was enriched in control samples and depleted in both the PEX and PEXG samples. Machine learning prediction with three supervised logistic regression binary classification tasks showed (1) POAG vs. control, with an 86% accuracy, (2) PEXG vs. control, with a 71% accuracy and (3) PEX vs. control, with an 86% accuracy. In conclusion, the analysis showed that the control (mean peak area 13.54 ± 6) had, on average, a higher total lipid content than the PEX, PEXG, and POAG AH samples. Elevations in Apolipoprotein A-I (APOA1) correlated with an increased abundance of PC lipid species in the AH of patients with PEXG. PC (18 : 0/18 : 2), PC (36 : 2), and PC (34 : 1e) are in low concentrations for PEX but are highly concentrated in PEXG, despite both having similar material deposits, suggesting that they are fundamentally different in composition

One-Year Changes in Optic Nerve Head Parameters in Recovered COVID-19 Patients

Background: The main purpose was to evaluate the changes in peripapillary retinal nerve fiber layer (RNFL) thickness and vessel density (VD) in post–COVID-19 patients during 12-month follow-up. Methods: In this prospective study, patients with COVID-19 who were attended in the Hospital Clinico San Carlos (Madrid, Spain) were included. All patients underwent a complete ophthalmological examination, optic nerve head optical coherence tomography (OCT), and OCT angiography (OCTA) using the Cirrus HD-OCT 5,000 with AngioPlex OCTA 1, 3, and 12 months after laboratory-confirmed diagnosis. Sociodemographic data, medical history, disease severity, and laboratory workup were registered. Results: A total of 180 eyes of 90 patients with SARS-CoV-2 infection were included; the mean age was 55.5 ± 8.9 years, and 46 patients (51%) were females. The mean visual acuity was 0.76 ± 0.16, and no abnormalities attributable to SARS-CoV-2 were detected in the ocular or fundus examination. No differences in the OCT and OCTA data were found between severity groups in each visit (all P > 0.05). Overall, there was a decrease in RNFL global thickness (P < 0.001) from the first to the last visit, and an increase in VD and flux index was noted in some sectors at the 12-month examination. A significant correlation was detected at 12 months between vascularization parameters and RNFL thickness. Conclusions: One year after SARS-CoV-2 infection, changes in peripapillary RNFL thickness and vascularization occur, possibly indicating a recovery in such parameters